Search

All lentiviruses encode a post-transcriptional transactivator, Rev, which mediates the export of viral mRNA from the nucleus to the cytoplasm and which is required for viral gene expression and viral replication. In the current study, we demonstrate that equine infectious anemia virus (EIAV), an equine lentivirus, encodes a second post-transcriptional transactivator that we designate Grev. Grev is encoded by a novel transcript with a single splicing event that was identified using reverse transcription-PCR (RT-PCR) and RNA-seq in EIAV-infected horse tissues and cells. Grev is about 18 kDa in size, comprises the first 18 amino acids (aa) of Gag protein together with the last 82 aa of Rev, and was detected in EIAV-infected cells. Similar to Rev, Grev is localized to the nucleus, and both are able to mediate the expression of Mat (a recently identified viral protein of unknown function from EIAV), but Rev can mediate the expression of EIAV Gag/Pol, while Grev cannot. We also demonstrate that Grev, similar to Rev, specifically binds to rev-responsive element 2 (RRE-2, located in the first exon of mat mRNAs) to promote nuclear export of mat mRNA via the chromosome region maintenance 1 (CRM1) pathway. However, unlike Rev, whose function depends on its multimerization, we could not detect multimerization of Grev using coimmunoprecipitation (co-IP) or bimolecular fluorescence complementation (BiFC) assays. Together, these data suggest that EIAV encodes two post-transcriptional transactivators, Rev and Grev, with similar, but not identical, functions.

Viral infection activates the type I interferons (IFNs) and cellular antiviral responses. Eukaryotic initiation factor 4A-III (eIF4A3) has been shown to promote influenza A virus (IAV) replication by promoting viral mRNA splicing and spliced mRNA nuclear export. Here, we identified eIF4A3 as a negative regulator of virus-triggered type I IFN induction. Our study found that eIF4A3 promoted multiple RNA viruses’ replication by binding to IFN regulatory factor 3 (IRF3) and impaired the interaction between tank-binding kinase 1 (TBK1) and IRF3, leading to attenuation of the phosphorylation of IRF3 by TBK1, the formation of IRF3 dimer, and the nuclear translocation of IRF3. This impaired its biological functions in the nucleus, which blocked IRF3 binding to interferon-stimulated response element (ISRE) and the interaction of IRF3 and CBP/p300, resulting in inhibiting the transcription of IFN-β and downstream IFN-stimulated genes (ISGs), thereby impairing innate antiviral immune responses against RNA viruses. These findings reveal a previously unknown function of eIF4A3 in host innate immunity and establish a mechanistic link between eIF4A3 and IRF3 activation that expands potential therapeutic strategies for viral infectious diseases. IMPORTANCE Production of type I IFN is pivotal for the cellular antiviral immunity. Virus infection leads to the activation of transcription factor IRF3 and subsequent production of type I IFN to eliminate viral infection. Thus, the regulation of IRF3 activity is an important way to affect type I IFN production. IRF3 activation requires phosphorylation, dimerization, and nuclear translocation. Here, we first reported that eIF4A3, a member of DEAD box family, served as a negative regulator of antiviral innate immune responses by inhibiting IRF3 activation. Mechanistically, eIF4A3 binds to IRF3 to impair the recruitment of IRF3 by TBK1, which is independent of eIF4A3 ATP binding, ATPase, and RNA helicase activities. Our study delineates a common mechanism of eIF4A3 promoting replication of different RNA viruses and provides important insights into the negative regulation of host antiviral innate immune responses against virus infections.

In Saccharomyces cerevisiae cells, dysfunction of the endoplasmic reticulum (ER), so-called ER stress, leads to conversion of HAC1 mRNA to the spliced form (HAC1i), which is translated into a transcription factor that drastically changes the gene expression profile. This cellular response ultimately enhances ER functions and is named the unfolded protein response (UPR). Artificial evocation of the UPR is therefore anticipated to increase productivity of beneficial materials on and in the ER. However, as demonstrated here, cells constitutively expressing HAC1i mRNA (HAC1i cells), which exhibited a strong UPR even under nonstress conditions, grew considerably slowly and frequently yielded fast-growing and low-UPR progeny. Intriguingly, growth of HAC1i cells was faster in the presence of weak ER stress that was induced by low concentrations of the ER stressor tunicamycin or by cellular expression of the ER-located version of green fluorescent protein (GFP). HAC1i cells producing ER-localized GFP stably exhibited a strong UPR activity, carried a highly expanded ER, and abundantly produced triglycerides and heterogenous carotenoids. We therefore propose that our findings provide a basis for metabolic engineering to generate cells producing valuable lipidic molecules. IMPORTANCE The UPR is thought to be a cellular response to cope with the accumulation of unfolded proteins in the ER. In S. cerevisiae cells, the UPR is severely repressed under nonstress conditions. The findings of this study shed light on the physiological significance of the tight regulation of the UPR. Constitutive UPR induction caused considerable growth retardation, which was partly rescued by the induction of weak ER stress. Therefore, we speculate that when the UPR is inappropriately induced in unstressed cells lacking aberrant ER client proteins, the UPR improperly impairs normal cellular functions. Another important point of this study was the generation of S. cerevisiae strains stably exhibiting a strong UPR activity and abundantly producing triglycerides and heterogenous carotenoids. We anticipate that our findings may be applied to produce valuable lipidic molecules using yeast cells as a potential next-generation technique.

SARS-CoV-2 antibody levels wane following two-doses of mRNA vaccination. An mRNA booster dose provides increased protection against hospitalization and death. We demonstrated that a booster dose provides a significant increase in the neutralization of the Beta, Delta and Omicron variants in addition to an increased neutralization of the vaccine strain. The total spike IgG measurements, obtained by using commercial kits that target the spike protein from the vaccine strain, may not reflect serum neutralization against variants of concern.

Iridoviruses are large DNA viruses which cause great economic losses to the aquaculture industry and serious threats to ecological diversity worldwide. Singapore grouper iridovirus (SGIV), a novel member of the genus Ranavirus, causes high mortality in grouper aquaculture. Previous work on genome annotation demonstrated that SGIV contained numerous uncharacterized or hypothetical open reading frames (ORFs), whose functions remained largely unknown. Here, we reported that the protein encoded by SGIV ORF131R (VP131) was localized predominantly within the endoplasmic reticulum (ER). Ectopic expression of GFP-VP131 significantly enhanced SGIV replication, while VP131 knockdown decreased viral infection in vitro, suggesting that VP131 functioned as a proviral factor during SGIV infection. Overexpression of GFP-VP131 inhibited the interferon (IFN)-1 promoter activity and mRNA level of IFN-related genes induced by poly(I:C), Epinephelus coioides cyclic GMP/AMP synthase (EccGAS)/stimulator of IFN genes (EcSTING), TANK-binding kinase 1 (EcTBK1), or melanoma differentiation-associated gene 5 (EcMDA5), whereas such activation induced by mitochondrial antiviral signaling protein (EcMAVS) was not affected. Moreover, VP131 interacted with EcSTING and degraded EcSTING through both the autophagy-lysosome pathway and ubiquitin-proteasome pathway, and targeted for the K63-linked ubiquitination. Of note, we also found that EcSTING significantly accelerated the formation of GFP-VP131 aggregates in co-transfected cells. Finally, GFP-VP131 inhibited EcSTING- or EcTBK1-induced antiviral activity upon red-spotted grouper nervous necrosis virus (RGNNV) infection. Together, our results demonstrated that the SGIV VP131 negatively regulated the IFN response by inhibiting EcSTING-EcTBK1 signaling for viral evasion. IMPORTANCE STING has been identified as a critical factor participating in the innate immune response which recruits and phosphorylates TBK1 and IFN regulatory factor 3 (IRF3) to induce IFN production and defend against viral infection. However, viruses also distort the STING-TBK1 pathway to negatively regulate the IFN response and facilitate viral replication. Here, we reported that SGIV VP131 interacted with EcSTING within the ER and degraded EcSTING, leading to the suppression of IFN production and the promotion of SGIV infection. These results for the first time demonstrated that fish iridovirus evaded the host antiviral response via abrogating the STING-TBK1 signaling pathway.

Despite having a highly reduced genome, Chlamydia trachomatis undergoes a complex developmental cycle in which the bacteria differentiate between the following two functionally and morphologically distinct forms: the infectious, nonreplicative elementary body (EB) and the noninfectious, replicative reticulate body (RB). The transitions between EBs and RBs are not mediated by division events that redistribute intracellular proteins. Rather, both primary (EB to RB) and secondary (RB to EB) differentiation likely require bulk protein turnover. One system for targeted protein degradation is the

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 6 million individuals worldwide and continues to spread in countries where vaccines are not yet widely available or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed to be essential for viral replication. We examined the master UPR sensor IRE1α kinase/RNase and its downstream transcription factor effector XBP1s, which is processed through an IRE1α-mediated mRNA splicing event, in human lung-derived cells infected with betacoronaviruses. We found that human respiratory coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome coronavirus (MERS-CoV), and murine coronavirus (MHV) all induce ER stress and strongly trigger the kinase and RNase activities of IRE1α as well as XBP1 splicing. In contrast, SARS-CoV-2 only partially activates IRE1α through autophosphorylation, but its RNase activity fails to splice XBP1. Moreover, while IRE1α was dispensable for replication in human cells for all coronaviruses tested, it was required for maximal expression of genes associated with several key cellular functions, including the interferon signaling pathway, during SARS-CoV-2 infection. Our data suggest that SARS-CoV-2 actively inhibits the RNase of autophosphorylated IRE1α, perhaps as a strategy to eliminate detection by the host immune system.

Krüppel-like factor 5 (KLF5) is critical in maintaining intestinal barrier function, and renal denervation (RDN) mitigates gut microbiota aberrations in rats with heart failure (HF). It is unclear whether intestinal KLF5 can be regulated by RDN and whether inhibiting intestinal KLF5 weakens the beneficial role of RDN on gut microbiota. Sprague-Dawley rats were distributed into a CG (sham transverse aortic constriction [TAC] and sham RDN), HF (induced by TAC), or RDN (underwent RDN after TAC) group or a CG.M, HF.M, or RDN.M group, which included the administration of the KLF5 inhibitor to the CG, HF, or RDN group, respectively. Transmission electron microscopy, mRNA, and protein expression of KLF5 and desmoglein 2 (DSG2) in jejunum and sequencing of the 16S rRNA gene in fecal samples were evaluated. KLF5 expression was lower in the RDN group than in the HF group (

All lentiviruses encode the accessory protein Rev, whose main biological function is to mediate the nuclear export of unspliced and incompletely spliced viral transcripts by binding to a viral cis-acting element (termed the Rev-responsive element, RRE) within the env-encoding region. Equine infectious anemia virus (EIAV) is a member of the lentivirus genus in the Retroviridae family and is considered an important model for the study of lentivirus pathogenesis. Here, we identified a novel transcript from the EIAV genome that encoded a viral protein, named Mat, with an unknown function. The transcript

Bovine viral diarrhea virus (BVDV) is the causative agent of the bovine viral diarrhea-mucosal disease, which is a leading cause of economic losses in the cattle industry worldwide. To date, many underlying mechanisms involved in BVDV-host interactions remain unclear, especially the functions of long noncoding RNAs (lncRNAs). In our previous study, the lncRNA expression profiles of BVDV-infected Madin-Darby bovine kidney (MDBK) cells were obtained by RNA-seq, and a significantly downregulated lncRNA IALNCR targeting MAPK8/JNK1 (a key regulatory factor of apoptosis) was identified through the lncRNA-mRNA coexpression network analysis. In this study, the function of IALNCR in regulating apoptosis to affect BVDV replication was further explored. Our results showed that BVDV infection-induced downregulation of the lncRNA IALNCR in the host cells could suppress the expression of MAPK8/JNK1 at both the mRNA and protein levels, thereby indirectly promoting the activation of caspase-3, leading to cell-autonomous apoptosis to antagonize BVDV replication. This was further confirmed by the small interfering RNA (siRNA)-mediated knockdown of the lncRNA IALNCR. However, the overexpression of the lncRNA IALNCR inhibited apoptosis and promoted BVDV replication. In conclusion, our findings demonstrated that the lncRNA IALNCR plays an important role in regulating host antiviral innate immunity against BVDV infection. IMPORTANCE Bovine viral diarrhea-mucosal disease caused by BVDV is an important viral disease in cattle, causing severe economic losses to the cattle industry worldwide. The molecular mechanisms of BVDV–host interactions are complex. To date, most studies focused only on how BVDV escapes host innate immunity. By contrast, how the host cell regulates anti-BVDV innate immune responses is rarely reported. In this study, a significantly downregulated lncRNA, with a potential function of inhibiting apoptosis (inhibiting apoptosis long noncoding RNA, IALNCR), was obtained from the lncRNA expression profiles of BVDV-infected cells and was experimentally evaluated for its function in regulating apoptosis and affecting BVDV replication. We demonstrated that downregulation of BVDV infection-induced lncRNA IALNCR displayed antiviral function by positively regulating the MAPK8/JNK1 pathway to promote cell apoptosis. Our data provided evidence that host lncRNAs regulate the innate immune response to BVDV infection.

Human papillomaviruses (HPVs) are highly infectious and cause the most common sexually transmitted viral infections. They induce hyperproliferation of squamous epithelial tissue, often forming warts. Virally encoded proteins reprogram gene expression and cell growth to create an optimal environment for viral replication. In addition to their normal roles in infection, functional alterations induced by viral proteins establish conditions that frequently contribute to human carcinogenesis. In fact, ~5% of human cancers are caused by HPVs, with virtually all cervical squamous cell carcinomas (CESC) and an increasing number of head and neck squamous cell carcinomas (HNSC) attributed to HPV infection. The Cancer Genome Atlas (TCGA) molecularly characterized thousands of primary human cancer samples in many cancer types, including CESC and HNSC, and created a comprehensive atlas of genomic, epigenomic, and transcriptomic data. This publicly available genome-wide information provides an unprecedented opportunity to expand the knowledge of the role that HPV plays in human carcinogenesis. While many tools exist to mine these data, few, if any, focus on the comparison of HPV-positive cancers with their HPV-negative counterparts or adjacent normal control tissue. We have constructed a suite of web-based tools, The HPV Induced Cancer Resource (THInCR), to utilize TCGA data for research related to HPV-induced CESC and HNSC. These tools allow investigators to gain greater biological and medical insights by exploring the impacts of HPV on cellular gene expression (mRNA and microRNA), altered gene methylation, and associations with patient survival and immune landscape features. These tools are accessible at https://thincr.ca/.

sRNAs are noncoding transcripts that play critical roles in posttranscriptional regulation in prokaryotes. In the intracellular bacterium Chlamydia, sRNAs have been identified, but functional studies have been limited to an E. coli heterologous system. We have developed an inducible sRNA overexpression system in Chlamydia trachomatis and used it to screen putative sRNAs for effects on the Chlamydia developmental cycle, which involves conversion between replicating (RB) and infectious (EB) chlamydial forms. Overexpression of 4 of 13 C. trachomatis sRNAs decreased production of infectious EBs. We performed detailed characterization of CtrR3 and CtrR7, the two sRNAs that caused the largest progeny defects in our screen. By quantifying chlamydial number and infectious progeny, and by visualizing chlamydial forms using electron microscopy, we showed that overexpression of CtrR3 prevented RB-to-EB conversion, whereas CtrR7 overexpression blocked bacterial replication. We also describe a workflow that allowed us to identify the mRNA targets of CtrR3 in Chlamydia. We first used MS2 aptamer affinity purification coupled with RNA sequencing as an unbiased approach to isolate interacting mRNAs. We then prioritized candidates based on sequence complementarity to the CtrR3 target recognition sequence, which we had identified with bioinformatic and mutational analyses. Finally, we tested putative targets with translational fusion assays in E. coli and C. trachomatis. Using this integrated approach, we provide experimental evidence that YtgB and CTL0389 are mRNA targets of CtrR3 in Chlamydia. These findings demonstrate how our C. trachomatis sRNA overexpression system can be used to investigate the functions and mRNA targets of chlamydial sRNAs.

DEAD-box helicase 5 (DDX5), a member of the DEAD/H-box helicases, is known to participate in all aspects of RNA metabolism. However, its regulatory effect in antiviral innate immunity during replication of influenza virus remains unclear. Herein, we found that human DDX5 promotes replication of influenza virus in A549 cells. Moreover, our results further revealed that DDX5 relies on its N terminus to interact with the nucleoprotein (NP) of influenza virus, which is independent of RNA. Of course, we also observed colocalization of DDX5 with NP in the context of transfection or infection. However, influenza virus infection had no significant effect on the protein expression and nucleocytoplasmic distribution of DDX5. Importantly, we found that DDX5 suppresses antiviral innate immunity induced by influenza virus infection. Mechanistically, DDX5 downregulated the mRNA levels of interferon beta (IFN-β), interleukin 6 (IL-6), and DHX58 via the METTL3-METTL14/YTHDF2 axis. We revealed that DDX5 bound antiviral transcripts and regulated immune responses through YTHDF2-dependent mRNA decay. Taken together, our data demonstrate that the DDX5/METTL3-METTL14/YTHDF2 axis regulates the replication of influenza A virus.

The emergence and prevalence of mcr-1 and its variants in humans, animals, and the environment pose a global public health threat. There is a pressing urgency to develop rapid and accurate methods to identify MCR-positive colistin-resistant bacteria in the clinical samples, providing a basis for subsequent effective antibiotic treatment.

Transcription of operons is initiated at the promoter of the first gene in the operon, continues through cistron junctions, and terminates at the end of the operon, generating a full-length mRNA. Here, we show that Rho-dependent termination of transcription occurs stochastically at a cistron junction, generating a stable mRNA that is shorter than the full-length mRNA.

Yes-associated protein 1 (YAP1) is indispensable for the development of mutant KRAS-driven pancreatic ductal adenocarcinoma (PDAC). High YAP1 mRNA is a prognostic marker for worse overall survival in patient samples; however, the regulatory mechanisms that mediate its overexpression are not well understood. YAP1 genetic alterations are rare in PDAC, suggesting that its dysregulation is likely not due to genetic events. HuR is an RNA-binding protein whose inhibition impacts many cancer-associated pathways, including the “conserved YAP1 signature” as demonstrated by gene set enrichment analysis. Screening publicly available and internal ribonucleoprotein immunoprecipitation (RNP-IP) RNA sequencing (RNA-Seq) data sets, we discovered that YAP1 is a high-confidence target, which was validated in vitro with independent RNP-IPs and 3′ untranslated region (UTR) binding assays. In accordance with our RNA sequencing analysis, transient inhibition (e.g., small interfering RNA [siRNA] and small-molecular inhibition) and CRISPR knockout of HuR significantly reduced expression of YAP1 and its transcriptional targets. We used these data to develop a HuR activity signature (HAS), in which high expression predicts significantly worse overall and disease-free survival in patient samples. Importantly, the signature strongly correlates with YAP1 mRNA expression. These findings highlight a novel mechanism of YAP1 regulation, which may explain how tumor cells maintain YAP1 mRNA expression at dynamic times during pancreatic tumorigenesis.

A molecule we termed Brugia malayi IL-5 receptor (IL-5R) binding protein (BmIL5Rbp; also known as Bm8757) was identified from B. malayi filarial worms and found to inhibit human interleukin-5 (IL-5) binding to its human receptor competitively. After the expression and purification of a recombinant BmIL5Rbp and generation of BmIL5Rbp-specific rabbit antibody, we localized the molecule on B. malayi worms through immunohistochemistry and immunoelectron microscopy. RNA interference (RNAi) was used to inhibit BmIL5Rbp mRNA and protein production. BmIL5Rbp was shown to localize to the cuticle of Brugia malayi and to be released in its excretory/secretory products. RNAi inhibited BmIL5Rbp mRNA production by 33%, reduced the surface protein expression by ~50%, and suppressed the release of BmIL5Rbp in the excretory/secretory products. RNAi has been used successfully to knock down the mRNA and protein expression of BmIL5Rbp in the early larval stages of B. malayi and provided a proof of principle for the local inhibition of the human IL-5R. These findings provide evidence that a parasite-encoded IL-5R antagonist may locally inhibit a vital host innate immune activation of IL-5 on eosinophils.

Gaining insight into the behavior of bacteria at the single cell level is important given that heterogeneous microenvironments strongly influence microbial physiology. The hybridization chain reaction (HCR) is a technique that provides in situ molecular signal amplification, enabling simultaneous mapping of multiple target RNAs at small spatial scales. To refine this method for biofilm applications, we designed and validated new probes to visualize expression of key catabolic genes in Pseudomonas aeruginosa aggregates. In addition to using existing probes for the dissimilatory nitrate reductase (narG), we developed probes for a terminal oxidase (ccoN1), nitrite reductase (nirS), nitrous oxide reductase (nosZ), and acetate kinase (ackA). These probes can be used to determine gene expression levels both in liquid culture and in biofilms. Using these probes, we quantified gene expression across oxygen gradients in aggregate populations grown using the agar block biofilm assay (ABBA). We observed distinct patterns of catabolic gene expression, with upregulation occurring in particular ABBA regions both within individual aggregates and over the aggregate population. Aerobic respiration (ccoN1) showed peak expression under oxic conditions, whereas fermentation (ackA) showed peak expression in the anoxic cores of high metabolic activity aggregates near the air-agar interface. Denitrification genes narG, nirS, and nosZ showed peak expression in hypoxic and anoxic regions, although nirS expression was much stronger in anoxic environments compared to other denitrification genes. These results reveal that the microenvironment correlates with catabolic gene expression in aggregates, and demonstrate the utility of HCR in unveiling cellular activities at the microscale in heterogeneous populations.ImportanceTo understand bacteria in diverse contexts we must understand the variations in behaviors and metabolisms they express spatiotemporally. Populations of bacteria are known to be heterogeneous, but the ways this variation manifests can be challenging to characterize due to technical limitations. By focusing on energy conservation, we demonstrate that HCR v3.0 can visualize nuances in gene expression, allowing us to understand how metabolism in Pseudomonas aeruginosa biofilms responds to microenvironmental variation at high spatial resolution. We validated probes for four catabolic genes: a constitutively expressed oxidase, acetate kinase, nitrite reductase, and nitrous oxide reductase. We showed that the genes for different modes of metabolism are expressed in overlapping but distinct subpopulations according to oxygen concentrations in a predictable fashion. The spatial transcriptomic technique described here has the potential to be used to map microbial activities across diverse environments.

The ability to rapidly change bacterial gene expression programs in response to environmental conditions is highly dependent on the efficient turnover of mRNA. While much is known about the regulation of gene expression at the transcriptional and translational levels, much less is known about the intermediate step of mRNA decay.

The Gram-negative pathogen Pasteurella multocida is the causative agent of many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. One mechanism by which bacteria regulate transcript abundance and protein production is riboregulation, which involves the interaction of a small RNA (sRNA) with a target mRNA to alter transcript stability and/or translational efficiency. This interaction often requires stabilization by an RNA-binding protein such as ProQ or Hfq. In Escherichia coli and a small number of other species, ProQ has been shown to play a critical role in stabilizing sRNA-mRNA interactions and preferentially binds to the 3′ stem-loop regions of the mRNA transcripts, characteristic of intrinsic transcriptional terminators. The aim of this study was to determine the role of ProQ in regulating P. multocida transcript abundance and identify the RNA targets to which it binds. We assessed differentially expressed transcripts in a proQ mutant and identified sites of direct ProQ-RNA interaction using in vivo UV-cross-linking and analysis of cDNA (CRAC). These analyses demonstrated that ProQ binds to, and stabilizes, ProQ-dependent sRNAs and transfer RNAs in P. multocida via adenosine-enriched, highly structured sequences. The binding of ProQ to two RNA molecules was characterized, and these analyses showed that ProQ bound within the coding sequence of the transcript PmVP161_1121, encoding an uncharacterized protein, and within the 3′ region of the putative sRNA Prrc13. IMPORTANCE Regulation in P. multocida involving the RNA-binding protein Hfq is required for hyaluronic acid capsule production and virulence. This study further expands our understanding of riboregulation by examining the role of a second RNA-binding protein, ProQ, in transcript regulation and abundance in P. multocida.

Regardless of the general model of translation in eukaryotic cells, a number of studies suggested that many mRNAs encode multiple proteins. Leaky scanning, which supplies ribosomes to downstream open reading frames (ORFs) by readthrough of upstream ORFs, has great potential to translate polycistronic mRNAs. However, the mRNA elements controlling leaky scanning and their biological relevance have rarely been elucidated, with exceptions such as the Kozak sequence. Here, we have analyzed the strategy of a plant RNA virus to translate three movement proteins from a single RNA molecule through leaky scanning. The in planta and in vitro results indicate thatthe significantly shorter 5′ untranslated region (UTR) of the most upstream ORF promotes leaky scanning, potentially fine-tuning the translation efficiency of the three proteins in a single RNA molecule to optimize viral propagation. Our results suggest that the remarkably short length of the leader sequence, like the Kozak sequence, is a translational regulatory element with a biologically important role, as previous studies have shown biochemically. IMPORTANCE Potexvirus, a group of plant viruses, infect a variety of crops, including cultivated crops. It has been thought that the three transition proteins that are essential for the cell-to-cell transfer of potexviruses are translated from two subgenomic RNAs, sgRNA1 and sgRNA2. However, sgRNA2 has not been clearly detected. In this study, we have shown that sgRNA1, but not sgRNA2, is the major translation template for the three movement proteins. In addition, we determined the transcription start site of sgRNA1 in flexiviruses and found that the efficiency of leaky scanning caused by the short 5′ UTR of sgRNA1, a widely conserved feature, regulates the translation of the three movement proteins. When we tested the infection of viruses with mutations introduced into the length of the 5′ UTR, we found that the movement efficiency of the virus was affected. Our results provide important additional information on the protein translation strategy of flexiviruses, including Potexvirus, and provide a basis for research on their control as well as the need to reevaluate the short 5′ UTR as a translational regulatory element with an important role in vivo.

Biomaterials offer unique properties that make them irreplaceable for next-generation applications. Fibrous proteins, such as various caterpillar silks and especially spider silk, have strength and toughness not found in human-made materials. In early studies, proteins containing long tandem repeats, such as major ampullate spidroin 1 (MaSp1) and flagelliform silk protein (FSLP), were produced using a large DNA template composed of many tandem repeats. The hierarchical DNA assembly of the DNA template is very time-consuming and labor-intensive, which makes the fibrous proteins difficult to study and engineer. In this study, we designed a circularized mRNA (cmRNA) employing the RNA cyclase ribozyme mechanism. cmRNAs encoding spider silk protein MaSp1 and FSLP were designed based on only one unit of the template sequence but provide ribosomes with a circular and infinite translation template for production of long peptides containing tandem repeats. Using this technique, cmRNAs of MaSp1 and FSLP were successfully generated with circularization efficiencies of 8.5% and 36.7%, respectively, which supported the production of recombinant MaSp1 and FSLP larger than 110 and 88 kDa, containing tens of repeat units. Western blot analysis and mass spectrometry confirmed the authenticity of MaSp1 and FSLP, which were produced at titers of 22.1 and 81.5 mg · liter(−1), respectively. IMPORTANCE Spider silk is a biomaterial with superior properties. However, its heterologous expression template is hard to construct. The cmRNA technique simplifies the construction and expression strategy by proving the ribosome a circular translation template for expression of long peptides containing tandem repeats. This revolutionary technique will allow researchers to easily build, study, and experiment with any fiber proteins with sequences either from natural genes or artificial designs. We expect a significantly accelerated development of fibrous protein-based biomaterials with the cmRNA technique.

The Gac/Rsm system is a global regulator of Pseudomonas aeruginosa gene expression. The primary effectors are RsmA and RsmF. Both are RNA-binding proteins that interact with target mRNAs to modulate protein synthesis. RsmA/RsmF recognize GGA sequences presented in the loop portion of stem-loop structures. For repressed targets, the GGA sites usually overlap the ribosome binding site (RBS) and RsmA/RsmF binding inhibits translation initiation. RsmA/RsmF activity is controlled by several small non-coding RNAs (sRNA) that sequester RsmA/RsmF from target mRNAs. The most important sequestering sRNAs are RsmY and RsmZ. Transcription of rsmY/rsmZ is directly controlled by the GacSA two-component regulatory system. GacSA activity is antagonized by RetS, a hybrid sensor kinase. In the absence of retS, rsmY/rsmZ transcription is derepressed and RsmA/RsmF are sequestered by RsmY/RsmZ. Gac/Rsm system homeostasis is tightly controlled by at least two mechanisms. First, direct binding of RsmA to the rsmA and rsmF mRNAs inhibits further synthesis of both proteins. Second, RsmA stimulates rsmY/rsmZ transcription through an undefined mechanism. In this study we demonstrate that RsmA stimulates rsmY/rsmZ transcription by directly inhibiting RetS synthesis. RetS protein levels are elevated 2.5-fold in an rsmA mutant. Epistasis experiments demonstrate that the rsmA requirement for rsmY/rsmZ transcription is entirely suppressed in an rsmA, retS double mutant. RsmA directly interacts with the retS mRNA and requires two distinct GGA sites, one of which overlaps the RBS. We propose a model wherein RsmA inhibits RetS synthesis to promote rsmY/rsmZ transcription and that this acts as a checkpoint to limit RsmA/RsmF availability. IMPORTANCE The Pseudomonas aeruginosa Gac/Rsm system controls ∼500 genes and governs a critical lifestyle switch by inversely regulating factors that favor acute or chronic colonization. Control of gene expression by the Gac/Rsm system is mediated through RsmA and RsmF, small RNA-binding proteins that interact with target mRNAs to inhibit or promote protein synthesis and/or mRNA stability. RsmA/RsmF activity is governed by two small non-coding RNAs (RsmY and RsmZ) that sequester RsmA/RsmF from target mRNAs. The GacSA two-component regulatory system plays a pivotal role in the Gac/Rsm system by controlling rsmYZ transcription. This study provides insight into the control of homeostasis by demonstrating that RsmA directly targets and inhibits expression of RetS, an orphan sensor kinase critical for rsmYZ transcription.

Hepatitis B virus (HBV) encodes a regulatory protein, termed HBx, that has been intensely studied in the past and shown to play a key role(s) in viral transcription and replication. In addition, a huge body of work exists in the literature related to signal transduction and possible mechanism(s) leading to hepatocarcinogenesis associated with infection. We have previously reported that HBV transcripts are modified by N6-methyladenosine (m6A) at the single consensus DRACH motif at nucleotides (nt) 1905 to 1909 in the epsilon structural element, and this m6A modification affects the HBV life cycle. In this study, we present evidence that additional variants of m6A (DRACH) motifs located within nt 1606 to 1809 correspond to the coding region of HBx mRNA and 3′ untranslated region (UTR) of other viral mRNAs. Using the mutants of additional m6A sites in nt 1606 to 1809 and a depletion strategy of m6A methyltransferases (METTL3/14) and reader proteins (YTHDFs), we show that m6A modification at nt 1616, located in the HBx coding region, regulates HBx protein expression. The HBx RNA and protein expression levels were notably increased by the silencing of m6A reader YTHDF2 and methyltransferases as well as the mutation of m6A sites in the HBx coding region. However, other viral protein expression levels were not affected by the m6A modification at nt 1616. Thus, m6A modifications in the HBx open reading frame (ORF) downregulate HBx protein expression, commonly seen during HBV transfections, transgenic mice, and natural infections of human hepatocytes. These studies identify the functional role of m6A modification in the subtle regulation of HBx protein expression consistent with its possible role in establishing chronic hepatitis. IMPORTANCE N6-methyladenosien (m6A) modifications recently have been implicated in the HBV life cycle. Previously, we observed that m6A modification occurs in the adenosine at nt 1907 of the HBV genome, and this modification regulates the viral life cycle. Here, we identified an additional m6A site located in nt 1616 of the HBV genome. This modification negatively affects HBx RNA and protein expression. In the absence of m6A methyltransferases (METTL3/14) and reader protein (YTHDF2), the HBx RNA and protein expression were increased. Using HBV mutants that lack m6A in the HBx coding region, we present the unique positional effects of m(6)A in the regulation of HBx protein expression.

The cGAS/STING/TBK1 (cyclic guanine monophosphate-AMP synthase/stimulator of interferon genes/Tank-binding kinase 1) innate immunity pathway is activated during human cytomegalovirus (HCMV) productive (lytic) replication in fully differentiated cells and during latency within incompletely differentiated myeloid cells. While multiple lytic-phase HCMV proteins neutralize steps along this pathway, none of them are expressed during latency. Here, we show that the latency-associated protein UL138 inhibits the cGAS/STING/TBK1 innate immunity pathway during transfections and infections, in fully differentiated cells and incompletely differentiated myeloid cells, and with loss of function and restoration of function approaches. UL138 inhibits the pathway downstream of STING but upstream of interferon regulatory factor 3 (IRF3) phosphorylation and NF-κB function and reduces the accumulation of interferon beta mRNA during both lytic and latent infections. IMPORTANCE While a cellular restriction versus viral countermeasure arms race between innate immunity and viral latency is expected, few examples have been documented. Our identification of the first HCMV latency protein that inactivates the cGAS/STING/TBK1 innate immune pathway opens the door to understanding how innate immunity, or its neutralization, impacts long-term persistence by HCMV and other latent viruses.

Long noncoding RNAs (lncRNAs) are frequently associated with broad modulation of gene expression and thus provide the cell with the ability to synchronize entire metabolic processes. We used transcriptomic approaches to investigate whether the most abundant human cytomegalovirus-encoded lncRNA, RNA2.7, has this characteristic. By comparing cells infected with wild-type virus (WT) to cells infected with RNA2.7 deletion mutants, RNA2.7 was implicated in regulating a large number of cellular genes late in lytic infection. Pathway analysis indicated that >100 of these genes are associated with promoting cell movement, and the 10 most highly regulated of these were validated in further experiments. Morphological analysis and live cell tracking of WT- and RNA2.7 mutant-infected cells indicated that RNA2.7 is involved in promoting the movement and detachment of infected cells late in infection, and plaque assays using sparse cell monolayers indicated that RNA2.7 is also involved in promoting cell-to-cell spread of virus. Consistent with the observation that upregulated mRNAs are relatively A+U-rich, which is a trait associated with transcript instability, and that they are also enriched in motifs associated with mRNA instability, transcriptional inhibition experiments on WT- and RNA2.7 mutant-infected cells showed that four upregulated transcripts lived longer in the presence of RNA2.7. These findings demonstrate that RNA2.7 is required for promoting cell movement and viral spread late in infection and suggest that this may be due to general stabilization of A+U-rich transcripts. IMPORTANCE In addition to messenger RNAs (mRNAs), the human genome encodes a large number of long noncoding RNAs (lncRNAs). Many lncRNAs that have been studied in detail are associated with broad modulation of gene expression and have important biological roles. Human cytomegalovirus, which is a large, clinically important DNA virus, specifies four lncRNAs, one of which (RNA2.7) is expressed at remarkably high levels during lytic infection. Our studies show that RNA2.7 is required for upregulating a large number of human genes, about 100 of which are associated with cell movement, and for promoting the movement of infected cells and the spread of virus from one cell to another. Further bioinformatic and experimental analyses indicated that RNA2.7 may exert these effects by stabilizing mRNAs that are relatively rich in A and U nucleotides. These findings increase our knowledge of how human cytomegalovirus regulates the infected cell to promote its own success.

Neutrophils are required for host resistance against Streptococcus pneumoniae, but their function declines with age. We previously found that CD73, an enzyme required for antimicrobial activity, is downregulated in neutrophils (also known as polymorphonuclear leukocytes [PMNs]) from aged mice. This study explored transcriptional changes in neutrophils induced by S. pneumoniae to identify pathways controlled by CD73 and dysregulated with age. Pure bone marrow-derived neutrophils isolated from wild-type (WT) young and old and CD73 knockout (CD73KO) young mice were mock challenged or infected with S. pneumoniae ex vivo. RNA sequencing (RNA-Seq) was performed to identify differentially expressed genes (DEGs). We found that infection triggered distinct global transcriptional changes across hosts that were strongest in CD73KO neutrophils. Surprisingly, there were more downregulated than upregulated genes in all groups upon infection. Downregulated DEGs indicated a dampening of immune responses in old and CD73KO hosts. Further analysis revealed that CD73KO neutrophils expressed higher numbers of long noncoding RNAs (lncRNAs) than those in WT controls. Predicted network analysis indicated that CD73KO-specific lncRNAs control several signaling pathways. We found that genes in the c-Jun N-terminal kinase (JNK)-mitogen-activated protein kinase (MAPK) pathway were upregulated upon infection in CD73KO mice and in WT old mice, but not in WT young mice. This corresponded to functional differences, as phosphorylation of the downstream AP-1 transcription factor component c-Jun was significantly higher in neutrophils from infected CD73KO mice and old mice. Importantly, inhibition of JNK/AP-1 rescued the ability of these neutrophils to kill S. pneumoniae. Together, our findings revealed that the ability of neutrophils to modify their gene expression to better adapt to bacterial infection is in part regulated by CD73 and declines with age.

The supply of testing equipment is vital in controlling the spread of SARS-CoV-2. We compared the diagnostic efficacy and tolerability of molded plastic (FinSwab; Valukumpu, Finland) versus flocked nylon (FLOQSwab; Copan, Italy) nasopharyngeal swabs in a clinical setting. Adults (n = 112) with suspected symptomatic COVID-19 infection underwent nasopharyngeal sampling with FinSwab and FLOQSwab from the same nostril at a drive-in coronavirus testing station. In a subset of 36 patients the samples were collected in a randomized order to evaluate the discomfort associated with sampling. SARS-CoV-2 and 16 other respiratory viruses, as well as human β-actin mRNA were analyzed by using reverse transcriptase PCR (RT-PCR) assays. Among the 112 patients (mean age, 38 [standard deviation (SD), 14] years) β-actin mRNA was found in all samples. There was no difference in the β-actin mRNA cycle threshold (CT) values between FinSwab (mean, 22.3; SD, 3.61) and FLOQSwab (mean, 22.1; SD, 3.50; P = 0.46) swabs. There were 31 virus-positive cases (26 rhinovirus, 4 SARS-CoV-2, and 1 coronavirus-OC43), 24 of which were positive in both swabs; 3 rhinovirus positives were only found in the FinSwab, and similarly 4 rhinovirus positives were only found in the FLOQSwab. Rhinovirus CT values were similar between swab types. Of the 36 patients, 22 (61%) tolerated the sampling with the FinSwab better than with the FLOQSwab (P = 0.065). The molded plastic nasopharyngeal swab (FinSwab) was comparable to the standard flocked swab in terms of efficacy for respiratory virus detection and tolerability of sampling. IMPORTANCE We demonstrate that a molded plastic swab is a valid alternative to conventional brush-like swabs in collection of a nasopharyngeal sample for virus diagnostics.

Iron is an essential element for the growth and survival of pathogenic bacteria; however, it is not fully understood how bacteria sense and respond to iron deficiency or excess. In this study, we show that xfeA in Xanthomonas oryzae pv. oryzicola senses extracytoplasmic iron and changes the hydrogen bonding network of ligand channel domains by adenosine-to-inosine (A-to-I) RNA editing. The frequency of A-to-I RNA editing during iron-deficient conditions increased by 76.87%, which facilitated the passage of iron through the XfeA outer membrane channel. When bacteria were subjected to high iron concentrations, the percentage of A-to-I editing in xfeA decreased, which reduced iron transport via XfeA. Furthermore, A-to-I RNA editing increased expression of multiple genes in the chemotaxis pathway, including methyl-accepting chemotaxis proteins (MCPs) that sense concentrations of exogenous ferrienterobactin (Fe-Ent) at the cytoplasmic membrane. A-to-I RNA editing helps X. oryzae pv. oryzicola move toward an iron-rich environment and supports our contention that editing in xfeA facilitates entry of a ferric siderophore. Overall, our results reveal a new signaling mechanism that bacteria use to adjust to iron concentrations. IMPORTANCE Adenosine-to-inosine (A-to-I) RNA editing, which is catalyzed by the adenosine deaminase RNA-specific family of enzymes, is a frequent posttranscriptional modification in metazoans. Research on A-to-I editing in bacteria is limited, and the importance of this editing is underestimated. In this study, we show that bacteria may use A-to-I editing as an alternative strategy to promote uptake of metabolic iron, and this form of editing can quickly and precisely modify RNA and subsequent protein sequences similar to an “on/off” switch. Thus, bacteria have the capacity to use a rapid switch-like mechanism to facilitate iron uptake and improve their competitiveness.

One of the many challenges faced by RNA viruses is the maintenance of their genomes during infections of host cells. Members of the family Tombusviridae are plus-strand RNA viruses with unmodified triphosphorylated genomic 5′ termini. The tombusvirus Carnation Italian ringspot virus was used to investigate how it protects its RNA genome from attack by 5′-end-targeting degradation enzymes. In vivo and in vitro assays were employed to determine the role of genomic RNA structure in conferring protection from the 5′-to-3′ exoribonuclease Xrn. The results revealed that (i) the CIRV RNA genome is more resistant to Xrn than its sg mRNAs, (ii) the genomic 5′-untranslated region (UTR) folds into a compact RNA structure that effectively and independently prevents Xrn access, (iii) the RNA structure limiting 5′ access is formed by secondary and tertiary interactions that function cooperatively, (iv) the structure is also able to block access of RNA pyrophosphohydrolase to the genomic 5′ terminus, and (v) the RNA structure does not stall an actively digesting Xrn. Based on its proficiency at impeding Xrn 5′ access, we have termed this 5′-terminal structure an Xrn-evading RNA, or xeRNA. These and other findings demonstrate that the 5′UTR of the CIRV RNA genome folds into a complex structural conformation that helps to protect its unmodified 5′ terminus from enzymatic decay during infections. IMPORTANCE The plus-strand RNA genomes of plant viruses in the large family Tombusviridae are not 5′ capped. Here, we explored how a species in the type genus Tombusvirus protects its genomic 5′ end from cellular nuclease attack. Our results revealed that the 5′-terminal sequence of the CIRV genome folds into a complex RNA structure that limits access of the 5′-to-3′ exoribonuclease Xrn, thereby protecting it from processive degradation. The RNA conformation also impeded access of RNA pyrophosphohydrolase, which converts 5′-triphosphorylated RNA termini into 5′-monophosphorylated forms, the preferred substrate for Xrn. This study represents the first report of a higher-order RNA structure in an RNA plant virus genome independently conferring resistance to 5′-end-attacking cellular enzymes.

Vaccinia virus produces two types of virions known as single-membraned intracellular mature virus (MV) and double-membraned extracellular enveloped virus (EV). EV production peaks earlier when initial MVs are further wrapped and secreted to spread infection within the host. However, late during infection, MVs accumulate intracellularly and become important for host-to-host transmission. The process that regulates this switch remains elusive and is thought to be influenced by host factors. Here, we examined the hypothesis that EV and MV production are regulated by the virus through expression of F13 and the MV-specific protein A26. By switching the promoters and altering the expression kinetics of F13 and A26, we demonstrate that A26 expression downregulates EV production and plaque size, thus limiting viral spread. This process correlates with A26 association with the MV surface protein A27 and exclusion of F13, thus reducing EV titers. Thus, MV maturation is controlled by the abundance of the viral A26 protein, independently of other factors, and is rate limiting for EV production. The A26 gene is conserved within vertebrate poxviruses but is strikingly lost in poxviruses known to be transmitted exclusively by biting arthropods. A26-mediated virus maturation thus has the appearance to be an ancient evolutionary adaptation to enhance transmission of poxviruses that has subsequently been lost from vector-adapted species, for which it may serve as a genetic signature. The existence of virus-regulated mechanisms to produce virions adapted to fulfill different functions represents a novel level of complexity in mammalian viruses with major impacts on evolution, adaptation, and transmission. IMPORTANCE Chordopoxviruses are mammalian viruses that uniquely produce a first type of virion adapted to spread within the host and a second type that enhances transmission between hosts, which can take place by multiple ways, including direct contact, respiratory droplets, oral/fecal routes, or via vectors. Both virion types are important to balance intrahost dissemination and interhost transmission, so virus maturation pathways must be tightly controlled. Here, we provide evidence that the abundance and kinetics of expression of the viral protein A26 regulates this process by preventing formation of the first form and shifting maturation toward the second form. A26 is expressed late after the initial wave of progeny virions is produced, so sufficient viral dissemination is ensured, and A26 provides virions with enhanced environmental stability. Conservation of A26 in all vertebrate poxviruses, but not in those transmitted exclusively via biting arthropods, reveals the importance of A26-controlled virus maturation for transmission routes involving environmental exposure.

Modified vaccinia virus Ankara (MVA) was derived by repeated passaging in chick fibroblasts, during which deletions and mutations rendered the virus unable to replicate in most mammalian cells. Marker rescue experiments demonstrated that the host range defect could be overcome by replacing DNA that had been deleted from near the left end of the genome. One virus isolate, however, recovered the ability to replicate in monkey BS-C-1 cells but not human cells without added DNA, suggesting that it arose from a spontaneous mutation. Here, we showed that variants with enhanced ability to replicate in BS-C-1 cells could be isolated by blind passaging of MVA and that in each there was a point mutation leading to an amino acid substitution in the D10 decapping enzyme. The sufficiency of these single mutations to enhance host range was confirmed by constructing recombinant viruses. The D10 mutations occurred at N- or C-terminal locations distal to the active site, suggesting an indirect effect on decapping or on another previously unknown role of D10. Although increased amounts of viral mRNA and proteins were found in BS-C-1 cells infected with the mutants compared to those with parental MVA, the increases were much less than the 1- to 2-log-higher virus yields. Nevertheless, a contributing role for diminished decapping in overcoming the host range defect was consistent with increased replication and viral protein synthesis in BS-C-1 cells infected with an MVA engineered to have active-site mutations that abrogate decapping activity entirely. Optimal decapping may vary depending on the biological context. IMPORTANCE Modified vaccinia virus Ankara (MVA) is an attenuated virus that is approved as a smallpox vaccine and is in clinical trials as a vector for other pathogens. The safety of MVA is due in large part to its inability to replicate in mammalian cells. Although host range restriction is considered a stable feature of the virus, we describe the occurrence of spontaneous mutations in MVA that increase replication considerably in monkey BS-C-1 cells but only slightly in human cells. The mutants contain single nucleotide changes that lead to amino acid substitutions in one of the two decapping enzymes. Although the spontaneous mutations are distant from the decapping enzyme active site, engineered active-site mutations also increased virus replication in BS-C-1 cells. The effects of these mutations on the immunogenicity of MVA vectors remain to be determined.

Toxin-antitoxin systems are widely distributed genetic modules typically featuring toxins that can inhibit bacterial growth and antitoxins that can reverse inhibition. Although Escherichia coli encodes 11 toxins with known or putative endoribonuclease activity, the targets of most of these toxins remain poorly characterized. Using a new RNA sequencing (RNA-seq) pipeline that enables the mapping and quantification of RNA cleavage with single-nucleotide resolution, we characterized the targets and specificities of 9 endoribonuclease toxins from E. coli. We found that these toxins use low-information cleavage motifs to cut a significant proportion of mRNAs in E. coli, but not tRNAs or the rRNAs from mature ribosomes. However, all the toxins, including those that are ribosome dependent and cleave only translated RNA, inhibit ribosome biogenesis. This inhibition likely results from the cleavage of ribosomal protein transcripts, which disrupts the stoichiometry and biogenesis of new ribosomes and causes the accumulation of aberrant ribosome precursors. Collectively, our results provide a comprehensive, global analysis of endoribonuclease-based toxin-antitoxin systems in E. coli and support the conclusion that, despite their diversity, each disrupts translation and ribosome biogenesis. IMPORTANCE Toxin-antitoxin (TA) systems are widespread genetic modules found in almost all bacteria that can regulate their growth and may play prominent roles in phage defense. Escherichia coli encodes 11 TA systems in which the toxin is a known or predicted endoribonuclease. The targets and cleavage specificities of these endoribonucleases have remained largely uncharacterized, precluding an understanding of how each impacts cell growth and an assessment of whether they have distinct or overlapping targets. Using a new and broadly applicable RNA-seq pipeline, we carried out a global analysis of 9 endoribonuclease toxins from E. coli. We found that each uses a relatively low-information cleavage motif to cut a large proportion of mRNAs in E. coli, but not tRNAs or mature rRNAs. Notably, although the precise set of targets varies, each toxin efficiently disrupts ribosome biogenesis, primarily by cleaving the mRNAs of ribosomal proteins. In sum, the analyses presented provide new, comprehensive insights into the cleavage specificities and targets of almost all endoribonuclease toxins in E. coli. Despite different specificities, our work reveals a striking commonality in function, as each toxin disrupts ribosome biogenesis and translation.

Plague-causing Yersinia pestis is transmitted through regurgitation when it forms a biofilm-mediated blockage in the foregut of its flea vector. This biofilm is composed of an extracellular polysaccharide substance (EPS) produced when cyclic-di-GMP (c-di-GMP) levels are elevated. The Y. pestis diguanylate cyclase enzymes HmsD and HmsT synthesize c-di-GMP. HmsD is required for biofilm blockage formation but contributes minimally to in vitro biofilms. HmsT, however, is necessary for in vitro biofilms and contributes to intermediate rates of biofilm blockage. C-di-GMP synthesis is regulated at the transcriptional and posttranscriptional levels. In this, the global RNA chaperone, Hfq, posttranscriptionally represses hmsT mRNA translation. How c-di-GMP levels and biofilm blockage formation is modulated by nutritional stimuli encountered in the flea gut is unknown. Here, the RNA-binding regulator protein CsrA, which controls c-di-GMP-mediated biofilm formation and central carbon metabolism responses in many Gammaproteobacteria, was assessed for its role in Y. pestis biofilm formation. We determined that CsrA was required for markedly greater c-di-GMP and EPS levels when Y. pestis was cultivated on alternative sugars implicated in flea biofilm blockage metabolism. Our assays, composed of mobility shifts, quantification of mRNA translation, stability, and abundance, and epistasis analyses of a csrA hfq double mutant strain substantiated that CsrA represses hfq mRNA translation, thereby alleviating Hfq-dependent repression of hmsT mRNA translation. Additionally, a csrA mutant exhibited intermediately reduced biofilm blockage rates, resembling an hmsT mutant. Hence, we reveal CsrA-mediated control of c-di-GMP synthesis in Y. pestis as a tiered, posttranscriptional regulatory process that enhances biofilm blockage-mediated transmission from fleas. IMPORTANCE Yersinia pestis, the bacterial agent of bubonic plague, produces a c-di-GMP-dependent biofilm-mediated blockage of the flea vector foregut to facilitate its transmission by flea bite. However, the intricate molecular regulatory processes that underlie c-di-GMP-dependent biofilm formation and thus, biofilm-mediated blockage in response to the nutritional environment of the flea are largely undefined. This study provides a novel mechanistic understanding of how CsrA transduces alternative sugar metabolism cues to induce c-di-GMP-dependent biofilm formation required for efficient Y. pestis regurgitative transmission through biofilm-mediated flea foregut blockage. The Y. pestis-flea interaction represents a unique, biologically relevant, in vivo perspective on the role of CsrA in biofilm regulation.

The study of gene expression in fungi has typically relied on measuring transcripts in populations of cells. A major disadvantage of this approach is that the transcripts’ spatial distribution and stochastic variation among individual cells within a clonal population is lost. Traditional fluorescence in situ hybridization techniques have been of limited use in fungi due to poor specificity and high background signal. Here, we report that in situ hybridization chain reaction (HCR), a method that employs split-initiator probes to trigger signal amplification upon mRNA-probe hybridization, is ideally suited for the imaging and quantification of low-abundance transcripts at single-cell resolution in the fungus Candida albicans. We show that HCR allows the absolute quantification of transcripts within a cell by microscopy as well as their relative quantification by flow cytometry. mRNA imaging also revealed the subcellular localization of specific transcripts. Furthermore, we establish that HCR is amenable to multiplexing by visualizing different transcripts in the same cell. Finally, we combine HCR with immunostaining to image specific mRNAs and proteins simultaneously within a single C. albicans cell. The fungus is a major pathogen in humans where it can colonize and invade mucosal surfaces and most internal organs. The technical development that we introduce, therefore, paves the way to study the patterns of expression of pathogenesis-associated C. albicans genes in infected organs at single-cell resolution. IMPORTANCE Tools to visualize and quantify transcripts at single-cell resolution have enabled the dissection of spatiotemporal patterns of gene expression in animal cells and tissues. Yet the accurate quantification of transcripts at single-cell resolution remains challenging for the much smaller microbial cells. Widespread phenomena such as stochastic variation in transcript levels among cells—even within a clonal population—seem to play important roles in the biology of many microorganisms. Investigating this process requires microbial cell-optimized procedures to image and measure mRNAs at single-molecule resolution. In this report, we adapt and expand in situ hybridization chain reaction (HCR) combined with split-initiator probes to visualize transcripts in the human-pathogenic fungus Candida albicans at high resolution.

The methylation of RNA at the N6 position of adenosine (m6A) orchestrates multiple biological processes to control development, differentiation, and cell cycle, as well as various aspects of the virus life cycle. How the m6A RNA modification pathway is regulated to finely tune these processes remains poorly understood. Here, we discovered the m6A reader YTHDF2 as a caspase substrate via proteome-wide prediction, followed by in vitro and in vivo validations. We further demonstrated that cleavage-resistant YTHDF2 blocks, while cleavage-mimicking YTHDF2 fragments promote, the replication of a common human oncogenic virus, Epstein-Barr virus (EBV). Intriguingly, our study revealed a feedback regulation between YTHDF2 and caspase-8 via m6A modification of CASP8 mRNA and YTHDF2 cleavage during EBV replication. Further, we discovered that caspases cleave multiple components within the m6A RNA modification pathway to benefit EBV replication. Our study establishes that caspase disarming of the m6A RNA modification machinery fosters EBV replication. IMPORTANCE The discovery of an N6-methyladenosine (m6A) RNA modification pathway has fundamentally altered our understanding of the central dogma of molecular biology. This pathway is controlled by methyltransferases (writers), demethylases (erasers), and specific m6A binding proteins (readers). Emerging studies have linked the m6A RNA modification pathway to the life cycle of various viruses. However, very little is known regarding how this pathway is subverted to benefit viral replication. In this study, we established an unexpected linkage between cellular caspases and the m6A modification pathway, which is critical to drive the reactivation of a common tumor virus, Epstein-Barr virus (EBV).

Spores of firmicute species contain 100s of mRNAs, whose major function in Bacillus subtilis is to provide ribonucleotides for new RNA synthesis when spores germinate. To determine if this is a general phenomenon, RNA was isolated from spores of multiple firmicute species and relative mRNA levels determined by transcriptome sequencing (RNA-seq). Determination of RNA levels in single spores allowed calculation of RNA nucleotides/spore, and assuming mRNA is 3% of spore RNA indicated that only ∼6% of spore mRNAs were present at >1/spore. Bacillus subtilis, Bacillus atrophaeus, and Clostridioides difficile spores had 49, 42, and 51 mRNAs at >1/spore, and numbers of mRNAs at ≥1/spore were ∼10 to 50% higher in Geobacillus stearothermophilus and Bacillus thuringiensis Al Hakam spores and ∼4-fold higher in Bacillus megaterium spores. In all species, some to many abundant spore mRNAs (i) were transcribed by RNA polymerase with forespore-specific σ factors, (ii) encoded proteins that were homologs of those encoded by abundant B. subtilis spore mRNAs and are proteins in dormant spores, and (iii) were likely transcribed in the mother cell compartment of the sporulating cell. Analysis of the coverage of RNA-seq reads on mRNAs from all species suggested that abundant spore mRNAs were fragmented, as was confirmed by reverse transcriptase quantitative PCR (RT-qPCR) analysis of abundant B. subtilis and C. difficile spore mRNAs. These data add to evidence indicating that the function of at least the great majority of mRNAs in all firmicute spores is to be degraded to generate ribonucleotides for new RNA synthesis when spores germinate. IMPORTANCE Only ∼6% of mRNAs in spores of six firmicute species are at ≥1 molecule/spore, many abundant spore mRNAs encode proteins similar to B. subtilis spore proteins, and some abundant B. subtilis and C. difficile spore mRNAs were fragmented. Most of the abundant B. subtilis and other Bacillales spore mRNAs are transcribed under the control of the forespore-specific RNA polymerase σ factors, F or G, and these results may stimulate transcription analyses in developing spores of species other than B. subtilis. These findings, plus the absence of key nucleotide biosynthetic enzymes in spores, suggest that firmicute spores' abundant mRNAs are not translated when spores germinate but instead are degraded to generate ribonucleotides for new RNA synthesis by the germinated spore.

To replicate efficiently and evade the antiviral immune response of the host, some viruses degrade host mRNA to induce host gene shutoff via encoding shutoff factors. In this study, we found that feline calicivirus (FCV) infection promotes the degradation of endogenous and exogenous mRNAs and induces host gene shutoff, which results in global inhibition of host protein synthesis. Screening assays revealed that proteinase-polymerase (PP) is a most effective factor in reducing mRNA expression. Moreover, PP from differently virulent strains of FCV could induce mRNA degradation. Further, we found that the key sites of the PP protein required for its proteinase activity are also essential for its shutoff activity but also required for viral replication. The mechanism analysis showed that PP mainly targets Pol II-transcribed RNA in a ribosome-, 5′ cap-, and 3′ poly(A) tail-independent manner. Moreover, purified glutathione S-transferase (GST)-PP fusion protein exhibits RNase activity in vitro in assays using green fluorescent protein (GFP) RNA transcribed in vitro as a substrate in the absence of other viral or cellular proteins. Finally, PP-induced shutoff requires host Xrn1 to complete further RNA degradation. This study provides a newly discovered strategy in which FCV PP protein induces host gene shutoff by promoting the degradation of host mRNAs. IMPORTANCE Virus infection-induced shutoff is the result of targeted or global manipulation of cellular gene expression and leads to efficient viral replication and immune evasion. FCV is a highly contagious pathogen that persistently infects cats. It is unknown how FCV blocks the host immune response and persistently exists in cats. In this study, we found that FCV infection promotes the degradation of host mRNAs and induces host gene shutoff via a common strategy. Further, PP protein for different FCV strains is a key factor that enhances mRNA degradation. An in vitro assay showed that the GST-PP fusion protein possesses RNase activity in the absence of other viral or cellular proteins. This study demonstrates that FCV induces host gene shutoff by promoting the degradation of host mRNAs, thereby introducing a potential mechanism by which FCV infection inhibits the immune response.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic human gammaherpesvirus and the causative agent of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). During reactivation, viral genes are expressed in a temporal manner. These lytic genes encode transactivators, core replication proteins, or structural proteins. During reactivation, other viral factors that are required for lytic replication are expressed. The most abundant viral transcript is the long noncoding RNA (lncRNA) known as polyadenylated nuclear (PAN) RNA. lncRNAs have diverse functions, including the regulation of gene expression and the immune response. PAN possesses two main cis-acting elements, the Mta response element (MRE) and the expression and nuclear retention element (ENE). While PAN has been demonstrated to be required for efficient viral replication, the function of these elements within PAN remains unclear. Our goal was to determine if the ENE of PAN is required in the context of infection. A KSHV bacmid containing a deletion of the 79-nucleotide (nt) ENE in PAN was generated to assess the effects of the ENE during viral replication. Our studies demonstrated that the ENE is not required for viral DNA synthesis, lytic gene expression, or the production of infectious virus. Although the ENE is not required for viral replication, we found that the ENE functions to retain PAN in the nucleus, and the absence of the ENE results in an increased accumulation of PAN in the cytoplasm. Furthermore, open reading frame 59 (ORF59), LANA, ORF57, H1.4, and H2A still retain the ability to bind to PAN in the absence of the ENE. Together, our data highlight how the ENE affects the nuclear retention of PAN but ultimately does not play an essential role during lytic replication. Our data suggest that PAN may have other functional domains apart from the ENE. IMPORTANCE KSHV is an oncogenic herpesvirus that establishes latency and exhibits episodes of reactivation. KSHV disease pathologies are most often associated with the lytic replication of the virus. PAN RNA is the most abundant viral transcript during the reactivation of KSHV and is required for viral replication. Deletion and knockdown of PAN resulted in defects in viral replication and reduced virion production in the absence of PAN RNA. To better understand how the cis elements within PAN may contribute to its function, we investigated if the ENE of PAN was necessary for viral replication. Although the ENE had previously been extensively studied with both biochemical and in vitro approaches, this is the first study to demonstrate the role of the ENE in the context of infection and that the ENE of PAN is not required for the lytic replication of KSHV.

The mammalian orthologue of ecdysoneless (ECD) protein is required for embryogenesis, cell cycle progression, and mitigation of endoplasmic reticulum stress. Here, we identified key components of the mRNA export complexes as binding partners of ECD and characterized the functional interaction of ECD with key mRNA export-related DEAD BOX protein helicase DDX39A. We find that ECD is involved in RNA export through its interaction with DDX39A. ECD knockdown (KD) blocks mRNA export from the nucleus to the cytoplasm, which is rescued by expression of full-length ECD but not an ECD mutant that is defective in interaction with DDX39A. We have previously shown that ECD protein is overexpressed in ErbB2+ breast cancers (BC). In this study, we extended the analyses to two publicly available BC mRNA The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data sets. In both data sets, ECD mRNA overexpression correlated with short patient survival, specifically ErbB2+ BC. In the METABRIC data set, ECD overexpression also correlated with poor patient survival in triple-negative breast cancer (TNBC). Furthermore, ECD KD in ErbB2+ BC cells led to a decrease in ErbB2 mRNA level due to a block in its nuclear export and was associated with impairment of oncogenic traits. These findings provide novel mechanistic insight into the physiological and pathological functions of ECD.

SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), is an RNA virus with a large genome that encodes multiple accessory proteins. While these accessory proteins are not required for growth in vitro, they can contribute to the pathogenicity of the virus., RNA viruses that replicate in the cytoplasm often disrupt nucleocytoplasmic transport to preferentially translate their own transcripts and prevent host antiviral responses. The Sarbecovirus accessory protein ORF6 has previously been shown to be a major inhibitor of interferon production in both severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we show SARS-CoV-2-infected cells display an elevated level of nuclear mRNA accumulation compared to mock-infected cells. We demonstrate that ORF6 is responsible for this nuclear imprisonment of host mRNA, and using a cotransfected reporter assay, we show this nuclear retention of mRNA blocks expression of newly transcribed mRNAs. ORF6’s nuclear entrapment of host mRNA is associated with its ability to copurify with the mRNA export factors, Rae1 and Nup98. These protein-protein interactions map to the C terminus of ORF6 and can be abolished by a single amino acid mutation in Met58. Overexpression of Rae1 restores reporter expression in the presence of SARS-CoV-2 ORF6. SARS-CoV ORF6 also interacts with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly copurifies with Rae1 and Nup98 and results in significantly reduced expression of reporter proteins compared to SARS-CoV ORF6, a potential mechanism for the delayed symptom onset and presymptomatic transmission uniquely associated with the SARS-CoV-2 pandemic. We also show that both SARS-CoV and SARS-CoV-2 ORF6 block nuclear import of a broad range of host proteins. Together, these data support a model in which ORF6 clogs the nuclear pore through its interactions with Rae1 and Nup98 to prevent both nuclear import and export, rendering host cells incapable of responding to SARS-CoV-2 infection.