Search

Accumulated experimental evidence has shown that viruses recruit the host intracellular machinery to establish infection. It has recently been shown that the potyvirus Turnip mosaic virus (TuMV) transits through the late endosome (LE) for viral genome replication, but it is still largely unknown how the viral replication vesicles labelled by the TuMV membrane protein 6K2 target LE. To further understand the underlying mechanism, we studied the involvement of the vacuolar sorting receptor (VSR) family proteins from Arabidopsis in this process. We now report the identification of VSR4 as a new host factor required for TuMV infection. VSR4 interacted specifically with TuMV 6K2 and was required for targeting of 6K2 to enlarged LE. Following overexpression of VSR4 or its recycling-defective mutant that accumulates in the early endosome (EE), 6K2 did not employ the conventional VSR-mediated EE to LE pathway, but targeted enlarged LE directly from cis-Golgi and viral replication was enhanced. In addition, VSR4 can be N-glycosylated and this is required for its stability and for monitoring 6K2 trafficking to enlarged LE. A non-glycosylated VSR4 mutant enhanced the dissociation of 6K2 from cis-Golgi, leading to the formation of punctate bodies that targeted enlarged LE and to more robust viral replication than with glycosylated VSR4. Finally, TuMV hijacks N-glycosylated VSR4 and protects VSR4 from degradation via the autophagy pathway to assist infection. Taken together, our results have identified a host factor VSR4 required for viral replication vesicles to target endosomes for optimal viral infection and shed new light on the role of N-glycosylation of a host factor in regulating viral infection., Author summary A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host endomembrane system to produce a membranous replication organelle. Recent reports suggest that the late endosome (LE) serves as a replication site for the potyvirus Turnip mosaic virus (TuMV), but the mechanism(s) by which TuMV replication vesicles target LE are far from being fully elucidated. Identification of the host factors involved in this transport process could lead to new strategies to combat TuMV infection. In this report, we provide evidence that TuMV replication depends on functional vesicle transport from cis-Golgi to the enlarged LE pathway that is mediated by a specific VSR family member, VSR4, from Arabidopsis. Knock out of VSR4 impaired the targeting of TuMV replication vesicles to enlarged LE and suppressed viral infection, and this process depends on the specific interaction between VSR4 and the viral replication vesicle-forming protein 6K2. We also showed that N-glycosylation of VSR4 modulates the targeting of TuMV replication vesicles to enlarged LE and enhances viral infection, thus contributing to our understanding of how TuMV manipulates host factors in order to establish optimal infection. These results may have implications for the role of VSR in other positive-strand RNA viruses.

Dengue virus (DENV) disruption of the innate immune response is critical to establish infection. DENV non-structural protein 5 (NS5) plays a central role in this disruption, such as antagonism of STAT2. We recently found that DENV serotype 2 (DENV2) NS5 interacts with Polymerase associated factor 1 complex (PAF1C). The primary members of PAF1C are PAF1, LEO1, CTR9, and CDC73. This nuclear complex is an emerging player in the immune response. It promotes the expression of many genes, including genes related to the antiviral, antimicrobial and inflammatory responses, through close association with the chromatin of these genes. Our previous work demonstrated that NS5 antagonizes PAF1C recruitment to immune response genes. However, it remains unknown if NS5 antagonism of PAF1C is complementary to its antagonism of STAT2. Here, we show that knockout of PAF1 enhances DENV2 infectious virion production. By comparing gene expression profiles in PAF1 and STAT2 knockout cells, we find that PAF1 is necessary to express immune response genes that are STAT2-independent. Finally, we mapped the viral determinants for the NS5-PAF1C protein interaction. We found that NS5 nuclear localization and the C-terminal region of the methyltransferase domain are required for its interaction with PAF1C. Mutation of these regions rescued the expression of PAF1-dependent immune response genes that are antagonized by NS5. In sum, our results support a role for PAF1C in restricting DENV2 replication that NS5 antagonizes through its protein interaction with PAF1C., Author summary Dengue virus (DENV) is a pathogen that infects nearly 400 million people a year and thus represents a major challenge for public health. Productive infection by DENV relies on the effective evasion of intrinsic antiviral defenses and is often accomplished through virus-host protein interactions. Here, we investigate the recently discovered interaction between DENV non-structural protein 5 (NS5) and the transcriptional regulator Polymerase associated factor 1 complex (PAF1C). Our work demonstrates PAF1C member PAF1 acts as an antiviral factor and inhibits DENV replication. In parallel, we identified immune response genes involved in intrinsic antiviral defense that depend on PAF1 for expression. We further identified the regions of NS5 required for the protein interaction with PAF1C. Breaking the NS5-PAF1C protein interaction restores the expression of PAF1-dependent immune response genes. Together, our work establishes the antiviral role of PAF1C in DENV infection and NS5 antagonism of PAF1-dependent gene expression through a virus-host protein interaction.

The cellular prion protein PrPC is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels of PrPC may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC expression. We cultured human U251-MG glioblastoma cells in the presence of 64’752 unique siRNAs targeting 21’584 annotated human genes, and measured PrPC using a one-pot fluorescence-resonance energy transfer immunoassay in 51’128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC. When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPC biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPC expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies., Author summary The cellular prion protein (PrPC) acts as both, the substrate for prion formation and mediator of prion toxicity during the progression of all prion diseases. Suppressing the levels of PrPC is a viable therapeutic strategy as PRNP null animals are resistant to prion disease and the knockout of PRNP is not associated with any severe phenotypes. Motivated by the scarcity of knowledge regarding the molecular regulators of PrPC biosynthesis and degradation, which might serve as valuable targets to control its expression, here, we present a cell-based genome wide RNAi screen in arrayed format. The screening effort led to the identification of 54 regulators, nine of which were confirmed by an independent CRISPR-based method. Among the final nine targets, we identified PUM1 as a regulator of PRNP mRNA by acting on the 3’UTR promoting its degradation. The newly identified factors involved in the life cycle of PrPC provided by our study may also represent themselves as therapeutic targets for the intervention of prion diseases.

Hepatitis C virus (HCV) infection induces the degradation and decreases the secretion of apolipoprotein B (ApoB). Impaired production and secretion of ApoB-containing lipoprotein is associated with an increase in hepatic steatosis. Therefore, HCV infection-induced degradation of ApoB may contribute to hepatic steatosis and decreased lipoprotein secretion, but the mechanism of HCV infection-induced ApoB degradation has not been completely elucidated. In this study, we found that the ApoB level in HCV-infected cells was regulated by proteasome-associated degradation but not autophagic degradation. ApoB was degraded by the 20S proteasome in a ubiquitin-independent manner. HCV induced the oxidation of ApoB via oxidative stress, and oxidized ApoB was recognized by the PSMA5 and PSMA6 subunits of the 20S proteasome for degradation. Further study showed that ApoB was degraded at endoplasmic reticulum (ER)-associated lipid droplets (LDs) and that the retrotranslocation and degradation of ApoB required Derlin-1 but not gp78 or p97. Moreover, we found that knockdown of ApoB before infection increased the cellular lipid content and enhanced HCV assembly. Overexpression of ApoB-50 inhibited lipid accumulation and repressed viral assembly in HCV-infected cells. Our study reveals a novel mechanism of ApoB degradation and lipid accumulation during HCV infection and might suggest new therapeutic strategies for hepatic steatosis., Author summary Hepatitis C virus (HCV) infection induces the degradation of apolipoprotein B (ApoB), which is the primary apolipoprotein in low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL). Impaired production and secretion of ApoB-containing lipoprotein is associated with an increase in hepatic steatosis. Thus, ApoB degradation might contribute to HCV infection-induced fatty liver. Here, we found that ApoB was not degraded through endoplasmic reticulum-associated degradation (ERAD) or autophagy, as reported previously. Instead, HCV infection induced ApoB oxidation through oxidative stress, and oxidatively damaged ApoB could be recognized and directly degraded by the 20S proteasome. We also found that ApoB was retrotranslocated from the endoplasmic reticulum (ER) to lipid droplets (LDs) for degradation. Through overexpression of ApoB-50, which can mediate the assembly and secretion of LDL and VLDL, we confirmed that ApoB degradation contributed to hepatocellular lipid accumulation induced by HCV infection. Additionally, expression of ApoB-50 impaired HCV production due to the observed decrease in lipid accumulation. In this study, we identified new mechanisms of ApoB degradation and HCV-induced lipid accumulation, and our findings might facilitate the development of novel therapeutic strategies for HCV infection-induced fatty liver.

The capsid (CA) lattice of the HIV-1 core plays a key role during infection. From the moment the core is released into the cytoplasm, it interacts with a range of cellular factors that, ultimately, direct the pre-integration complex to the integration site. For integration to occur, the CA lattice must disassemble. Early uncoating or a failure to do so has detrimental effects on virus infectivity, indicating that an optimal stability of the viral core is crucial for infection. Here, we introduced cysteine residues into HIV-1 CA in order to induce disulphide bond formation and engineer hyper-stable mutants that are slower or unable to uncoat, and then followed their replication. From a panel of mutants, we identified three with increased capsid stability in cells and found that, whilst the M68C/E212C mutant had a 5-fold reduction in reverse transcription, two mutants, A14C/E45C and E180C, were able to reverse transcribe to approximately WT levels in cycling cells. Moreover, these mutants only had a 5-fold reduction in 2-LTR circle production, suggesting that not only could reverse transcription complete in hyper-stable cores, but that the nascent viral cDNA could enter the nuclear compartment. Furthermore, we observed A14C/E45C mutant capsid in nuclear and chromatin-associated fractions implying that the hyper-stable cores themselves entered the nucleus. Immunofluorescence studies revealed that although the A14C/E45C mutant capsid reached the nuclear pore with the same kinetics as wild type capsid, it was then retained at the pore in association with Nup153. Crucially, infection with the hyper-stable mutants did not promote CPSF6 re-localisation to nuclear speckles, despite the mutant capsids being competent for CPSF6 binding. These observations suggest that hyper-stable cores are not able to uncoat, or remodel, enough to pass through or dissociate from the nuclear pore and integrate successfully. This, is turn, highlights the importance of capsid lattice flexibility for nuclear entry. In conclusion, we hypothesise that during a productive infection, a capsid remodelling step takes place at the nuclear pore that releases the core complex from Nup153, and relays it to CPSF6, which then localises it to chromatin ready for integration., Author summary The mature viral core of human immunodeficiency virus (HIV) consists of a highly organised lattice formed by capsid molecules that encloses the viral RNA and viral enzymes. This lattice is crucial during the early stages of viral replication, as it has to break down–uncoat–at the right time and place in order for the viral DNA to integrate successfully. Lentiviruses, like HIV, can infect non-dividing cells and are able to access the host cell DNA by entering the nucleus through nuclear pores. Until recently, uncoating was thought to occur in the cytoplasm as the whole core was thought too large to pass through the nuclear pore. However, lately it has been suggested that uncoating might occur at the nuclear pore or even inside the nucleus and the site of uncoating is currently hotly debated. By investigating HIV mutants with an increased lattice stability, we have shown that lattice flexibility is crucial for nuclear entry. Provocatively, we observed hyper-stable mutant capsid in nuclear and chromatin-associated fractions suggesting that uncoating is not required for nuclear entry. Nonetheless, microscopy experiments suggested that these hyper-stable mutants were retained on the inner side of the nuclear pore, and were impaired for downstream events in the nucleus, including CPSF6 accumulation in nuclear speckles, leading to a severe infectivity defect. Therefore, we believe that an essential uncoating, or capsid lattice remodelling event normally takes place at the nuclear pore.

Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1α and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release (~1,000-fold). Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection., Author summary SARS-CoV-2 is the novel coronavirus responsible for the COVID-19 pandemic which has resulted in over 150 million cases since the end of 2019. Most people infected with the virus will experience mild to moderate respiratory illness and recover without any special treatment. However, older people, and those with underlying medical problems like chronic respiratory disease are more likely to develop a serious illness. So far, more than 3 million people have died of COVID-19. Unfortunately, there is no specific medication for this viral disease. In order to produce viral proteins and to replicate their genetic information, all coronaviruses use a cellular structure known as the endoplasmic reticulum or ER. However, the massive production and modification of viral proteins stresses the ER and this activates a compensatory cellular response that tries to reduce ER protein levels. This is termed the unfolded protein response or UPR. We believe that coronaviruses take advantage of the activation of the UPR to enhance their replication. The UPR is also activated in some types of cancer and neurodegenerative disorders and UPR inhibitor drugs have been developed to tackle these diseases. Here, we show also that these compounds can significantly reduce SARS-CoV-2 replication in human lung cells.

Tyrosine phosphatases are often weaponized by bacteria colonizing mucosal barriers to manipulate host cell signal transduction pathways. Porphyromonas gingivalis is a periodontal pathogen and emerging oncopathogen which interferes with gingival epithelial cell proliferation and migration, and induces a partial epithelial mesenchymal transition. P. gingivalis produces two tyrosine phosphatases, and we show here that the low molecular weight tyrosine phosphatase, Ltp1, is secreted within gingival epithelial cells and translocates to the nucleus. An ltp1 mutant of P. gingivalis showed a diminished ability to induce epithelial cell migration and proliferation. Ltp1 was also required for the transcriptional upregulation of Regulator of Growth and Cell Cycle (RGCC), one of the most differentially expressed genes in epithelial cells resulting from P. gingivalis infection. A phosphoarray and siRNA showed that P. gingivalis controlled RGCC expression through Akt, which was activated by phosphorylation on S473. Akt activation is opposed by PTEN, and P. gingivalis decreased the amount of PTEN in epithelial cells. Ectopically expressed Ltp1 bound to PTEN, and reduced phosphorylation of PTEN at Y336 which controls proteasomal degradation. Ltp-1 induced loss of PTEN stability was prevented by chemical inhibition of the proteasome. Knockdown of RGCC suppressed upregulation of Zeb2 and mesenchymal markers by P. gingivalis. RGCC inhibition was also accompanied by a reduction in production of the proinflammatory cytokine IL-6 in response to P. gingivalis. Elevated IL-6 levels can contribute to periodontal destruction, and the ltp1 mutant of P. gingivalis incited less bone loss compared to the parental strain in a murine model of periodontal disease. These results show that P. gingivalis can deliver Ltp1 within gingival epithelial cells, and establish PTEN as the target for Ltp1 phosphatase activity. Disruption of the Akt1/RGCC signaling axis by Ltp1 facilitates P. gingivalis-induced increases in epithelial cell migration, proliferation, EMT and inflammatory cytokine production., Author summary Bacteria colonizing the oral cavity can induce inflammatory destruction of the periodontal tissues, and are increasingly associated with oral squamous cell carcinoma. P. gingivalis, a major periodontal pathogen, can subvert epithelial pathways that control important physiological processes relating to innate immunity and cell fate; however, little is known about the effector molecules. Here we show that P. gingivalis can deliver a tyrosine phosphatase, Ltp1, within epithelial cells, and Ltp1 phosphatase activity destabilizes PTEN, a negative regulator of Akt1 signaling. The production of RGCC is thus increased and this leads to increased epithelial cell migration, proliferation, a partial mesenchymal phenotype and inflammatory cytokine production. Ltp1 phosphatase activity thus provides a mechanistic basis for a number of P. gingivalis properties that contribute to disease. Indeed, an Ltp1-deficient mutant was less pathogenic in a murine model of periodontitis. These results contribute to deciphering the pathophysiological events that underlie oral bacterial diseases that initiate at mucosal barriers.

LINE-1 (L1) retrotransposons are autonomous transposable elements that can affect gene expression and genome integrity. Potential consequences of exogenous viral infections for L1 activity have not been studied to date. Here, we report that hepatitis C virus (HCV) infection causes a significant increase of endogenous L1-encoded ORF1 protein (L1ORF1p) levels and translocation of L1ORF1p to HCV assembly sites at lipid droplets. HCV replication interferes with retrotransposition of engineered L1 reporter elements, which correlates with HCV RNA-induced formation of stress granules and can be partially rescued by knockdown of the stress granule protein G3BP1. Upon HCV infection, L1ORF1p localizes to stress granules, associates with HCV core in an RNA-dependent manner and translocates to lipid droplets. While HCV infection has a negative effect on L1 mobilization, L1ORF1p neither restricts nor promotes HCV infection. In summary, our data demonstrate that HCV infection causes an increase of endogenous L1 protein levels and that the observed restriction of retrotransposition of engineered L1 reporter elements is caused by sequestration of L1ORF1p in HCV-induced stress granules., Author summary Members of the Long Interspersed Nuclear Element 1 (LINE-1, L1) class of retrotransposons account for ~17% of the human genome and include ~100–150 intact L1 loci that are still functional. L1 mobilization is known to affect genomic integrity, thereby leading to disease-causing mutations, but little is known about the impact of exogenous viral infections on L1 and vice versa. While L1 retrotransposition is controlled by various mechanisms including CpG methylation, hypomethylation of L1 has been observed in hepatocellular carcinoma tissues of hepatitis C virus (HCV)-infected patients. Here, we demonstrate molecular interactions between HCV and L1 elements. HCV infection stably increases cellular levels of the L1-encoded ORF1 protein (L1ORF1p). HCV core and L1ORF1p interact in ribonucleoprotein complexes that traffic to lipid droplets. Despite its redistribution to HCV assembly sites, L1ORF1p is dispensable for HCV infection. In contrast, retrotransposition of engineered L1 reporter elements is restricted by HCV, correlating with an increased formation of L1ORF1p-containing cytoplasmic stress granules. Thus, our data provide first insights into the molecular interplay of endogenous transposable elements and exogenous viruses that might contribute to disease progression in vivo.

Both Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) establish the persistent, life-long infection primarily at the latent status, and associate with certain types of tumors, such as B cell lymphomas, especially in immuno-compromised individuals including people living with HIV (PLWH). Lytic reactivation of these viruses can be employed to kill tumor cells harboring latently infected viral episomes through the viral cytopathic effects and the subsequent antiviral immune responses. In this study, we identified that polo-like kinase 1 (PLK1) is induced by KSHV de novo infection as well as lytic switch from KSHV latency. We further demonstrated that PLK1 depletion or inhibition facilitates KSHV reactivation and promotes cell death of KSHV-infected lymphoma cells. Mechanistically, PLK1 regulates Myc that is critical to both maintenance of KSHV latency and support of cell survival, and preferentially affects the level of H3K27me3 inactive mark both globally and at certain loci of KSHV viral episomes. Furthremore, we recognized that PLK1 inhibition synergizes with STAT3 inhibition to efficiently induce KSHV reactivation. We also confirmed that PLK1 depletion or inhibition yields the similar effect on EBV lytic reactivation and cell death of EBV-infected lymphoma cells. Lastly, we noticed that PLK1 in B cells is elevated in the context of HIV infection and caused by HIV Nef protein to favor KSHV/EBV latency., Author summary KSHV is a large double-stranded DNA virus belonging to human gamma-herpesviruses and capable of establishing the long-term persistent infection, which causes cancers under immunocompromised states, such as acquired immunodeficiency syndrome (AIDS). There are currently still no effective therapies to specifically treat KSHV-associated tumors, nor eliminate its persistent infection. In this study, we illustrated that PLK1 is upregulated by KSHV, which in return suppresses KSHV lytic infection. We further demonstrated that PLK1 is a novel host target that can be inhibited pharmacologically to benefit the viral oncolysis for promotion of KSHV lytic reactivation and elimination of KSHV-positive lymphoma cells, particularly for people living with HIV (PLWH). PLK1 inhibitors also exerted the similar effect on EBV that is the other member of human gamma-herpesvirus.

Platelet-derived growth factor receptor alpha (PDGFR��) serves as an entry receptor for the human cytomegalovirus (HCMV), and soluble PDGFR��-Fc can neutralize HCMV at a halfmaximal effective concentration (EC50) of about 10 ng/ml. While this indicates a potential for usage as an HCMV entry inhibitor PDGFR��-Fc can also bind the physiological ligands of PDGFR�� (PDGFs), which likely interferes with the respective signaling pathways and represents a potential source of side effects. Therefore, we tested the hypothesis that interference with PDGF signaling can be prevented by mutations in PDGFR��-Fc or combinations thereof, without losing the inhibitory potential for HCMV. To this aim, a targeted mutagenesis approach was chosen. The mutations were quantitatively tested in biological assays for interference with PDGF-dependent signaling as well as inhibition of HCMV infection and biochemically for reduced affinity to PDGF-BB, facilitating quantification of PDGFR��-Fc selectivity for HCMV inhibition. Mutation of Ile 139 to Glu and Tyr 206 to Ser strongly reduced the affinity for PDGF-BB and hence interference with PDGF-dependent signaling. Inhibition of HCMV infection was less affected, thus increasing the selectivity by factor 4 and 8, respectively. Surprisingly, the combination of these mutations had an additive effect on binding of PDGF-BB but not on inhibition of HCMV, resulting in a synergistic 260fold increase of selectivity. In addition, a recently reported mutation, Val 242 to Lys, was included in the analysis. PDGFR��-Fc with this mutation was fully effective at blocking HCMV entry and had a drastically reduced affinity for PDGF-BB. Combining Val 242 to Lys with Ile 139 to Glu and/or Tyr 206 to Ser further reduced PDGF ligand binding beyond detection. In conclusion, this targeted mutagenesis approach identified combinations of mutations in PDGFR��-Fc that prevent interference with PDGF-BB but maintain inhibition of HCMV, which qualifies such mutants as candidates for the development of HCMV entry inhibitors., publishedVersion

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrPC, into a plethora of assemblies, PrPSc, associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrPSc structures. However, the degree to which intra- and inter-strain PrPSc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrPSc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrPSc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrPSc particles. We found that each strain had a continuum of PrPSc assemblies, with strong correlation between PrPSc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrPSc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrPSc took place at a hydrodynamic radius (Rh) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrPSc subpopulations and demonstrate that while PrPSc quaternary structure is a major contributor to PrPSc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrPSc particles from maintaining proteinase K resistance below an Rh of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype., Author summary Prion diseases are neurodegenerative diseases that include bovine spongiform encephalopathy (BSE or mad cow disease) in cattle, chronic wasting disease (CWD) in cervids and Creutzfeldt-Jakob disease (CJD) in humans. These diseases are caused by self-propagated misfolding and aggregation of the naturally occurring prion protein. Variations in the structure of prion aggregates are associated with distinct disease phenotypes, but how this prion structural heterogeneity translates into clinical presentation has been difficult to determine, largely because it is technically difficult to isolate and characterize the full range of prion structures from prion-infected brain. Here, we overcame this challenge by using a versatile fractionation technique, one that is strikingly unexplored in neurodegenerative research, and present the most detailed description, to date, of strain-specific prion subpopulations. We found that prion quaternary structure was a major contributor to structural heterogeneity. We also discovered that all prion strains studied underwent a significant structural change resulting in two distinctive subpopulations whose proportions correlated with the strain phenotype. Our work provides new insights into the molecular basis of prion strain variation and is a proof of concept that can be applied to other protein misfolding neurodegenerative disorders.

Bartonella T4SS effector BepC was reported to mediate internalization of big Bartonella aggregates into host cells by modulating F-actin polymerization. After that, BepC was indicated to induce host cell fragmentation, an interesting cell phenotype that is characterized by failure of rear-end retraction during cell migration, and subsequent dragging and fragmentation of cells. Here, we found that expression of BepC resulted in significant stress fiber formation and contractile cell morphology, which depended on combination of the N-terminus FIC (filamentation induced by c-AMP) domain and C-terminus BID (Bartonella intracellular delivery) domain of BepC. The FIC domain played a key role in BepC-induced stress fiber formation and cell fragmentation because deletion of FIC signature motif or mutation of two conserved amino acid residues abolished BepC-induced cell fragmentation. Immunoprecipitation confirmed the interaction of BepC with GEF-H1 (a microtubule-associated RhoA guanosine exchange factor), and siRNA-mediated depletion of GEF-H1 prevented BepC-induced stress fiber formation. Interaction with BepC caused the dissociation of GEF-H1 from microtubules and activation of RhoA to induce formation of stress fibers. The ROCK (Rho-associated protein kinase) inhibitor Y27632 completely blocked BepC effects on stress fiber formation and cell contractility. Moreover, stress fiber formation by BepC increased the stability of focal adhesions, which consequently impeded rear-edge detachment. Overall, our study revealed that BepC-induced stress fiber formation was achieved through the GEF-H1/RhoA/ROCK pathway., Author summary Intracellular pathogens modulate host cell actin cytoskeleton by secreting an array of effector molecules to ensure their cell invasion and intracellular survival. The zoonotic pathogen Bartonella spp trigger massive F-actin polymerization of host cells resulting the internalization of large bacterial aggregates (called “invasome” structure), which is dependent on a functional VirB/VirD4 type IV secretion system (T4SS) and its translocated Bep effector proteins. Here, we have used cell infection and ectopic expression assay to identify that Bartonella T4SS effector BepC induces stress fiber formation in infected host cells. However, BepC also disrupts the balance of stress fiber formation and focal adhesion maturation, and eventually causes cell fragmentation. Using immunoprecipitation and RNAi approaches, we identify GEF-H1 is the host factor targeted by BepC. Interaction with BepC induces the release of GEF-H1 from microtubules to plasma membrane and subsequently activates RhoA-ROCK to induce stress fiber formation. These findings shed light on our understanding of how Bartonella invade host cell and establish infection.

During infection, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) directly manipulate various aspects of host cell function through the translocation of type III secretion system (T3SS) effector proteins directly into the host cell. Many T3SS effector proteins are enzymes that mediate post-translational modifications of host proteins, such as the glycosyltransferase NleB1, which transfers a single N-acetylglucosamine (GlcNAc) to arginine residues, creating an Arg-GlcNAc linkage. NleB1 glycosylates death-domain containing proteins including FADD, TRADD and RIPK1 to block host cell death. The NleB1 paralogue, NleB2, is found in many EPEC and EHEC strains but to date its enzymatic activity has not been described. Using in vitro glycosylation assays combined with mass spectrometry, we found that NleB2 can utilize multiple sugar donors including UDP-glucose, UDP-GlcNAc and UDP-galactose during glycosylation of the death domain protein, RIPK1. Sugar donor competition assays demonstrated that UDP-glucose was the preferred substrate of NleB2 and peptide sequencing identified the glycosylation site within RIPK1 as Arg603, indicating that NleB2 catalyses arginine glucosylation. We also confirmed that NleB2 catalysed arginine-hexose modification of Flag-RIPK1 during infection of HEK293T cells with EPEC E2348/69. Using site-directed mutagenesis and in vitro glycosylation assays, we identified that residue Ser252 in NleB2 contributes to the specificity of this distinct catalytic activity. Substitution of Ser252 in NleB2 to Gly, or substitution of the corresponding Gly255 in NleB1 to Ser switches sugar donor preference between UDP-GlcNAc and UDP-glucose. However, this switch did not affect the ability of the NleB variants to inhibit inflammatory or cell death signalling during HeLa cell transfection or EPEC infection. NleB2 is thus the first identified bacterial Arg-glucose transferase that, similar to the NleB1 Arg-GlcNAc transferase, inhibits host protein function by arginine glycosylation., Author summary Bacterial gut pathogens including enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC), manipulate host cell function by using a type III secretion system to inject ‘effector’ proteins directly into the host cell cytoplasm. We and others have shown that many of these effectors are novel enzymes, including NleB1, which transfers a single N-acetylglucosamine (GlcNAc) sugar to arginine residues, mediating Arg-GlcNAc glycosylation. Here, we found that a close homologue of NleB1 that is also present in EPEC and EHEC termed NleB2, uses a different sugar during glycosylation. We demonstrated that in contrast to NleB1, the preferred nucleotide-sugar substrate of NleB2 is UDP-glucose and we identified the amino acid residue within NleB2 that dictates this unique catalytic activity. Substitution of this residue in NleB2 and NleB1 switches the sugar donor usage of these enzymes but does not affect their ability to inhibit host cell signalling. Thus, NleB2 is the first identified bacterial arginine-glucose transferase, an activity which has previously only been described in plants and algae.

During internalization and trafficking, human papillomavirus (HPV) moves from the cell surface to the endosome where the transmembrane protease γ-secretase promotes insertion of the viral L2 capsid protein into the endosome membrane. Protrusion of L2 through the endosome membrane into the cytosol allows the recruitment of cytosolic host factors that target the virus to the Golgi en route for productive infection. How endosome-localized HPV is delivered to γ-secretase, a decisive infection step, is unclear. Here we demonstrate that cytosolic p120 catenin, likely via an unidentified transmembrane protein, interacts with HPV at early time-points during viral internalization and trafficking. In the endosome, p120 is not required for low pH-dependent disassembly of the HPV L1 capsid protein from the incoming virion. Rather, p120 is required for HPV to interact with γ-secretase–an interaction that ensures the virus is transported along a productive route. Our findings clarify an enigmatic HPV infection step and provide critical insights into HPV infection that may lead to new therapeutic strategies against HPV-induced diseases., Author summary Human papillomavirus (HPV) is the primary cause of cervical, anogenital, and oropharyngeal cancers. Despite the significant impact of HPV on human health, there is limited understanding of how this small DNA virus traffics through a host cell to cause infection. This work identifies the role of the cellular factor p120 catenin in routing the virus along a productive entry pathway. Specifically, we propose that p120 targets HPV to the transmembrane protein γ-secretase, a critical step of viral entry. This work thus provides insights into the intracellular transport mechanisms of HPV and identifies a potential therapeutic target for HPV treatment.

TRIM5α is a key cross-species barrier to retroviral infection, with certain TRIM5 alleles conferring increased risk of HIV-1 infection in humans. TRIM5α is best known as a species-specific restriction factor that directly inhibits the viral life cycle. Additionally, it is also a pattern-recognition receptor (PRR) that activates inflammatory signaling. How TRIM5α carries out its multi-faceted actions in antiviral defense remains incompletely understood. Here, we show that proteins required for autophagy, a cellular self-digestion pathway, play an important role in TRIM5α’s function as a PRR. Genetic depletion of proteins involved in all stages of the autophagy pathway prevented TRIM5α-driven expression of NF-κB and AP1 responsive genes. One of these genes is the preeminent antiviral cytokine interferon β (IFN-β), whose TRIM5-dependent expression was lost in cells lacking the autophagy proteins ATG7, BECN1, and ULK1. Moreover, we found that the ability of TRIM5α to stimulate IFN-β expression in response to recognition of a TRIM5α-restricted HIV-1 capsid mutant (P90A) was abrogated in cells lacking autophagy factors. Stimulation of human macrophage-like cells with the P90A virus protected them against subsequent infection with an otherwise resistant wild type HIV-1 in a manner requiring TRIM5α, BECN1, and ULK1. Mechanistically, TRIM5α was attenuated in its ability to activate the kinase TAK1 in autophagy deficient cells, and both BECN1 and ATG7 contributed to the assembly of TRIM5α-TAK1 complexes. These data demonstrate a non-canonical role for the autophagy machinery in assembling antiviral signaling complexes and in establishing a TRIM5α-dependent antiviral state., Author summary TRIM5α is an antiretroviral protein that employs multiple mechanisms to protect cells against infection. Previous studies have linked TRIM5α to autophagy, a cytoplasmic quality control pathway with numerous roles in immunity, raising the possibility that TRIM5α engages autophagy in antiviral defense. This concept has been controversial, since TRIM5α’s best-known role as a directly acting antiretroviral effector is autophagy independent. However, retroviral restriction is only one aspect of TRIM5α function. We demonstrate that autophagy is crucial to another TRIM5α action: its role as a pattern-recognition receptor. We show that autophagy machinery is required for TRIM5α to transduce antiviral signaling and to establish an antiviral state. Our data indicate that autophagy provides TRIM5α with a platform upon which to activate antiviral responses.

Establishing latent infection but retaining the capability to reactivate in certain circumstance is an ingenious tactic for retroviruses to persist in vivo while evading host immune surveillance. Many evidences indicate that Human T-cell leukemia virus type 1 (HTLV-1) is not completely silent in vivo. However, signals that trigger HTLV-1 latency-reactivation switching remain poorly understood. Here, we show that aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, plays a critical role in HTLV-1 plus-strand expression. Importantly, HTLV-1 reactivation could be tunably manipulated by modulating the level of AHR ligands. Mechanistically, activated AHR binds to HTLV-1 LTR dioxin response element (DRE) site (CACGCATAT) and drives plus-strand transcription. On the other hand, persistent activation of nuclear factor kappa B (NF-κB) pathway constitutes one key prerequisite for AHR overexpression in HTLV-1 infected T-cells, setting the stage for the advent of AHR signaling. Our findings suggest that HTLV-1 might achieve its reactivation in vivo when encountering environmental, dietary, microbial and metabolic cues that induce sufficient AHR signaling., Author summary HTLV-1 is considered largely latent in vivo because viral products were rarely detected in freshly isolated PBMCs of infected individuals. However, the existence of strong HTLV-1-specific immune response in most infected individuals suggests that the virus should not be completely silent in vivo. Since viral gene expression plays a critical role in cell transformation and de novo infection, a novel insight into where and how HTLV-1 achieves its reactivation in vivo is essential for developing new therapeutic approaches. AHR is a ligand-activated transcription factor that regulates intricate transcriptional programs in response to environmental, dietary, microbial and metabolic cues. It has been reported that AHR is constitutively overexpressed in HTLV-1-infected T-cells. Nevertheless, the functional role of AHR in HTLV-1 pathogenesis is still obscure. In this study, we show that activated AHR can directly bind to HTLV-1 LTR DRE site (CACGCATAT) and drive HTLV-1 plus-strand transcription. Importantly, HTLV-1 latency-reactivation-latency switching could be manipulated in MT-1 cells by adding and removing additional kynurenine (a well-known AHR ligand). Moreover, we explicate that the persistent NF-κB activation is critical for AHR overexpression in HTLV-1-infected T-cells. These results imply that constitutive AHR overexpression in infected T-cells endues HTLV-1 the potential to reactivate from latency when the level of AHR ligands reaches a certain threshold. Accordingly, we propose that HTLV-1 might achieve its reactivation in certain parts of the body that are prone to accumulate AHR ligands.

The genomic instability associated with adult T cell leukemia/lymphoma (ATL) is causally linked to Tax, the HTLV-1 viral oncoprotein, but the underlying mechanism is not fully understood. We have previously shown that Tax hijacks and aberrantly activates ring finger protein 8 (RNF8) — a lysine 63 (K63)-specific ubiquitin E3 ligase critical for DNA double-strand break (DSB) repair signaling — to assemble K63-linked polyubiquitin chains (K63-pUbs) in the cytosol. Tax and the cytosolic K63-pUbs, in turn, initiate additional recruitment of linear ubiquitin assembly complex (LUBAC) to produce hybrid K63-M1 pUbs, which trigger a kinase cascade that leads to canonical IKK:NF-κB activation. Here we demonstrate that HTLV-1-infected cells are impaired in DNA damage response (DDR). This impairment correlates with the induction of microscopically visible nuclear speckles by Tax known as the Tax-speckle structures (TSS), which act as pseudo DNA damage signaling scaffolds that sequester DDR factors such as BRCA1, DNA-PK, and MDC1. We show that TSS co-localize with Tax, RNF8 and K63-pUbs, and their formation depends on RNF8. Tax mutants defective or attenuated in inducing K63-pUb assembly are deficient or tempered in TSS induction and DDR impairment. Finally, our results indicate that loss of RNF8 expression reduces HTLV-1 viral gene expression and frequently occurs in ATL cells. Thus, during HTLV-1 infection, Tax activates RNF8 to assemble nuclear K63-pUbs that sequester DDR factors in Tax speckles, disrupting DDR signaling and DSB repair. Down-regulation of RNF8 expression is positively selected during infection and progression to disease, and further exacerbates the genomic instability of ATL., Author summary Approximately 3–5% of HTLV-1-infected individuals develop an intractable malignancy called adult T cell leukemia/lymphoma (ATL) decades after infection. Unlike other leukemia, ATL is characterized by extensive genomic instability. Here we show that the genomic instability of ATL is associated with the hijacking and aberrant activation of a molecule known as ring finger protein 8 (RNF8) by HTLV-1 for viral replication. RNF8 is crucial for initiating the cellular DNA damage response (DDR) required for the repair of DNA double-strand breaks (DSBs), the most deleterious DNA damage. Its dysregulation in HTLV-1-infected cells results in the formation of pseudo DNA damage signaling scaffolds known as Tax speckle structures that sequester critical repair factors, causing an inability to repair DSBs efficiently. We have further found that loss of RNF8 expression reduces HTLV-1 viral replication and frequently occurs in ATL of all types. This likely facilitates the immune evasion of virus-infected cells, but degrades their ability to repair DSBs and exacerbates the genomic instability of ATL cells. Since DDR defects impact cancer response to DNA-damaging radiation and chemotherapies, RNF8 deficiency in ATL may be exploited for disease treatment.

The retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I and melanoma differentiation-associated gene 5 (MDA5), sense cytoplasmic viral RNA and initiate innate antiviral responses. How RIG-I and MDA5 are differentially regulated remains enigmatic. In this study, we identified the guanylate-binding protein (GBP) and zinc-finger FYVE domain-containing protein ZFYVE1 as a negative regulator of MDA5- but not RIG-I-mediated innate antiviral responses. ZFYVE1-deficiency promoted MDA5- but not RIG-I-mediated transcription of downstream antiviral genes. Comparing to wild-type mice, Zfyve1-/- mice were significantly protected from lethality induced by encephalomyocarditis virus (EMCV) that is sensed by MDA5, whereas Zfyve1-/- and Zfyve1+/+ mice were comparable to death induced by vesicular stomatitis virus (VSV) that is sensed by RIG-I. Mechanistically, ZFYVE1 interacted with MDA5 but not RIG-I. ZFYVE1 bound to viral RNA and decreased the ligand binding and oligomerization of MDA5. These findings suggest that ZFYVE1 acts as a specific negative regulator of MDA5-mediated innate immune responses by inhibiting its ligand binding and oligomerization., Author summary RIG-I and MDA5 are the main cytosolic sensors for invaded viral RNA. How these sensors are differentially regulated is largely unknown. In this study, we identified ZFYVE1 as a specific regulator of MDA5- but not RIG-I-mediated antiviral responses. ZFYVE1-deficiency promotes antiviral immune responses and renders the mice less susceptible to EMCV-induced death. ZFYVE1 interacts with MDA5 and viral dsRNA, and inhibits the ligand binding and oligomerization of MDA5. Our study reveals a negative regulatory mechanism for keeping MDA5 inactive in un-infected cells, which contributes to our understanding on how innate antiviral responses are delicately regulated to avoid immune damage.

Latent Epstein-Barr virus (EBV) infection is strongly associated with several malignancies, including B-cell lymphomas and epithelial tumors. EBNA1 is a key antigen expressed in all EBV-associated tumors during latency that is required for maintenance of the EBV episome DNA and the regulation of viral gene transcription. However, the mechanism utilized by EBV to maintain latent infection at the levels of posttranslational regulation remains largely unclear. Here, we report that EBNA1 contains two SUMO-interacting motifs (SIM2 and SIM3), and mutation of SIM2, but not SIM3, dramatically disrupts the EBNA1 dimerization, while SIM3 contributes to the polySUMO2 modification of EBNA1 at lysine 477 in vitro. Proteomic and immunoprecipitation analyses further reveal that the SIM3 motif is required for the EBNA1-mediated inhibitory effects on SUMO2-modified STUB1, SUMO2-mediated degradation of USP7, and SUMO1-modified KAP1. Deletion of the EBNASIM motif leads to functional loss of both EBNA1-mediated viral episome maintenance and lytic gene silencing. Importantly, hypoxic stress induces the SUMO2 modification of EBNA1, and in turn the dissociation of EBNA1 with STUB1, KAP1 and USP7 to increase the SUMO1 modification of both STUB1 and KAP1 for reactivation of lytic replication. Therefore, the EBNA1SIM motif plays an essential role in EBV latency and is a potential therapeutic target against EBV-associated cancers., Author summary The Small Ubiquitin-related modifier (SUMO) modification of proteins is a reversible post-translational regulation involved in control of gene transcription, among other functions. Epstein-Barr virus (EBV) infects most people worldwide and contributes to the development of several types of cancers due to its ability to induce cell proliferation and survival. EBNA1 is expressed in all forms of EBV-associated tumors. In this study, we found that EBNA1 contains a SUMO-interacting motif (SIM) named EBNA1SIM, which is required for EBNA1 to exert inhibitory effects on a SUMO2-modified complex (SC2) including STUB1, KAP1 and USP7. Disruption of EBNA1SIM leads to loss of both EBNA1-mediated viral episome maintenance and lytic gene silencing. Importantly, hypoxia-mediated reactivation of viral lytic replication induces the EBNA1 dissociation from STUB1 in the SC2 complex. This discovery not only opens a new insight on the interplay between host and virus, but it also provides a therapeutic target specific against EBV-associated cancers.

Recent efforts have been paid to identify previously unrecognized HIV-1 latency-promoting genes (LPGs) that can potentially be targeted for eradication of HIV-1 latent reservoirs. From our earlier orthologous RNAi screens of host factors regulating HIV-1 replication, we identified that the nucleolar protein NOP2/NSUN1, a m5C RNA methyltransferase (MTase), is an HIV-1 restriction factor. Loss- and gain-of-function analyses confirmed that NOP2 restricts HIV-1 replication. Depletion of NOP2 promotes the reactivation of latently infected HIV-1 proviruses in multiple cell lines as well as primary CD4+ T cells, alone or in combination with latency-reversing agents (LRAs). Mechanistically, NOP2 associates with HIV-1 5’ LTR, interacts with HIV-1 TAR RNA by competing with HIV-1 Tat protein, as well as contributes to TAR m5C methylation. RNA MTase catalytic domain (MTD) of NOP2 mediates its competition with Tat and binding with TAR. Overall, these findings verified that NOP2 suppresses HIV-1 transcription and promotes viral latency., Author summary Host machinery plays a significant role in the regulation of HIV persistent infection. Identification of novel host factors kin to HIV replication and characterization of their molecular mechanisms are crucial for not only improving the fundamental understanding of host-HIV infections but also providing new drug targets for the development of novel anti-HIV therapies. The impacts of NOP2/Sun (NSUN) m5C RNA methyltransferaes (MTases) on HIV-1 infection are mainly elusive. Here, we confirmed that NOP2, NSUN m5C RNA MTase is a novel host factor suppressing HIV-1 transcription. We also identified that the potential mechanism underlying NOP2’s antiviral activities is through its competition with HIV-1 Tat protein for TAR RNA binding, which involves its RNA MTase catalytic motifs. Indeed, we also showed that NOP2 contributes to m5C methylation of TAR RNA. These findings provide new insight into the impacts of RNA MTases on HIV-1 latency.

Chromatin dynamics regulated by epigenetic modification is crucial in genome stability and gene expression. Various epigenetic mechanisms have been identified in the pathogenesis of human diseases. Here, we examined the effects of ten epigenetic agents on pseudorabies virus (PRV) infection by using GFP-reporter assays. Inhibitors of bromodomain protein 4 (BRD4), which receives much more attention in cancer than viral infection, was found to exhibit substantial anti-viral activity against PRV as well as a range of DNA and RNA viruses. We further demonstrated that BRD4 inhibition boosted a robust innate immune response. BRD4 inhibition also de-compacted chromatin structure and induced the DNA damage response, thereby triggering the activation of cGAS-mediated innate immunity and increasing host resistance to viral infection both in vitro and in vivo. Mechanistically, the inhibitory effect of BRD4 inhibition on viral infection was mainly attributed to the attenuation of viral attachment. Our findings reveal a unique mechanism through which BRD4 inhibition restrains viral infection and points to its potent therapeutic value for viral infectious diseases., Author summary BRD4 has been well investigated in tumorigenesis for its contribution to chromatin remodeling and gene transcription. BRD4 inhibitors are used as promising chemotherapeutic drugs for cancer therapy. Here, we show a unique mechanism through which BRD4 inhibition broadly inhibits attachment of DNA and RNA viruses through DNA damage-dependent antiviral innate immune activation via the cGAS-STING pathway, in both cell culture and an animal model. STING-associated innate immune signaling has been considered to be a new possibility for cancer therapy, and STING agonists have been tested in early clinical trials. Our data identify BRD4 inhibitors as a potent therapy not only for viral infection but also for cancer immunotherapy.

West Nile virus (WNV) belongs to the Flaviviridae family and has emerged as a significant cause of viral encephalitis in birds and animals including humans. WNV replication directly induces neuronal injury, followed by neuronal cell death. We previously showed that accumulation of ubiquitinated protein aggregates was involved in neuronal cell death in the WNV-infected mouse brain. In this study, we attempted to elucidate the mechanisms of the accumulation of protein aggregates in the WNV-infected cells. To identify the viral factor inducing the accumulation of ubiquitinated proteins, intracellular accumulation of ubiquitinated proteins was examined in the cells expressing the viral protein. Expression of capsid (C) protein induced the accumulation, while mutations at residues L51 and A52 in C protein abrogated the accumulation. Wild-type (WT) or mutant WNV in which mutations were introduced into the residues was inoculated into human neuroblastoma cells. The expression levels of LC3-II, an autophagy-related protein, and AMP-activated protein kinase (AMPK), an autophagy inducer, were reduced in the cells infected with WT WNV, while the reduction was not observed in the cells infected with WNV with the mutations in C protein. Similarly, ubiquitination and degradation of AMPK were only observed in the cells infected with WT WNV. In the cells expressing C protein, AMPK was co-precipitated with C protein and mutations in L51 and A52 reduced the interaction. Although the viral replication was not affected, the accumulation of ubiquitinated proteins in brain and neurological symptoms were attenuated in the mouse inoculated with WNV with the mutations in C protein as compared with that with WT WNV. Taken together, ubiquitination and degradation of AMPK by C protein resulted in the inhibition of autophagy and the accumulation of protein aggregates, which contributes to the development of neurological disease., Author summary The elimination of ubiquitinated protein aggregates from neuronal cells occurs via protein degradation systems such as autophagy or proteasome. Previously, we determined that WNV infection induces the accumulation of ubiquitinated proteins in neuronal cells. However, the detailed mechanisms underlying this accumulation and its association with pathogenicity in the brain remain unclear. Here, we demonstrated that residues L51 and A52 of WNV C protein are responsible for the accumulation of the ubiquitinated proteins. The two C protein residues promoted the interaction between C protein and the autophagy-inducing enzyme AMPK, resulting in ubiquitination and degradation of AMPK and inhibition of autophagy. A mutation in WNV that impaired the AMPK-C protein interaction attenuated the accumulation of ubiquitinated proteins and neurological symptoms in mice. Our findings are the first to describe the regulation of AMPK by a viral protein and represent a step forward in understanding of molecular mechanisms of the neuropathogenesis of WNV infection.

Mediator of IRF3 activation (MITA, also known as stimulator of interferon genes, STING) senses the second messenger cyclic GMP-AMP (cGAMP) which is synthesized upon DNA virus infection and activates innate antiviral immune response. It has been demonstrated that the activity of MITA is delicately regulated by various post-translational modifications including polyubiquitination. In this study, we identified the deubiquitinating enzyme USP44 as a positive regulator of MITA. USP44 is recruited to MITA following DNA virus infection and removes K48-linked polyubiquitin moieties from MITA at K236, therefore prevents MITA from proteasome mediated degradation. USP44-deficiency results in acceleration of HSV-1-induced degradation of MITA and reduced induction of type I interferons (IFNs) and proinflammatory cytokines. Consistently, Usp44-/- mice are more susceptible to HSV-1 infection as indicated by higher tissue viral titers, greater tissue damage and lower survival rate. These findings suggest that USP44 plays a specific and critical role in the regulation of innate immune response against DNA viruses., Author summary Cyclic GMP-AMP synthase (cGAS) senses cytosolic dsDNA and initiates signal transductions, leading to activation of innate immune response. MITA is the key adaptor protein downstream of cGAS and plays a critical role in cGAS-mediated signaling. The activity of MITA is tightly regulated by various post-translational modifications including polyubiquitination and deubiquitination. Here we found that the deubiquitinating enzymes USP44 associates with MITA and removes the K48-linked polyubiquitin chains from MITA, therefore maintains the stability of MITA after DNA virus infection. Deficiency of USP44 results in accelerated degradation of MITA, impaired induction of type I IFNs and proinflammatory cytokines, and increased viral replication. These findings suggest that USP44 is a positive regulator of MITA and plays an important role in the regulation of innate immune response against DNA viruses.

It is known that the non-structural protein (NSs) of Toscana virus (TOSV), an emergent sandfly-borne virus causing meningitis or more severe central nervous system injuries in humans, exerts its function triggering RIG-I for degradation in a proteasome-dependent manner, thus breaking off the IFN-β production. The non-structural protein of different members of Bunyavirales has recently appeared as a fundamental protagonist in immunity evasion through ubiquitination-mediated protein degradation targets. We showed that TOSV NSs has an E3 ubiquitin ligase activity, mapping at the carboxy-terminal domain and also involving the amino-terminal of the protein. Indeed, neither the amino- (NSsΔN) nor the carboxy- (NSsΔC) terminal-deleted mutants of TOSV NSs were able to cause ubiquitin-mediated proteasome degradation of RIG-I. Moreover, the addition of the C-terminus of TOSV NSs to the homologous protein of the Sandfly Fever Naples Virus, belonging to the same genus and unable to inhibit IFN-β activity, conferred new properties to this protein, favoring RIG-I ubiquitination and its degradation. NSs lost its antagonistic activity to IFN when one of the terminal residues was missing. Therefore, we showed that NSs could behave as an atypical RING between RING (RBR) E3 ubiquitin ligases. This is the first report which identified the E3 ubiquitin ligase activity in a viral protein among negative strand RNA viruses., Author summary Toscana virus is an emergent sandfly-borne virus mainly transmitted to humans by phlebotomine sandflies, which can cause meningitis or more severe central nervous system injuries in some subjects. As many other RNA viruses, it counteracts IFN-β expression by its non-structural protein. Our results expanded our knowledge about the molecular mechanisms by which TOSV exerts its activity as an E3 ubiquitin ligase. This is the first example of a viral protein presenting this activity among negative-strand RNA viruses. Thus, the recognition of this activity and its substrates among viruses are of primary importance to understand how viruses can alter their fitness by the ubiquitin pathway and provide an attractive target for the development of antiviral therapies.

Mouse APOBEC3 (mA3) inhibits murine leukemia virus (MuLV) replication by a deamination-independent mechanism in which the reverse transcription is considered the main target process. However, other steps in virus replication that can be targeted by mA3 have not been examined. We have investigated the possible effect of mA3 on MuLV protease-mediated processes and found that mA3 binds both mature viral protease and Pr180gag-pol precursor polyprotein. Using replication-competent MuLVs, we also show that mA3 inhibits the processing of Pr65 Gag precursor. Furthermore, we demonstrate that the autoprocessing of Pr180gag-pol is impeded by mA3, resulting in reduced production of mature viral protease. This reduction appears to link with the above inefficient Pr65gag processing in the presence of mA3. Two major isoforms of mA3, exon 5-containing and -lacking ones, equally exhibit this antiviral activity. Importantly, physiologically expressed levels of mA3 impedes both Pr180gag-pol autocatalysis and Pr65gag processing. This blockade is independent of the deaminase activity and requires the C-terminal region of mA3. These results suggest that the above impairment of Pr180gag-pol autoprocessing may significantly contribute to the deaminase-independent antiretroviral activity exerted by mA3., Author summary Soon after the identification of the polynucleotide cytidine deaminase APOBEC3 as a host restriction factor against vif-deficient HIV, it was noticed that deamination-independent mechanisms are involved in the inhibition of viral replication in addition to the deaminase-dependent mechanism. We previously showed that mouse APOBEC3 (mA3) physiologically restricted mouse retrovirus replication in their natural hosts without causing significant G-to-A hypermutations. Inhibition of reverse transcription is reported to be the most plausible mechanism for the deamination-independent antiretroviral function. However, it remains unknown whether the inhibition of reverse transcription is the only way to explain the whole picture of deamination-independent antiviral activity exerted by APOBEC3. Here we show that mA3 targets the autoprocessing of Pr180gag-pol polyprotein. This activity does not require the deaminase catalytic center and mainly exerted by the C-terminal half of mA3. mA3 physically interacts with murine retroviral protease and its precursor Pr180gag-pol. mA3-induced disruption of the autocatalytic Pr180gag-pol cleavage leads to a significant reduction of mature viral protease, resulting in the inhibition of Pr65gag processing to mature Gag proteins. As the Pr180gag-pol autoprocessing is necessary for the maturation of other viral enzymes including the reverse transcriptase, its inhibition by host APOBEC3 may precede the previously described impairment of reverse transcription. Our discovery may lead to the development of novel antiretroviral drugs through the future identification of detailed molecular interfaces between retroviral Gag-Pol polyprotein and APOBEC3.

Primary effusion lymphoma (PEL) is an aggressive B-cell malignancy without effective treatment, and caused by the infection of Kaposi’s sarcoma-associated herpesvirus (KSHV), predominantly in its latent form. Previously we showed that the SUMO2-interacting motif within the viral latency-associated nuclear antigen (LANASIM) is essential for establishment and maintenance of KSHV latency. Here, we developed a luciferase based live-cell reporter system to screen inhibitors selectively targeting the interaction between LANASIM and SUMO2. Cambogin, a bioactive natural product isolated from the Garcinia genus (a traditional herbal medicine used for cancer treatment), was obtained from the reporter system screening to efficiently inhibit the association of SUMO2 with LANASIM, in turn reducing the viral episome DNA copy number for establishment and maintenance of KSHV latent infection at a low concentration (nM). Importantly, Cambogin treatments not only specifically inhibited proliferation of KSHV-latently infected cells in vitro, but also induced regression of PEL tumors in a xenograft mouse model. This study has identified Cambogin as a novel therapeutic agent for treating PEL as well as eliminating persistent infection of oncogenic herpesvirus., Author summary Primary effusion lymphoma is a common AIDS-associated malignancy caused by infection with Kaposi’s sarcoma-associated herpesvirus (KSHV), and is currently absence of efficient and specific treatment. Natural product from herbal medicines is a major source of drug discovery for the treatment of a variety of diseases. In this study, the authors demonstrated that Cambogin, a polycyclic polyprenylated acylphloroglucinols (PPAPs) isolated from the branches of Garcinia esculenta (a tropical evergreen tree and traditional cancer treatment across Southern Asia), is a potent and effective inhibitor of KSHV-latently infected cells at a low concentration (nM) in vitro and in vivo, through targeting viral LANASIM-SUMO2 interaction.

RSK1, a downstream kinase of the MAPK pathway, has been shown to regulate multiple cellular processes and is essential for lytic replication of a variety of viruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV). Besides phosphorylation, it is not known whether other post-translational modifications play an important role in regulating RSK1 function. We demonstrate that RSK1 undergoes robust SUMOylation during KSHV lytic replication at lysine residues K110, K335, and K421. SUMO modification does not alter RSK1 activation and kinase activity upon KSHV ORF45 co-expression, but affects RSK1 downstream substrate phosphorylation. Compared to wild-type RSK1, the overall phosphorylation level of RxRxxS*/T* motif is significantly declined in RSK1K110/335/421R expressing cells. Specifically, SUMOylation deficient RSK1 cannot efficiently phosphorylate eIF4B. Sequence analysis showed that eIF4B has one SUMO-interacting motif (SIM) between the amino acid position 166 and 170 (166IRVDV170), which mediates the association between eIF4B and RSK1 through SUMO-SIM interaction. These results indicate that SUMOylation regulates the phosphorylation of RSK1 downstream substrates, which is required for efficient KSHV lytic replication., Author summary RSK1 is an essential celluar kinase for lytic replication of Kaposi’s sarcoma-associated herpesvirus (KSHV). Besides phosphorylation, it is not known whether other post-translational modifications play an important role in regulating RSK1 function. Here, we found that RSK1 is SUMOylated at lysine residues K110, K335, and K421, which is enhanced by KSHV lytic replication. RSK1 SUMOylation is required for effient KSHV lytic replication and mutations on its SUMOylation sites leads to reduced lytic gene expressions and impaired progeny virus production. Interestingly, SUMO modification does not alter RSK1 activation and kinase activity upon KSHV ORF45 co-expression, but affects RSK1 downstream substrate phosphorylation. Specifically, SUMOylation deficient RSK1 cannot efficiently phosphorylate eIF4B since SUMO-SIM interaction mediates the association between eIF4B and RSK1. These results indicate that SUMOylation regulates the phosphorylation of RSK1 downstream substrates, which is required for efficient KSHV lytic replication.

Under RNA virus infection, retinoic acid-inducible gene I (RIG-I) in host cells recognizes viral RNA and activates the expression of type I IFN. To investigate the roles of protein methyltransferases and demethylases in RIG-I antiviral signaling pathway, we screened all the known related enzymes with a siRNA library and identified LSD1 as a positive regulator for RIG-I signaling. Exogenous expression of LSD1 enhances RIG-I signaling activated by virus stimulation, whereas its deficiency restricts it. LSD1 interacts with RIG-I, promotes its K63-linked polyubiquitination and interaction with VISA/MAVS. Interestingly, LSD1 exerts its function in antiviral response not dependent on its demethylase activity but through enhancing the interaction between RIG-I with E3 ligases, especially TRIM25. Furthermore, we provide in vivo evidence that LSD1 increases antiviral gene expression and inhibits viral replication. Taken together, our findings demonstrate that LSD1 is a positive regulator of signaling pathway triggered by RNA-virus through mediating RIG-I polyubiquitination., Author summary RIG-I signaling pathway is critical for human cells to defend from RNA virus infection, such as SARS-CoV-2, influenza virus, and Vesicular Stomatitis Virus (VSV). LSD1 is a histone demethylase regulating transcription. The current study reveals a novel function of LSD1 in regulating the activation of RIG-I signaling pathway. LSD1 interacts with RIG-I and promotes RIG-I poly-ubiquitination independent of its demethylase activity. LSD1 facilitates the interaction between RIG-I and its ubiquitin E3 ligase TRIM25, which is crucial for recruitment of downstream proteins. The mice with LSD1 deficiency are susceptible to virus infection and have lower survival rate. Taken together, our findings demonstrate a novel molecular mechanism for regulating the anti-viral RIG-I signaling pathway.

The presumed DNA helicase encoded by ORF44 of Kaposi’s sarcoma-associated herpesvirus (KSHV) plays a crucial role in unwinding viral double-stranded DNA and initiating DNA replication during lytic reactivation. However, the regulatory mechanism of KSHV ORF44 has not been fully elucidated. In a previous study, we identified that N-Myc downstream regulated gene 1 (NDRG1), a host scaffold protein, facilitates viral genome replication by interacting with proliferating cell nuclear antigen (PCNA) and the latent viral protein latency-associated nuclear antigen (LANA) during viral latency. In the present study, we further demonstrated that NDRG1 can interact with KSHV ORF44 during viral lytic replication. We also found that the mRNA and protein levels of NDRG1 were significantly increased by KSHV ORF50-encoded replication and transcription activator (RTA). Remarkably, knockdown of NDRG1 greatly decreased the protein level of ORF44 and impaired viral lytic replication. Interestingly, NDRG1 enhanced the stability of ORF44 and inhibited its ubiquitin-proteasome-mediated degradation by reducing the polyubiquitination of the lysine residues at positions 79 and 368 in ORF44. In summary, NDRG1 is a novel binding partner of ORF44 and facilitates viral lytic replication by maintaining the stability of ORF44. This study provides new insight into the mechanisms underlying KSHV lytic replication., Author summary During lytic replication, KSHV ORF44 unwinds viral DNA and initiates DNA replication. Here, we report that the host protein NDRG1, a novel ORF44 binding partner, is significantly upregulated during viral lytic replication and facilitates this process. Mechanistically, NDRG1 can increase the stability of ORF44, impairing the polyubiquitination of the lysine residues at positions 79 and 368 in ORF44, thus inhibiting ubiquitin-proteasome-mediated degradation of ORF44. Our study demonstrates that NDRG1 plays an important role in KSHV lytic replication and may thus constitute a promising therapeutic target for KSHV infection.

Porphyromonas gingivalis is a major pathogen in severe and chronic manifestations of periodontal disease, which is one of the most common infections of humans. A central feature of P. gingivalis pathogenicity is dysregulation of innate immunity at the gingival epithelial interface; however, the molecular basis underlying P. gingivalis–dependent abrogation of epithelial barrier function remains unknown. Gingival epithelial cells express junctional adhesion molecule (JAM1), a tight junction–associated protein, and JAM1 homodimers regulate epithelial barrier function. Here we show that Arg-specific or Lys-specific cysteine proteases (gingipains) secreted by P. gingivalis can specifically degrade JAM1 at K134 and R234 in gingival epithelial cells, resulting in permeability of the gingival epithelium to 40 kDa dextran, lipopolysaccharide (LPS), and proteoglycan (PGN). A P. gingivalis strain lacking gingipains was impaired in degradation of JAM1. Knockdown of JAM1 in monolayer cells and a three-dimensional multilayered tissue model also increased permeability to LPS, PGN, and gingipains. Inversely, overexpression of JAM1 in epithelial cells prevented penetration by these agents following P. gingivalis infection. Our findings strongly suggest that P. gingivalis gingipains disrupt barrier function of stratified squamous epithelium via degradation of JAM1, allowing bacterial virulence factors to penetrate into subepithelial tissues., Author summary Periodontal diseases, which are among the most common infections of humans, are characterized by gingival inflammation and destruction of the hard and soft tissues that support the tooth, eventually causing tooth loss. Porphyromonas gingivalis is a major pathogen in periodontal diseases. Infection of gingival epithelial cells by P. gingivalis increases epithelial permeability. However, the molecular mechanism and pathological significance of P. gingivalis–dependent barrier dysfunction in human gingival epithelium remain unknown. In this study, we developed a three-dimensional multilayered tissue model of gingival epithelium infected by P. gingivalis and used it to monitor penetration of bacterial products derived from P. gingivalis and other bacteria. We found that P. gingivalis proteases, called gingipains, have a potent and specific ability to degrade JAM1, which regulates epithelial barrier function. Mechanistically, gingipains degrade mature form of JAM1 on the plasma membrane, increasing penetration of 40 kDa dextran, lipopolysaccharide, peptidoglycan, and gingipains. Our study provides new insights into the etiological role of P. gingivalis, leading to periodontal destruction.

Mediator of IRF3 activation (MITA, also named as STING/ERIS/MPYS/TMEM173), is essential to DNA virus- or cytosolic DNA-triggered innate immune responses. In this study, we demonstrated the negative regulatory role of RING-finger protein (RNF) 90 in innate immune responses targeting MITA. RNF90 promoted K48-linked ubiquitination of MITA and its proteasome-dependent degradation. Overexpression of RNF90 inhibited HSV-1- or cytosolic DNA-induced immune responses whereas RNF90 knockdown had the opposite effects. Moreover, RNF90-deficient bone marrow-derived dendritic cells (BMDCs), bone marrow-derived macrophages (BMMs) and mouse embryonic fibroblasts (MEFs) exhibited increased DNA virus- or cytosolic DNA-triggered signaling and RNF90 deficiency protected mice from DNA virus infection. Taken together, our findings suggested a novel function of RNF90 in innate immunity., Author summary After the invasion of viruses, the genetic materials, RNA and DNA generated by the viruses, could be recognized by the cytosolic RNA or DNA sensors and then these sensors will initiate the innate immune signaling pathway and protect the host from viral invasion. However, excessive immune responses may cause tissue damage and do harm to the host. Therefore, the innate immune responses should be tightly controlled and turned off timely. MITA is a key adaptor in DNA-sensor signaling pathway. Here, we find that RNF90, a known E3 ubiquitin-protein ligase, interacted with MITA, enhanced the ubiquitination of MITA and promoted the degradation of MITA. RNF90-defient mice showed increased innate immune responses upon DNA virus HSV-1 infection. Our research revealed a new negative regulatory mechanism of the host to manipulate the antiviral innate immune responses.

Recognition of viral RNA by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I and MDA5, initiates innate antiviral responses. Although regulation of RLR-mediated signal transduction has been extensively investigated, how the recognition of viral RNA by RLRs is regulated remains enigmatic. In this study, we identified heterogeneous nuclear ribonucleoprotein M (hnRNPM) as a negative regulator of RLR-mediated signaling. Overexpression of hnRNPM markedly inhibited RNA virus-triggered innate immune responses. Conversely, hnRNPM-deficiency increased viral RNA-triggered innate immune responses and inhibited replication of RNA viruses. Viral infection caused translocation of hnRNPM from the nucleus to the cytoplasm. hnRNPM interacted with RIG-I and MDA5, and impaired the binding of the RLRs to viral RNA, leading to inhibition of innate antiviral response. Our findings suggest that hnRNPM acts as an important decoy for excessive innate antiviral immune response., Author summary Infection by virus, such as the RNA virus Sendai virus, induces the host cells to express proteins that mediate antiviral immune responses. Upon infections, the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) detects the intracellular viral RNA and initiates innate immune responses. Although the regulation of RLR-mediated signal transduction has been extensively investigated, how the recognition of viral RNA by RLRs is regulated remains enigmatic. In this study, we found that a protein called hnRNPM plays an important role in the process of antiviral immune response. hnRNPM does this by impairing the binding of the RLRs to viral RNA. Our results suggest that hnRNPM is an inhibitor of RNA virus-induced signaling which provides a critical control mechanism of viral RNA sensing for the host to avoid excessive and harmful immune response.

It is important that bacterium can coordinately deliver several effectors into host cells to disturb the cellular progress during infection, however, the precise role of effectors in host cell cytosol remains to be resolved. In this study, we identified a new bacterial virulence effector from pathogenic Edwardsiella piscicida, which presents conserved crystal structure to thioredoxin family members and is defined as a thioredoxin-like protein (Trxlp). Unlike the classical bacterial thioredoxins, Trxlp can be translocated into host cells, mimicking endogenous thioredoxin to abrogate ASK1 homophilic interaction and phosphorylation, then suppressing the phosphorylation of downstream Erk1/2- and p38-MAPK signaling cascades. Moreover, Trxlp-mediated inhibition of ASK1-Erk/p38-MAPK axis promotes the pathogenesis of E. piscicida in zebrafish larvae infection model. Taken together, these data provide insights into the mechanism underlying the bacterial thioredoxin as a virulence effector in downmodulating the innate immune responses during E. piscicida infection., Author summary Thioredoxin (Trx) is universally conserved thiol-oxidoreductase that regulates numerous cellular pathways under thiol-based redox control in both prokaryotic and eukaryotic organisms. Despite its central importance, the mechanism of bacterial Trx recognizes its target proteins in host cellular signaling remains unknown. Here, we uncover a bacterial thioredoxin-like protein that can be translocated into host cells and mimic the endogenous TRX1 to target ASK1-MAPK signaling, finally facilitating bacterial pathogenesis. This work expands our understanding of bacterial thioredoxins in manipulating host innate immunity.

Toxoplasma gondii is an apicomplexan parasite with the ability to use foodborne, zoonotic, and congenital routes of transmission that causes severe disease in immunocompromised patients. The parasites harbor a lysosome-like organelle, termed the "Vacuolar Compartment/Plant-Like Vacuole" (VAC/PLV), which plays an important role in maintaining the lytic cycle and virulence of T. gondii. The VAC supplies proteolytic enzymes that contribute to the maturation of invasion effectors and that digest autophagosomes and endocytosed host proteins. Previous work identified a T. gondii ortholog of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) that localized to the VAC. Here, we show that TgCRT is a membrane transporter that is functionally similar to PfCRT. We also genetically ablate TgCRT and reveal that the TgCRT protein plays a key role in maintaining the integrity of the parasite’s endolysosomal system by controlling morphology of the VAC. When TgCRT is absent, the VAC dramatically increases in volume by ~15-fold and overlaps with adjacent endosome-like compartments. Presumably to reduce aberrant swelling, transcription and translation of endolysosomal proteases are decreased in ΔTgCRT parasites. Expression of subtilisin protease 1 is significantly reduced, which impedes trimming of microneme proteins, and significantly decreases parasite invasion. Chemical or genetic inhibition of proteolysis within the VAC reverses these effects, reducing VAC size and partially restoring integrity of the endolysosomal system, microneme protein trimming, and invasion. Taken together, these findings reveal for the first time a physiological role of TgCRT in substrate transport that impacts VAC volume and the integrity of the endolysosomal system in T. gondii., Author summary Toxoplasma gondii is an obligate intracellular protozoan parasite that belongs to the phylum Apicomplexa and that infects virtually all warm-blooded organisms. Approximately one-third of the human population is infected with Toxoplasma. Toxoplasma invades host cells using processed invasion effectors. A lysosome-like organelle (VAC) is involved in refining these invasion effectors to reach their final forms. A T. gondii ortholog of the malarial chloroquine resistance transporter protein (TgCRT) was found to be localized to the VAC membrane. Although the mutated version of the malarial chloroquine resistance transporter (PfCRT) has been shown to confer resistance to chloroquine treatment, its physiologic function remains poorly understood. Comparison between the related PfCRT and TgCRT facilitates definition of the physiologic role of CRT proteins. Here, we report that TgCRT plays a key role in affecting the integrity and proteolytic activity of the VAC and adjacent organelles, the secretion of invasion effectors, and parasite invasion and virulence. To relieve osmotic stress caused by VAC swelling when TgCRT is deleted, parasites repress proteolysis within this organelle to decrease solute accumulation, which then has secondary effects on parasite invasion. Our findings highlight a common function for PfCRT and TgCRT in mediating small solute transport to affect apicomplexan parasite vacuolar size and function.

Measles virus (MeV) is a highly contagious, re-emerging, major human pathogen. Replication requires a viral RNA-dependent RNA polymerase (RdRP) consisting of the large (L) polymerase protein complexed with the homo-tetrameric phosphoprotein (P). In addition, P mediates interaction with the nucleoprotein (N)-encapsidated viral RNA genome. The nature of the P:L interface and RdRP negotiation of the ribonucleoprotein template are poorly understood. Based on biochemical interface mapping, swapping of the central P tetramerization domain (OD) for yeast GCN4, and functional assays, we demonstrate that the MeV P-to-L interface is bipartite, comprising a coiled-coil microdomain proximal to the OD and an unoccupied face of the triangular prism-shaped C-terminal P X-domain (P-XD), which is distinct from the known P-XD face that binds N-tail. Mixed null-mutant P tetramers regained L-binding competence in a ratio-dependent manner and fully reclaimed bioactivity in minireplicon assays and recombinant MeV, demonstrating that the individual L-binding interface elements are physically and mechanistically distinct. P-XD binding competence to L and N was likewise trans-complementable, which, combined with mathematical modeling, enabled the mechanistic characterization of P through two- and stoichiometrically-controlled three-way complementations. Only one each of the four XDs per P tetramer must be L or N binding-competent for bioactivity, but interaction of the same P-XD with L and N was mutually exclusive, and L binding superseded engaging N. Mixed P tetramers with a single, designated L binding-competent P-XD caused significant RdRP hyperactivity, outlining a model of iterative resolution and reformation of the P-XD:L interface regulating polymerase mobility., Author summary MeV belongs to the order of non-segmented negative polarity RNA viruses, which includes devastating human pathogens. While all feature encapsidated RNA genomes and P-L type polymerase complexes, insight into the intermolecular interactions within the polymerase hetero-oligomer and between the polymerase complex and the RNA-encapsidating N protein is rudimentary. Our mapping of the MeV P-to-L interaction revealed a bipartite interface with physically and mechanistically distinct contact zones, which provided a unique experimental platform to dissect the stoichiometry and dynamics of P interactions with L and N through functional trans-complementation assays in minireplicon settings and, ensuring physiological significance, recombinant virions. The identification and functional characterization of a novel L-binding face on the P-XD triangular prism, distinct from the side contacting N-tail, places P-XD at the center of a regulatory mechanism that controls the dynamics of polymerase advancement along the encapsidated genome through iterative separation and restoration of P-XD interaction with L. These observations and the high structural homology of polymerase components within the Paramyxoviridae recommend the P-XD:L protein-protein interface as premier target for directed drug discovery against emerging and re-emerging paramyxoviruses.

Cyclic GMP-AMP synthase (cGAS) senses viral DNA in the cytosol and then catalyzes synthesis of the second messenger cGAMP, which activates the ER-localized adaptor protein Mediator of IRF3 Activator (MITA) to initiate innate antiviral response. Human cytomegalovirus (HCMV) proteins can antagonize host immune responses to promote latent infection. Here, we identified HCMV UL42 as a negative regulator of cGAS/MITA-dependent antiviral response. UL42-deficiency enhances HCMV-induced production of type I interferons (IFNs) and downstream antiviral genes. Consistently, wild-type HCMV replicates more efficiently than UL42-deficient HCMV. UL42 interacts with both cGAS and MITA. UL42 inhibits DNA binding, oligomerization and enzymatic activity of cGAS. UL42 also impairs translocation of MITA from the ER to perinuclear punctate structures, which is required for MITA activation, by facilitating p62/LC3B-mediated degradation of translocon-associated protein β (TRAPβ). These results suggest that UL42 can antagonize innate immune response to HCMV by targeting the core components of viral DNA-triggered signaling pathways., Author summary Recognition of viral DNA by the cytosolic DNA sensor cGAS and subsequent induction of type I IFNs via the cGAS-MITA signaling axis are important for host antiviral innate immunity. The human cytomegalovirus (HCMV) causes complications in immunodeficient populations and is a major cause of birth defects. It is known that HCMV suppresses innate immunity, which is pivotal for establishing immune evasion and latent infection. In this study, we found that HCMV protein UL42 inhibits innate antiviral responses thus promotes HCMV replication. UL42 functions by targeting cGAS and MITA through distinct mechanisms. UL42 inhibits cGAS activation by interrupting its DNA binding and oligomerization, while it targets MITA by interfering trafficking of MITA from the ER to perinuclear punctate structures, a process required for MITA activation. These findings defined an important mechanism for HCMV immune evasion, which may provide a therapeutic target for the treatment of HCMV infection.

Streptococcus equi subsp. zooepidemicus (SEZ) is a zoonotic pathogen capable of causing meningitis in humans. The mechanisms that enable pathogens to traverse the blood-brain barrier (BBB) are incompletely understood. Here, we investigated the role of a newly identified Fic domain-containing protein, BifA, in SEZ virulence. BifA was required for SEZ to cross the BBB and to cause meningitis in mice. BifA also enhanced SEZ translocation across human Brain Microvascular Endothelial Cell (hBMEC) monolayers. Purified BifA or its Fic domain-containing C-terminus alone were able to enter into hBMECs, leading to disruption of monolayer barrier integrity. A SILAC-based proteomic screen revealed that BifA binds moesin. BifA’s Fic domain was required for its binding to this regulator of host cell cytoskeletal processes. BifA treatment of hBMECs led to moesin phosphorylation and downstream RhoA activation. Inhibition of moesin activation or moesin depletion in hBMEC monolayers abrogated BifA-mediated increases in barrier permeability and SEZ’s capacity to translocate across monolayers. Thus, BifA activation of moesin appears to constitute a key mechanism by which SEZ disrupts endothelial monolayer integrity to penetrate the BBB., Author summary Streptococcus equi subsp. zooepidemicus (SEZ) is an important animal pathogen and can cause meningitis in humans. Little is known about how this Group C streptococcal species penetrates the blood-brain barrier (BBB). We identified bifA, a gene that is critical for SEZ to cause meningitis in mice and to penetrate a human brain endothelial monolayer in a tissue culture model. BifA’s Fic domain enables the protein to enter into endothelial monolayers and to bind to moesin, a cytoskeletal regulatory protein, leading to its activation. Preventing moesin activation abolished BifA-induced barrier leakiness and SEZ’s capacity to penetrate a monolayer barrier. Together, our findings suggest that SEZ meningitis depends on BifA, a Fic-domain protein that manipulates moesin-dependent signaling to modulate BBB permeability.

Plant viruses have evolved multiple strategies to overcome host defense to establish an infection. Here, we identified two components of a host mitogen-activated protein kinase (MAPK) cascade, MKK2 and MPK4, as bona fide targets of the βC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNV). βC1 interacts with the kinase domain of MKK2 and inhibits its activity. In vivo, βC1 suppresses flagellin-induced MAPK activation and downstream responses by targeting MKK2. Furthermore, βC1 also interacts with MPK4 and inhibits its kinase activity. TYLCCNV infection induces the activation of the MAPK cascade, mutation in MKK2 or MPK4 renders the plant more susceptible to TYLCCNV, and can complement the lack of βC1. This work shows for the first time that a plant virus both activates and suppresses a MAPK cascade, and the discovery of the ability of βC1 to selectively interfere with the host MAPK activation illustrates a novel virulence function and counter-host defense mechanism of geminiviruses., Author summary We identified a plant MAPK cascade that contributes to the defense against geminiviruses. Rapid activation of MAPKs was observed upon geminivirus infection. The βC1 protein of geminivirus betasatellite interacts with MKK2 and MPK4 and inhibits their kinase activity. Also, βC1 suppresses flagellin-induced MAPK activation and downstream responses. Knocking out two components of this cascade, MKK2 or MPK4 in tobacco renders the plant more susceptible to geminivirus infection and virus infection of geminivirus without betasatellite induced typical virus symptom in MKK2 or MPK4 knockout tobacco, indicating that MKK2 or MPK4 mutant can complement the lack of βC1 in virus infection. This study unveils MAPK cascades as novel players in anti-viral defense and illustrates how plant viruses have in turn evolved to suppress this pathway.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), and the neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein persistently activates the NF-κB pathway to enhance the proliferation and survival of HTLV-1 infected T cells. Lysine 63 (K63)-linked polyubiquitination of Tax provides an important regulatory mechanism that promotes Tax-mediated interaction with the IKK complex and activation of NF-κB; however, the host proteins regulating Tax ubiquitination are largely unknown. To identify new Tax interacting proteins that may regulate its ubiquitination we conducted a yeast two-hybrid screen using Tax as bait. This screen yielded the E3/E4 ubiquitin conjugation factor UBE4B as a novel binding partner for Tax. Here, we confirmed the interaction between Tax and UBE4B in mammalian cells by co-immunoprecipitation assays and demonstrated colocalization by proximity ligation assay and confocal microscopy. Overexpression of UBE4B specifically enhanced Tax-induced NF-κB activation, whereas knockdown of UBE4B impaired Tax-induced NF-κB activation and the induction of NF-κB target genes in T cells and ATLL cell lines. Furthermore, depletion of UBE4B with shRNA resulted in apoptotic cell death and diminished the proliferation of ATLL cell lines. Finally, overexpression of UBE4B enhanced Tax polyubiquitination, and knockdown or CRISPR/Cas9-mediated knockout of UBE4B attenuated both K48- and K63-linked polyubiquitination of Tax. Collectively, these results implicate UBE4B in HTLV-1 Tax polyubiquitination and downstream NF-κB activation., Author summary Infection with the retrovirus HTLV-1 leads to the development of either CD4+CD25+ leukemia/lymphoma (ATLL) or a demyelinating neuroinflammatory disease (HAM/TSP) in a subset of infected individuals. The HTLV-1 Tax protein is a regulatory protein which regulates viral gene expression and persistently activates cellular signaling pathways such as NF-κB to drive the clonal expansion and longevity of HTLV-1 infected CD4+ T cells. Polyubiquitination of Tax is a key mechanism of NF-κB activation by assembling and activating IκB kinase (IKK) signaling complexes; however, the host factors regulating Tax ubiquitination have remained elusive. Here, we have identified the E3/E4 ubiquitin conjugation factor UBE4B as a novel Tax binding protein that promotes both K48- and K63-linked polyubiquitination of Tax. Knockdown or knockout of UBE4B impairs Tax-induced NF-κB activation and triggers apoptosis of HTLV-1-transformed cells. Therefore, UBE4B is an integral host factor that supports HTLV-1 Tax polyubiquitination, NF-κB activation and cell survival.

SAMHD1 is a host restriction factor that functions to restrict both retroviruses and DNA viruses, based on its nuclear deoxynucleotide triphosphate (dNTP) hydrolase activity that limits availability of intracellular dNTP pools. In the present study, we demonstrate that SAMHD1 expression was increased following human cytomegalovirus (HCMV) infection, with only a modest effect on infectious virus production. SAMHD1 was rapidly phosphorylated at residue T592 after infection by cellular cyclin-dependent kinases, especially Cdk2, and by the viral kinase pUL97, resulting in a significant fraction of phosho-SAMHD1 being relocalized to the cytoplasm of infected fibroblasts, in association with viral particles and dense bodies. Thus, our findings indicate that HCMV-dependent SAMHD1 cytoplasmic delocalization and inactivation may represent a potential novel mechanism of HCMV evasion from host antiviral restriction activities., Author summary Human cytomegalovirus (HCMV) is among the most widespread viruses worldwide, causing severe systemic illness in individuals without a fully competent immune system (e.g., transplanted patients and infants). Therefore, host immune response is crucial to control HCMV replication and dissemination throughout the organism. Some frontline proteins belonging to the innate immune system, known as restriction factors, are already expressed and active even before a pathogen infects a cell and are able to mediate a cell-intrinsic resistance to infections. Among them is SAMHD1, a nuclear deoxynucleotide triphosphate (dNTP) hydrolase that limits the availability of intracellular dNTPs, essential components of growing DNA molecules, including those of replicating viruses. We thus investigated SAMHD1 ability to counteract HCMV infection. We show that SAMHD1 expression was upregulated after infection, but with only a modest effect in limiting viral replication. This was mainly due to HCMV-induced phosphorylation at Threonine-592 (the key residue known to negatively regulate viral restriction), to phosphoT592-SAMHD1 relocalization to the cytoplasm of infected cells, and to its association with viral particles. The unprecedented observation of relocation of phosphoT592-SAMHD1 outside the nucleus may affect SAMHD1 function upon HCMV infection, and thus might represent a novel HCMV-mediated immune evasion strategy that will need further investigations.