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Background: Uncoating of the HIV-1 core plays a critical role during early post-fusion stages of infection but is poorly understood. Microscopy-based assays are unable to easily distinguish between intact and partially uncoated viral cores. Results: In this study, we used 5-ethynyl uridine (EU) to label viral-associated RNA during HIV production. At early time points after infection with EU-labeled virions, the viral-associated RNA was stained with an EU-specific dye and was detected by confocal microscopy together with viral proteins. We observed that detection of the viral-associated RNA was specific for EU-labeled virions, was detected only after viral fusion with target cells, and occurred after an initial opening of the core. In vitro staining of cores showed that the opening of the core allowed the small molecule dye, but not RNase A or antibodies, inside. Also, staining of the viral-associated RNA, which is co-localized with nucleocapsid, decays over time after viral infection. The decay rate of RNA staining is dependent on capsid (CA) stability, which was altered by CA mutations or a small molecule inducer of HIV-1 uncoating. While the staining of EU-labeled RNA was not affected by inhibition of reverse transcription, the kinetics of core opening of different CA mutants correlated with initiation of reverse transcription. Analysis of the E45A CA mutant suggests that initial core opening is independent of complete capsid disassembly. Conclusions: Taken together, our results establish a novel RNA accessibility-based assay that detects an early event in HIV-1 uncoating and can be used to further define this process. [ABSTRACT FROM AUTHOR]

TRIM5 is a restriction factor that blocks retrovirus infection soon after the virion core enters the cell cytoplasm. Restriction activity is targeted to the virion core via recognition of the capsid protein lattice that encases the viral genomic RNA. In common with all of the many TRIM family members, TRIM5 has RING, B-box, and coiled-coil domains. As an E3 ubiquitin ligase TRIM5 cooperates with the heterodimeric E2, UBC13/UEV1A, to activate the TAK1 (MAP3K7) kinase, NF-κB and AP-1 signaling, and the transcription of inflammatory cytokines and chemokines. TAK1, UBC13, and UEV1A all contribute to TRIM5-mediated retrovirus restriction activity. Interaction of the carboxy-terminal PRYSPRY or cyclophilin domains of TRIM5 with the retroviral capsid lattice stimulates the formation of a complementary lattice by TRIM5, with greatly increased TRIM5 E3 activity, and host cell signal transduction. Structural and biochemical studies on TRIM5 have opened a much needed window on how the innate immune system detects the distinct molecular features of HIV-1 and other retroviruses. [Copyright &y& Elsevier]

Background: Certain post-translational modifications to histones, including H3K4me3, as well as binding sites for the transcription factor STAT1, predict the site of integration of exogenous gamma-retroviruses with great accuracy and cell-type specificity. Statistical methods that were used to identify chromatin features that predict exogenous gamma-retrovirus integration site selection were exploited here to determine whether cell type-specific chromatin markers are enriched in the vicinity of endogenous retroviruses (ERVs). Results: Among retro-elements in the human genome, the gamma-retrovirus HERV-H was highly associated with H3K4me3, though this association was only observed in embryonic stem (ES) cells (p < 10-300) and, to a lesser extent, in induced pluripotent stem (iPS) cells. No significant association was observed in nearly 40 differentiated cell types, nor was any association observed with other retro-elements. Similar strong association was observed between HERV-H and the binding sites within ES cells for the pluripotency transcription factors NANOG, OCT4, and SOX2. NANOG binding sites were located within the HERV-H 5′LTR itself. OCT4 and SOX2 binding sites were within 1 kB and 2 kB of the 5′LTR, respectively. In keeping with these observations, HERV-H RNA constituted 2% of all poly A RNA in ES cells. As ES cells progressed down a differentiation pathway, the levels of HERV-H RNA decreased progressively. RNA-Seq datasets showed HERV-H transcripts to be over 5 kB in length and to have the structure 5′LTR-gag-pro-3′LTR, with no evidence of splicing and no intact open reading frames. Conclusion: The developmental regulation of HERV-H expression, the association of HERV-H with binding sites for pluripotency transcription factors, and the extremely high levels of HERV-H RNA in human ES cells suggest that HERV-H contributes to pluripotency in human cells. Proximity of HERV-H to binding sites for pluripotency transcription factors within ES cells might be due to retention of the same chromatin features that determined the site of integration of the ancestral, exogenous, gamma-retrovirus that gave rise to HERV-H in the distant past. Retention of these markers, or, alternatively, recruitment of them to the site of the established provirus, may have acted post-integration to fix the provirus within the germ-line of the host species. Either way, HERV-H RNA provides a specific marker for pluripotency in human cells. [ABSTRACT FROM AUTHOR]

Primate lentivirus nef is required for sustained virus replication in vivo and accelerated progression to AIDS. While exploring the mechanism by which Nef increases the infectivity of cell-free virions, we investigated a functional link between Nef and Env. Since we failed to detect an effect of Nef on the quantity of virion-associated Env, we searched for qualitative changes by examining whether Nef alters HIV-1 sensitivity to agents that target distinct features of Env. Nef conferred as much as 50- fold resistance to 2F5 and 4E10, two potent neutralizing monoclonal antibodies (nAbs) that target the membrane proximal external region (MPER) of TMgp41. In contrast, Nef had no effect on HIV-1 neutralization by MPER-specific nAb Z13e1, by the peptide inhibitor T20, nor by a panel of nAbs and other reagents targeting gp120. Resistance to neutralization by 2F5 and 4E10 was observed with Nef from a diverse range of HIV-1 and SIV isolates, as well as with HIV-1 virions bearing Env from CCR5- and CXCR4-tropic viruses, clade B and C viruses, or primary isolates. Functional analysis of a panel of Nef mutants revealed that this activity requires Nef myristoylation but that it is genetically separable from other Nef functions such as the ability to enhance virus infectivity and to downregulate CD4. Glycosylated-Gag from MoMLV substituted for Nef in conferring resistance to 2F5 and 4E10, indicating that this activity is conserved in a retrovirus that does not encode Nef. Given the reported membrane-dependence of MPER-recognition by 2F5 and 4E10, in contrast to the membrane-independence of Z13e1, the data here is consistent with a model in which Nef alters MPER recognition in the context of the virion membrane. Indeed, Nef and Glycosylated-Gag decreased the efficiency of virion capture by 2F5 and 4E10, but not by other nAbs. These studies demonstrate that Nef protects lentiviruses from one of the most broadly-acting classes of neutralizing antibodies. This newly discovered activity for Nef has important implications for anti-HIV-1 immunity and AIDS pathogenesis. [ABSTRACT FROM AUTHOR]

TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice and several reports indicate that TRIM5 has a role in signal transduction, but the precise mechanism of restriction is unknown. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13-UEV1A (also known as UBE2N-UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NFκB signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13-UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-κB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13-UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice. [ABSTRACT FROM AUTHOR]

Plasma cells daily secrete their own mass in antibodies, which fold and assemble in the endoplasmic reticulum (ER). To reach these levels, cells require pERp1, a novel lymphocyte-specific small ER-resident protein, which attains expression levels as high as BiP when B cells differentiate into plasma cells. Although pERp1 has no homology with known ER proteins, it does contain a CXXC motif typical for oxidoreductases. In steady state, the CXXC cysteines are locked by two parallel disulfide bonds with a downstream C(X)6C motif, and pERpi displays only modest oxidoreductase activity. pERp1 emerged as a dedicated folding factor for IgM, associating with both heavy and light chains and promoting assembly and secretion of mature IgM. [ABSTRACT FROM AUTHOR]

The isolation of human embryonic and somatic stem cells of different types has made it possible to design novel gene and cell replacement therapies. Vectors derived from retro/lentiviruses are used to stably introduce genes into stem cells and their progeny. However, the permissivity to retroviral infection varies among cell types. We previously showed that hematopoietic stem cells are poorly permissive to human immunodeficiency virus (HIV)-derived vectors and that pharmacological inhibition of the proteasome strongly enhances gene transfer. Here we report that the proteasome limits lentiviral gene transfer in all stem cell types tested, including embryonic, mesenchymal, and neural, of both human and mouse origin. Remarkably, this inhibitory activity was sharply reduced upon differentiation of the stem cells, suggesting that it represents a novel feature of the stem cell/immature progenitor phenotype. Proteasome-mediated inhibition was specific for lentiviral vectors and occurred at a postentry infection step. It was not mediated by activation of nuclear factor-κB, a major signaling pathway modulated by the proteasome, and did not correlate with high proteasome activity. Interaction of the virion core with cyclophilin A was required to maximize the effect of proteasome inhibitor on the infection pathway. These findings are relevant to uncover new mediators of HIV gene transfer and help in designing more effective protocols for the genetic modification of stem cells. [ABSTRACT FROM AUTHOR]

Retroviruses are obligate intracellular parasites that have coevolved with their hosts for millions of years. It is therefore not surprising that retroviruses take advantage of numerous host factors during their life cycle. In addition to positive cellular factors that are of use to the virus, host cells have also evolved intracellular proteins to antagonize the retroviral replication cycle. Such inhibitory cellular factors have been called retroviral restriction factors. Recently, several such restriction factors have been cloned, including Friend virus susceptibility factor 1, apolipoprotein B mRNA-editing enzyme catalytic proteins 3F and 3G, and ZAP. Here, we review the explosion of publications from the past 2 years concerning TRIM5, a host factor that potently inhibits HIV-1 and other retroviruses. [ABSTRACT FROM AUTHOR]

The peptidyl-prolyl isomerase cyclophilin A (CypA) binds a proline-rich loop on the surface of HIV-1 capsid (CA). This interaction increases HIV-1 infectivity in humans but promotes an anti-HIV-1 restriction activity in non-human primates. Efforts to understand these paradoxical effects of cyclophilin, along with more targeted approaches to uncover the genetic basis for HIV-1 restriction, led to the discovery of TRIM5 (tripartite motif protein 5), a CA-specific receptor for the retroviral core. The ensuing TRIM5 publication flurry established a paradigm of innate immunity in which the protein lattice of an invading retroviral core, rather than double-stranded RNA or lipopolysaccharide, is recognized by a multimeric, cytoplasmic receptor. CypA modulates HIV-1 virion core detection by this class of innate pattern recognition molecule, apparently by inducing subtle shifts in CA conformation. [Copyright &y& Elsevier]

The peptidyl-prolyl isomerase cyclophilin A (CypA) embraces an exposed, proline-rich loop on HIV-1 capsid (CA) and renders reverse transcription complexes resistant to an antiviral activity in human cells. A CypA fusion with TRIM5 that is unique to New World owl monkeys also targets HIV-1 CA, but this interaction potently inhibits infection. A similar block to HIV-1 infection in Old World monkeys is attributable to the a isoform of the TRIM5 orthologue in these species. To determine whether HIV-1 restriction by Old World monkey TRIM5α is modulated by the CA-CypA interaction, RNA interference was used to disrupt CypA in cells from African green monkeys and rhesus macaques. HIV-1 infectivity increased in response to CypA knock-down to the same extent that it increased in response to TRIMS knock-down. CypA knock-down eliminated the HIV-1 stimulatory effect of cyclosporin A (CsA), a competitive inhibitor of the CypA-CA interaction, or of CA mutants that block binding to CypA but caused no change in titer of retroviruses that don't interact with CypA. Simultaneous knock-down of both CypA and TRIMS caused minimal additional increase in titer, suggesting that CypA inhibits HIV-1 replication in these cells because it is required for CA recognition by TRIMSa. Finally, CsA increased HIV-1 titer in otherwise nonrestrictive feline cells but only after these cells were transduced with Old World monkey TRIM5α. Thus, CypA is required for HIV-1 restriction by Old World monkey orthologues of TRIM5α. [ABSTRACT FROM AUTHOR]

Capsid (CA)-specific restrictions are determinants of retroviral tropism in mammalian cells. One such restriction, human Ref1, targets strains of murine leukemia virus bearing an arginine at CA residue 110 (N-MLV), resulting in decreased accumulation of viral cDNA. The cellular factors accounting for Reft activity are unknown. As2O3 increases N-MLV titer in Ref1-positive cells, possibly by counteracting Ref1. Restriction factor saturation experiments suggest that Reft may also target human immunodeficiency virus type 1 (HIV-1), but only if its CA is not bound to the cellular protein cyclophilin A (CypA). As a step towards understanding the genetic determinants of Ref1, we subjected Ref1-positive TE671 cells to three sequential rounds of selection with N-MLV reporter viruses. We isolated a subclone, 17H1, that was permissive for N-MLV infection and therefore deficient in Reft activity. Stimulation of N-MLV replication by As2O3 was attenuated in 17H1, confirming that the drug acts by overcoming Ref1 activity. HIV-1 infection of 17H1 cells was resistant to disruption of the CA-CypA interaction, demonstrating that Ref1 restricts CypA-free HIV-1. Our results suggest that interaction with CypA evolved to protect HIV-1 from this human antiviral activity. [ABSTRACT FROM AUTHOR]

In Old World primates, TRIM5-a confers a potent block to human immunodeficiency virus type 1 (HIV-1) infection that acts after virus entry into cells. Cyclophilin A (CypA) binding to viral capsid protects HIV-1 from a similar activity in human cells. Among New World primates, only owl monkeys exhibit post-entry restriction of HIV-1 (ref. 1). Paradoxically, the barrier to HIV-1 in owl monkey cells is released by capsid mutants or drugs that disrupt capsid interaction with CypA. Here we show that knockdown of owl monkey CypA by RNA interference (RNAi) correlates with suppression of anti-HIV-1 activity. However, reintroduction of CypA protein to RNAi-treated cells did not restore antiviral activity. A search for additional RNAi targets unearthed TRIMCyp, an RNAi-responsive messenger RNA encoding a TRIM5-CypA fusion protein. TRIMCyp accounts for post-entry restriction of HIV-1 in owl monkeys and blocks HIV-1 infection when transferred to otherwise infectable human or rat cells. It seems that TRIMCyp arose after the divergence of New and Old World primates when a LINE-1 retrotransposon catalysed the insertion of a CypA complementary DNA into the TRIM5 locus. This is the first vertebrate example of a chimaeric gene generated by this mechanism of exon shuffling. [ABSTRACT FROM AUTHOR]

Cyclophilin A (CypA) was determined to interact with apoptosis-inducing factor (AIF) by mass spectroscopy, coimmunoprecipitation, pull-down assays, and molecular modeling. During the initial, caspase-independent stage of chromatin condensation that accompanies apoptosis, AIF and CypA were found to coimmunolocalize in the nucleus. Recombinant AIF and CypA proteins synergized in vitro in the degradation of plasmid DNA, as well as in the capacity to induce DNA loss in purified nuclei. The apoptogenic cooperation between AIF and CypA did not rely on the CypA peptidyl-prolyl cis-trans isomerase activity. In Cyp-expressing cells, AIF overexpression augmented apoptotic chromatinolysis. The AIF-dependent large-scale DNA fragmentation was less pronounced in CypA knockout cells as compared to controls. AIF mutants lacking the CypA-binding domain were inefficient apoptosis sensitizers in transfection experiments. Moreover, AIF failed to sensitize CypA knockout cells to apoptosis induction, and this defect in the AIF response was reversed by reintroduction of the CypA gene into CypA-deficient cells. In summary, AIF and CypA collaborate in chromatinolysis.Oncogene (2004) 23, 1514-1521. doi:10.1038/sj.onc.1207279 Published online 12 January 2004 [ABSTRACT FROM AUTHOR]

Many mammalian species express restriction factors that confer host resistance to retroviral infection. Here we show that HIV-1 sensitivity to restriction factors is modulated by cyclophilin A (CypA), a host cell protein that binds the HIV-1 capsid protein (CA). In certain nonhuman primate cells, the CA-CypA interaction is essential for restriction: HIV-1 infectivity is increased >100-fold by cyclosporin A (CsA), a competitive inhibitor of the interaction, or by an HIV-1 CA mutation that disrupts CypA binding. Conversely, disruption of CA-CypA interaction in human cells reveals that CypA protects HIV-1 from the Ref-1 restriction factor. These findings suggest that HIV-1 has co-opted a host cell protein to counteract restriction factors expressed by human cells and that this adaptation can confer sensitivity to restriction in unnatural hosts. Manipulation of HIV-1 CA recognition by restriction factors promises to advance animal models and new therapeutic strategies for HIV-1 and AIDS. [ABSTRACT FROM AUTHOR]

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein binds most members of the cyclophilin family of peptidyl-prolyl isomerases. Of 15 known human cyclophilins, cyclophilin A (CypA) has been the focus of investigation because it was detected in HIV-1 virions. To determine whether CypA promotes HIV-1 replication, we deleted the gene encoding CypA (PPIA) in human CD4+ T cells by homologous recombination. HIV-1 replication in PPIA-/- cells was decreased and not inhibited further by cyclosporin or gag mutations that disrupt Gag's interaction with cyclophilins, indicating that no other cyclophilin family members promote HIV-1 replication. The defective replication phenotype was specific for wild-type HIV-1 since HIV-2/SIV isolates, as well as HIV-1 bearing a gag mutation that confers cyclosporin resistance, replicated the same in PPIA+/+ and PPIA-/- cells. Stable re-expression of CypA in PPIA-/- cells restored HIV-1 replication to an extent that correlated with steady-state levels of CypA. Finally, virions from PPIA-/- cells possessed no obvious biochemical abnormalities but were less infectious than virions from wild-type cells. These data formally demonstrate that CypA regulates the infectivity of HIV-1 virions. [ABSTRACT FROM AUTHOR]

The molecular checkpoints driving T cell activation and cytokine responses in ulcerative colitis (UC) are incompletely understood. Here, we studied the Tec kinase ITK in UC. We analyzed patients with inflammatory bowel disease (n = 223) and evaluated ITK activity as well as the functional effects of cyclosporine-A (CsA). In addition, 3 independent murine colitis models were used to investigate the functional role of ITK. Finally, the activity of ITK was blocked via pharmacological inhibitors and genetically engineered mice. Readout parameters were mini-endoscopy, histopathology, mucosal T cell apoptosis, and cytokine production. We found an expansion of pITK-expressing mucosal CD4+ T cells in UC rather than Crohn's disease that correlated with disease severity. CsA suppressed activation of ITK in cultured CD4+ T cells and calcineurin-containing microclusters adjacent to the T cell receptor signaling complex. Functionally, the capacity of CsA to suppress activity of experimental colitis was critically dependent on ITK. Genetic inactivation of Itk via gene targeting or induction of allele-sensitive Itk mutants prevented experimental colitis in 3 colitis models, and treatment with pharmacological ITK blockers suppressed established colitis. In addition, ITK controlled apoptosis and activation of mucosal Th2 and Th17 lymphocytes via NFATc2 signaling pathways. ITK activation was detected in UC and could be down-regulated in cultured T cells by CsA administration. Selective targeting of ITK emerges as an attractive approach for treatment of chronic intestinal inflammation and potentially UC by driving resolution of mucosal inflammation. [Display omitted] ITK is highly activated in patients with ulcerative colitis only and cyclosporine-A suppresses ITK activity. Therapeutic modulation of the cyclosporine-A target ITK might be helpful for optimized therapy in ulcerative colitis, as inactivation of ITK leads to resolution of intestinal mucosal inflammation. [ABSTRACT FROM AUTHOR]

Cyclophilin A (CypA) binds to the HIV-1 capsid to facilitate reverse transcription and nuclear entry and counter the antiviral activity of TRIM5a. Interestingly, recent studies suggest that the capsid enters the nucleus of an infected cell and uncoats prior to integration. We have previously reported that the capsid protein regulates HIV-1 integration. Therefore, we probed whether CypA-capsid interaction also regulates this post-nuclear entry step. First, we challenged CypA-expressing (CypA+/+) and CypA-depleted (CypA-/-) cells with HIV-1 and quantified the levels of provirus. CypA-depletion significantly reduced integration, an effect that was independent of CypA's effect on reverse transcription, nuclear entry, and the presence or absence of TRIM5a. In addition, cyclosporin A, an inhibitor that disrupts CypA-capsid binding, inhibited proviral integration in CypA+/+ cells but not in CypA-/- cells. HIV-1 capsid mutants (G89V and P90A) deficient in CypA binding were also blocked at the integration step in CypA+/+ cells but not in CypA-/- cells. Then, to understand the mechanism, we assessed the integration activity of the HIV-1 preintegration complexes (PICs) extracted from acutely infected cells. PICs from CypA-/- cells retained lower integration activity in vitro compared to those from CypA+/+ cells. PICs from cells depleted of both CypA and TRIM5a also had lower activity, suggesting that CypA's effect on PIC was independent of TRIM5a. Finally, CypA protein specifically stimulated PIC activity, as this effect was significantly blocked by CsA. Collectively, these results provide strong evidence that CypA directly promotes HIV-1 integration, a previously unknown role of this host factor in the nucleus of an infected cell. IMPORTANCE Interaction between the HIV-1 capsid and host cellular factors is essential for infection. However, the molecular details and functional consequences of viral-host factor interactions during HIV-1 infection are not fully understood. Over 30 years ago, Cyclophilin A (CypA) was identified as the first host protein to bind to the HIV-1 capsid. Now it is established that CypA-capsid interaction promotes reverse transcription and nuclear entry of HIV-1. In addition, CypA blocks TRIM5a-mediated restriction of HIV-1. In this report, we show that CypA promotes the post-nuclear entry step of HIV-1 integration by binding to the viral capsid. Notably, we show that CypA stimulates the viral DNA integration activity of the HIV-1 preintegration complex. Collectively, our studies identify a novel role of CypA during the early steps of HIV-1 infection. This new knowledge is important because recent reports suggest that an operationally intact HIV-1 capsid enters the nucleus of an infected cell. [ABSTRACT FROM AUTHOR]

Host plasma membrane protein SERINC5 is incorporated into budding retrovirus particles where it blocks subsequent entry into susceptible target cells. Three structurally unrelated proteins encoded by diverse retroviruses, human immunodeficiency virus type 1 (HIV-1) Nef, equine infectious anemia virus (EIAV) S2, and ecotropic murine leukemia virus (MLV) GlycoGag, disrupt SERINC5 antiviral activity by redirecting SERINC5 from the site of virion assembly on the plasma membrane to an internal RAB7+ endosomal compartment. Pseudotyping retroviruses with particular glycoproteins, e.g., vesicular stomatitis virus glycoprotein (VSV G), renders the infectivity of particles resistant to inhibition by virion-associated SERINC5. To better understand viral determinants for SERINC5-sensitivity, the effect of SERINC5 was assessed using HIV-1, MLV, and Mason-Pfizer monkey virus (M-PMV) virion cores, pseudotyped with glycoproteins from Arenavirus, Coronavirus, Filovirus, Rhabdovirus, Paramyxovirus, and Orthomyxovirus genera. SERINC5 restricted virions pseudotyped with glycoproteins from several retroviruses, an orthomyxovirus, a rhabdovirus, a paramyxovirus, and an arenavirus. Infectivity of particles pseudotyped with HIV-1, amphotropic-MLV (A-MLV), or influenza A virus (IAV) glycoproteins, was decreased by SERINC5, whether the core was provided by HIV-1, MLV, or M-PMV. In contrast, particles pseudotyped with glycoproteins from M-PMV, parainfluenza virus 5 (PIV5), or rabies virus (RABV) were sensitive to SERINC5, but only with particular retroviral cores. Resistance to SERINC5 did not correlate with reduced SERINC5 incorporation into particles, route of viral entry, or absolute infectivity of the pseudotyped virions. These findings indicate that some non-retroviruses may be sensitive to SERINC5 and that, in addition to the viral glycoprotein, the retroviral core influences sensitivity to SERINC5. [ABSTRACT FROM AUTHOR]

• COVID-19 is typified by SARS-CoV-2 replication and an excessive inflammatory response. • PTC299 is a small orally bioavailable DHODH inhibitor that blocks pyrimidine synthesis. • PTC299 is a potent inhibitor of the replication of SARS-CoV-2 and other RNA viruses. • PTC299 also reduces the production of cytokines associated with COVID-19 inflammation. • PTC299 may be a promising therapeutic for COVID-19. The coronavirus disease 2019 (COVID-19) pandemic has created an urgent need for therapeutics that inhibit the SARS−COV-2 virus and suppress the fulminant inflammation characteristic of advanced illness. Here, we describe the anti−COVID-19 potential of PTC299, an orally bioavailable compound that is a potent inhibitor of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme of the de novo pyrimidine nucleotide biosynthesis pathway. In tissue culture, PTC299 manifests robust, dose-dependent, and DHODH-dependent inhibition of SARS−COV-2 replication (EC 50 range, 2.0–31.6 nM) with a selectivity index >3,800. PTC299 also blocked replication of other RNA viruses, including Ebola virus. Consistent with known DHODH requirements for immunomodulatory cytokine production, PTC299 inhibited the production of interleukin (IL)-6, IL-17A (also called IL-17), IL-17 F, and vascular endothelial growth factor (VEGF) in tissue culture models. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and previously established favorable pharmacokinetic and human safety profiles render PTC299 a promising therapeutic for COVID-19. [ABSTRACT FROM AUTHOR]

Adipocytes deficient in fatty acid synthase (iAdFASNKO) emit signals that mimic cold exposure to enhance the appearance of thermogenic beige adipocytes in mouse inguinal white adipose tissues (iWATs). Both cold exposure and iAdFASNKO upregulate the sympathetic nerve fiber (SNF) modulator Neuregulin 4 (Nrg4), activate SNFs, and require adipocyte cyclic AMP/protein kinase A (cAMP/PKA) signaling for beige adipocyte appearance, as it is blocked by adipocyte Gsα deficiency. Surprisingly, however, in contrast to cold-exposed mice, neither iWAT denervation nor Nrg4 loss attenuated adipocyte browning in iAdFASNKO mice. Single-cell transcriptomic analysis of iWAT stromal cells revealed increased macrophages displaying gene expression signatures of the alternately activated type in iAdFASNKO mice, and their depletion abrogated iWAT beiging. Altogether, these findings reveal that divergent cellular pathways are sufficient to cause adipocyte browning. Importantly, adipocyte signaling to enhance alternatively activated macrophages in iAdFASNKO mice is associated with enhanced adipose thermogenesis independent of the sympathetic neuron involvement this process requires in the cold. • Light sheet microscopy reveals nerve fibers abutting beige adipocytes in iWAT • iWAT denervation blocks cold-induced but not adipocyte FASNKO-induced iWAT beiging • cAMP/PKA pathway in adipocytes mediates iWAT beiging in adipocyte FASNKO mice • Macrophages but not innervation are required for beiging in adipocyte FASNKO mice Henriques et al. show an alternative pathway to enhance thermogenesis through an adipocyte cAMP/PKA axis in denervated iWAT. Signals emanating from this pathway generate M2-type macrophages associated with iWAT browning. [ABSTRACT FROM AUTHOR]

For highly pathogenic viruses, reporter assays that can be rapidly performed are critically needed to identify potentially functional mutations for further study under maximal containment (e.g., biosafety level 4 [BSL-4]). The Ebola virus nucleoprotein (NP) plays multiple essential roles during the viral life cycle, yet few tools exist to study the protein under BSL-2 or equivalent containment. Therefore, we adapted reporter assays to measure NP oligomerization and virion-like particle (VLP) production in live cells and further measured transcription and replication using established minigenome assays. As a proof-of-concept, we examined the NP-R111C substitution, which emerged during the 2013–2016 Western African Ebola virus disease epidemic and rose to high frequency. NP-R111C slightly increased NP oligomerization and VLP budding but slightly decreased transcription and replication. By contrast, a synthetic charge-reversal mutant, NP-R111E, greatly increased oligomerization but abrogated transcription and replication. These results are intriguing in light of recent structures of NP oligomers, which reveal that the neighboring residue, K110, forms a salt bridge with E349 on adjacent NP molecules. By developing and utilizing multiple reporter assays, we find that the NP-111 position mediates a complex interplay between NP's roles in protein structure, virion budding, and transcription and replication. [ABSTRACT FROM AUTHOR]

The Ebola virus (EBOV) envelope glycoprotein (GP) mediates the fusion of the virion membrane with the membrane of susceptible target cells during infection. While proteolytic cleavage of GP by endosomal cathepsins and binding of the cellular receptor Niemann-Pick C1 protein (NPC1) are essential steps for virus entry, the detailed mechanisms by which these events promote membrane fusion remain unknown. Here, we applied single-molecule Förster resonance energy transfer (smFRET) imaging to investigate the structural dynamics of the EBOV GP trimeric ectodomain, and the functional transmembrane protein on the surface of pseudovirions. We show that in both contexts, pre-fusion GP is dynamic and samples multiple conformations. Removal of the glycan cap and NPC1 binding shift the conformational equilibrium, suggesting stabilization of conformations relevant to viral fusion. Furthermore, several neutralizing antibodies enrich alternative conformational states. This suggests that these antibodies neutralize EBOV by restricting access to GP conformations relevant to fusion. This work demonstrates previously unobserved dynamics of pre-fusion EBOV GP and presents a platform with heightened sensitivity to conformational changes for the study of GP function and antibody-mediated neutralization. [ABSTRACT FROM AUTHOR]

Intron-containing RNA expressed from the HIV-1 provirus activates type 1 interferon in primary human blood cells, including CD4+ T cells, macrophages, and dendritic cells. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with short hairpin RNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte-derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the interferon-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with nontargetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant 2-CARD domain-deletion or phosphomimetic point mutations, indicates that IFIH1 (MDA5) filament formation, dephosphorylation, and association with MAVS are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 (MDA5) and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1 knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes by HIV-1. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 (MDA5), over two orders of magnitude, was revealed by formaldehyde cross-linking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is the innate immune receptor for intron-containing RNA from the HIV-1 provirus and that IFIH1 potentially contributes to chronic inflammation in people living with HIV-1, even in the presence of effective antiretroviral therapy. [ABSTRACT FROM AUTHOR]

HIV-1-infected people who take drugs that suppress viremia to undetectable levels are protected from developing AIDS. Nonetheless, HIV-1 establishes proviruses in long-lived CD4+ memory T cells, and perhaps other cell types, that preclude elimination of the virus even after years of continuous antiviral therapy. Here we show that the HIV-1 provirus activates innate immune signaling in isolated dendritic cells, macrophages, and CD4+ T cells. Immune activation requires transcription from the HIV-1 provirus and expression of CRM1-dependent, Rev-dependent, RRE-containing, unspliced HIV-1 RNA. If rev is provided in trans, all HIV-1 coding sequences are dispensable for activation except those cis-acting sequences required for replication or splicing. Our results indicate that the complex, post-transcriptional regulation intrinsic to HIV-1 RNA is detected by the innate immune system as a danger signal, and that drugs which disrupt HIV-1 transcription or HIV-1 RNA metabolism would add qualitative benefit to current antiviral drug regimens. During HIV infection, antiviral therapy can suppress viraemia to undetectable levels and hinder the progression towards AIDS; however the HIV-1 provirus can remain in long-lived CD4+ memory T cells. Here the authors show that intronic RNA from the HIV-1 provirus can induce type I interferon and inflammatory cytokine production. [ABSTRACT FROM AUTHOR]

The article focuses on tripartite motif-containing protein 21 (TRIM21) which is found to degrade virion capsid. The author states that the TRIM21 binds the adenovirus capsid in the cytoplasm and triggers capsid degradation by the proteasome. He note that neutralizing antibodies are believed to block interaction of the virion with cognate cell surface receptors on susceptible target cells.

Interaction between the Ebola virus envelope glycoprotein (GP) and the endosomal membrane is an essential step during virus entry into the cell. Acidic pH and Ca2+ have been implicated in mediating the GP-membrane interaction. However, the molecular mechanism by which these environmental factors regulate the conformational changes that enable engagement of GP with the target membrane is unknown. Here, we apply fluorescence correlation spectroscopy (FCS) and single-molecule Förster resonance energy transfer (smFRET) imaging to elucidate how the acidic pH, Ca2+ and anionic phospholipids in the late endosome promote GP-membrane interaction, thereby facilitating virus entry. We find that bis(monoacylglycero)phosphate (BMP), which is specific to the late endosome, is especially critical in determining the Ca2+-dependence of the GP-membrane interaction. Molecular dynamics (MD) simulations suggested residues in GP that sense pH and induce conformational changes that make the fusion loop available for insertion into the membrane. We similarly confirm residues in the fusion loop that mediate GP's interaction with Ca2+, which likely promotes local conformational changes in the fusion loop and mediates electrostatic interactions with the anionic phospholipids. Collectively, our results provide a mechanistic understanding of how the environment of the late endosome regulates the timing and efficiency of virus entry. Author summary: Ebola virus causes disease in humans with high fatality. A better understanding of how Ebola virus enters cells is critical to inform the development of novel therapeutic and preventative measures. The viral glycoprotein present on the surface of the virus mediates attachment to cells and subsequent entry through a poorly understood mechanism involving fusion of viral and cellular membranes. Here, we employ computational and experimental biophysical techniques to understand how the Ebola glycoprotein senses chemical cues in its environment, such as pH, calcium ions, and specific lipid species to ensure that entry occurs at the right time and place. Our results specify elements of the glycoprotein that control its structure under changing physiological environments. [ABSTRACT FROM AUTHOR]

The HIV-1 genome encodes a small number of proteins with structural, enzymatic, regulatory, and accessory functions. These viral proteins interact with a number of host factors to promote the early and late stages of HIV-1 infection. During the early stages of infection, interactions between the viral proteins and host factors enable HIV-1 to enter the target cell, traverse the cytosol, dock at the nuclear pore, gain access to the nucleus, and integrate into the host genome. Similarly, the viral proteins recruit another set of host factors during the late stages of infection to orchestrate HIV-1 transcription, translation, assembly, and release of progeny virions. Among the host factors implicated in HIV-1 infection, Cyclophilin A (CypA) was identified as the first host factor to be packaged within HIV-1 particles. It is now well established that CypA promotes HIV-1 infection by directly binding to the viral capsid. Mechanistic models to pinpoint CypA's role have spanned from an effect in the producer cell to the early steps of infection in the target cell. In this review, we will describe our understanding of the role(s) of CypA in HIV-1 infection, highlight the current knowledge gaps, and discuss the potential role of this host factor in the post-nuclear entry steps of HIV-1 infection. [ABSTRACT FROM AUTHOR]