Search

Positive-strand RNA viruses encompass a variety of established and emerging eukaryotic pathogens. Their genome replication is confined to specialized cytoplasmic membrane compartments known as replication organelles (ROs). These ROs derive from host membranes, transformed into distinct structures such as invaginated spherules or intricate membrane networks including single- and/or double-membrane vesicles. ROs play a vital role in orchestrating viral RNA synthesis and evading detection by innate immune sensors of the host. In recent years, groundbreaking cryo–electron microscopy studies conducted with several prototypic viruses have significantly advanced our understanding of RO structure and function. Notably, these studies unveiled the presence of crown-shaped multimeric viral protein complexes that seem to actively participate in viral RNA synthesis and regulate the release of newly synthesized RNA into the cytosol for translation and packaging. These findings have shed light on novel viral functions and fascinating macromolecular complexes that delineate promising new avenues for future research. [ABSTRACT FROM AUTHOR]

The Mononegavirales, or non-segmented negative-sense RNA viruses (nsNSVs), includes significant human pathogens, such as respiratory syncytial virus, parainfluenza virus, measles virus, Ebola virus, and rabies virus. Although these viruses differ widely in their pathogenic properties, they are united by each having a genome consisting of a single strand of negative-sense RNA. Consistent with their shared genome structure, the nsNSVs have evolved similar ways to transcribe their genome into mRNAs and replicate it to produce new genomes. Importantly, both mRNA transcription and genome replication are performed by a single virus-encoded polymerase. A fundamental and intriguing question is: how does the nsNSV polymerase commit to being either an mRNA transcriptase or a replicase? The polymerase must become committed to one process or the other either before it interacts with the genome template or in its initial interactions with the promoter sequence at the 3´ end of the genomic RNA. This review examines the biochemical, molecular biology, and structural biology data regarding the first steps of transcription and RNA replication that have been gathered over several decades for different families of nsNSVs. These findings are discussed in relation to possible models that could explain how an nsNSV polymerase initiates and commits to either transcription or genome replication. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) is a Flavivirus that causes the most prevalent arthropod-borne viral disease. Clinical manifestation of DENV infection ranges from asymptomatic to severe symptoms that can lead to death. Unfortunately, no antiviral treatments against DENV are currently available. In order to identify novel DENV inhibitors, we screened a library of 1,604 chemically diversified fragment-based compounds using DENV reporter viruses that allowed quantification of viral replication in infected cells. Following a validation screening, the two best inhibitor candidates were N-phenylpyridine-3-carboxamide (NPP3C) and 6-acetyl-1H-indazole (6A1HI). The half maximal effective concentration of NPP3C and 6A1H1 against DENV were 7.1 μM and 6.5 μM, respectively. 6A1H1 decreased infectious DENV particle production up to 1,000-fold without any cytotoxicity at the used concentrations. While 6A1HI was DENV-specific, NPP3C also inhibited the replication of other flaviviruses such as West Nile virus and Zika virus. Structure-activity relationship (SAR) studies with 151 analogues revealed key structural elements of NPP3C and 6A1HI required for their antiviral activity. Time-of-drug-addition experiments identified a postentry step as a target of these compounds. Consistently, using a DENV subgenomic replicon, we demonstrated that these compounds specifically impede the viral RNA replication step and exhibit a high genetic barrier-to-resistance. In contrast, viral RNA translation and the de novo biogenesis of DENV replication organelles were not affected. Overall, our data unveil NPP3C and 6A1H1 as novel DENV inhibitors. The information revealed by our SAR studies will help chemically optimize NPP3C and 6A1H1 in order to improve their anti-flaviviral potency and to challenge them in in vivo models.

Group B enteroviruses, including coxsackievirus B3 (CVB3), can persistently infect cardiac tissue and cause dilated cardiomyopathy. Persistence is linked to 5' terminal deletions of viral genomic RNAs that have been detected together with minor populations of full-length genomes in human infections. In this study, we explored the functions and interactions of the different viral RNA forms found in persistently infected patients and their putative role(s) in pathogenesis. Since enterovirus cardiac pathogenesis is linked to the viral proteinase 2A, we investigated the effect of different terminal genomic RNA deletions on 2A activity. We discovered that 5' terminal deletions in CVB3 genomic RNAs decreased the levels of 2A proteinase activity but could not abrogate it. Using newly generated viral reporters encoding nano-luciferase, we found that 5' terminal deletions resulted in decreased levels of viral protein and RNA synthesis in singly transfected cardiomyocyte cultures. Unexpectedly, when full-length and terminally deleted forms were cotransfected into cardiomyocytes, a cooperative interaction was observed, leading to increased viral RNA and protein production. However, when viral infections were carried out in cells harboring 5' terminally deleted CVB3 RNAs, a decrease in infectious particle production was observed. Our results provide a possible explanation for the necessity of full-length viral genomes during persistent infection, as they would stimulate efficient viral replication compared to that of the deleted genomes alone. To avoid high levels of viral particle production that would trigger cellular immune activation and host cell death, the terminally deleted RNA forms act to limit the production of viral particles, possibly as trans-dominant inhibitors. IMPORTANCE Enteroviruses like coxsackievirus B3 are able to initiate acute infections of cardiac tissue and, in some cases, to establish a long-term persistent infection that can lead to serious disease sequelae, including dilated cardiomyopathy. Previous studies have demonstrated the presence of 5' terminally deleted forms of enterovirus RNAs in heart tissues derived from patients with dilated cardiomyopathy. These deleted RNAs are found in association with very low levels of full-length enterovirus genomic RNAs, an interaction that may facilitate continued persistence while limiting virus particle production. Even in the absence of detectable infectious virus particle production, these deleted viral RNA forms express viral proteinases at levels capable of causing viral pathology. Our studies provide mechanistic insights into how full-length and deleted forms of enterovirus RNA cooperate to stimulate viral protein and RNA synthesis without stimulating infectious viral particle production. They also highlight the importance of targeting enteroviral proteinases to inhibit viral replication while at the same time limiting the long-term pathologies they trigger.

An RNA polymerase ribozyme that was obtained by directed evolution can propagate a functional RNA through repeated rounds of replication and selection, thereby enabling Darwinian evolution. Earlier versions of the polymerase did not have sufficient copying fidelity to propagate functional information, but a new variant with improved fidelity can replicate the hammerhead ribozyme through reciprocal synthesis of both the hammerhead and its complement, with the products then being selected for RNA-cleavage activity. Two evolutionary lineages were carried out in parallel, using either the prior low-fidelity or the newer high-fidelity polymerase. The former lineage quickly lost hammerhead functionality as the population diverged toward random sequences, whereas the latter evolved new hammerhead variants with improved fitness compared to the starting RNA. The increase in fitness was attributable to specific mutations that improved the replicability of the hammerhead, counterbalanced by a small decrease in hammerhead activity. Deep sequencing analysis was used to follow the course of evolution, revealing the emergence of a succession of variants that progressively diverged from the starting hammerhead as fitness increased. This study demonstrates the critical importance of replication fidelity for maintaining heritable information in an RNA-based evolving system, such as is thought to have existed during the early history of life on Earth. Attempts to recreate RNA-based life in the laboratory must achieve further improvements in replication fidelity to enable the fully autonomous Darwinian evolution of RNA enzymes as complex as the polymerase itself. [ABSTRACT FROM AUTHOR]

ABSTRACT Chikungunya virus (CHIKV) is an enveloped, positive-sense RNA virus that has re-emerged to cause millions of human infections worldwide. In humans, acute CHIKV infection causes fever and severe muscle and joint pain. Chronic and debilitating arthritis and joint pain can persist for months to years. To date, there are no approved antivirals against CHIKV. Recently, the ribonucleoside analog 4′-fluorouridine (4′-FlU) was reported as a highly potent orally available inhibitor of SARS-CoV-2, respiratory syncytial virus, and influenza virus replication. In this study, we assessed 4′-FlU’s potency and breadth of inhibition against a panel of alphaviruses including CHIKV, and found that it broadly suppressed alphavirus production in cell culture. 4′-FlU acted on the viral RNA replication step, and the first 4 hours post-infection were the critical time for its antiviral effect. In vitro replication assays identified nsP4 as the target of inhibition. In vivo, treatment with 4′-FlU reduced disease signs, inflammatory responses, and viral tissue burden in mouse models of CHIKV and Mayaro virus infection. Treatment initiated at 2 hours post-infection was most effective; however, treatment initiated as late as 24–48 hours post-infection produced measurable antiviral effects in the CHIKV mouse model. 4′-FlU showed effective oral delivery in our mouse model and resulted in the accumulation of both 4′-FlU and its bioactive triphosphate form in tissues relevant to arthritogenic alphavirus pathogenesis. Together, our data indicate that 4′-FlU inhibits CHIKV infection in vitro and in vivo and is a promising oral therapeutic candidate against CHIKV infection.IMPORTANCEAlphaviruses including chikungunya virus (CHIKV) are mosquito-borne positive-strand RNA viruses that can cause various diseases in humans. Although compounds that inhibit CHIKV and other alphaviruses have been identified in vitro, there are no licensed antivirals against CHIKV. Here, we investigated a ribonucleoside analog, 4′-fluorouridine (4′-FlU), and demonstrated that it inhibited infectious virus production by several alphaviruses in vitro and reduced virus burden in mouse models of CHIKV and Mayaro virus infection. Our studies also indicated that 4′-FlU treatment reduced CHIKV-induced footpad swelling and reduced the production of pro-inflammatory cytokines. Inhibition in the mouse model correlated with effective oral delivery of 4′-FlU and accumulation of both 4′-FlU and its bioactive form in relevant tissues. In summary, 4′-FlU exhibits potential as a novel anti-alphavirus agent targeting the replication of viral RNA.

Hepatitis E Virus (HEV) is a positively oriented RNA virus having a 7.2 kb genome. HEV consists of three open reading frames (ORF1-3). Of these, ORF1 codes for the enzymes Methyltransferase (Mtase), Papain-like cysteine protease (PCP), RNA helicase, and RNA-dependent RNA polymerase (RdRp). Unavailability of a vaccine or effective drug against HEV and considering the side effects associated with the off-label use of ribavirin (RBV) and pegylated interferons, an alternative approach is required by the modulation of specific enzymes to prevent the infection. HEV helicase is involved in unwinding the double-stranded RNA, RNA processing, transcriptional regulation, and pre-mRNA processing. Therefore, we screened FDA-approved compounds from the ZINC15 database against the modelled 3D structure of HEV helicase and found that methotrexate and compound A (Pubchem ID BTB07890) inhibit the NTPase and dsRNA unwinding activity leading to inhibition of HEV RNA replication. This may be further authenticated by in vivo study. [ABSTRACT FROM AUTHOR]

Hepatitis E Virus (HEV) is a positively oriented RNA virus having a 7.2 kb genome. HEV consists of three open reading frames (ORF1-3). Of these, ORF1 codes for the enzymes Methyltransferase (Mtase), Papain-like cysteine protease (PCP), RNA helicase, and RNA-dependent RNA polymerase (RdRp). Unavailability of a vaccine or effective drug against HEV and considering the side effects associated with the off-label use of ribavirin (RBV) and pegylated interferons, an alternative approach is required by the modulation of specific enzymes to prevent the infection. HEV helicase is involved in unwinding the double-stranded RNA, RNA processing, transcriptional regulation, and pre-mRNA processing. Therefore, we screened FDA-approved compounds from the ZINC15 database against the modelled 3D structure of HEV helicase and found that methotrexate and compound A (Pubchem ID BTB07890) inhibit the NTPase and dsRNA unwinding activity leading to inhibition of HEV RNA replication. This may be further authenticated by in vivo study.

In this study, we characterized the role of host cell protein tyrosyl-DNA phosphodiesterase 2 (TDP2) activity, also known as VPg unlinkase, in picornavirus infections in a human cell model of infection. TDP2/VPg unlinkase is used by picornaviruses to remove the small polypeptide, VPg (Virus Protein genome-linked, the primer for viral RNA synthesis), from virus genomic RNA. We utilized a CRISPR/Cas-9-generated TDP2 knock out (KO) human retinal pigment epithelial-1 (hRPE-1) cell line, in addition to the wild type (WT) counterpart for our studies. We determined that in the absence of TDP2, virus growth kinetics for two enteroviruses (poliovirus and coxsackievirus B3) were delayed by about 2 h. Virus titers were reduced by ~2 log10 units for poliovirus and 0.5 log10 units for coxsackievirus at 4 hours post-infection (hpi), and by ~1 log10 unit at 6 hpi for poliovirus. However, virus titers were nearly indistinguishable from those of control cells by the end of the infectious cycle. We determined that this was not the result of an alternative source of VPg unlinkase activity being activated in the absence of TPD2 at late times of infection. Viral protein production in TDP2 KO cells was also substantially reduced at 4 hpi for poliovirus infection, consistent with the observed growth kinetics delay, but reached normal levels by 6 hpi. Interestingly, this result differs somewhat from what has been reported previously for the TDP2 KO mouse cell model, suggesting that either cell type or species-specific differences might be playing a role in the observed phenotype. We also determined that catalytically inactive TDP2 does not rescue the growth defect, confirming that TDP2 5' phosphodiesterase activity is required for efficient virus replication. Importantly, we show for the first time that polysomes can assemble efficiently on VPg-linked RNA after the initial round of translation in a cell culture model, but both positive and negative strand RNA production is impaired in the absence of TDP2 at mid-times of infection, indicating that the presence of VPg on the viral RNA affects a step in the replication cycle downstream of translation (e.g., RNA synthesis). In agreement with this conclusion, we found that double-stranded RNA production (a marker of viral RNA synthesis) is delayed in TDP2 KO RPE-1 cells. Moreover, we show that premature encapsidation of nascent, VPg-linked RNA is not responsible for the observed virus growth defect. Our studies provide the first lines of evidence to suggest that either negative- or positive-strand RNA synthesis (or both) is a likely candidate for the step that requires the removal of VPg from the RNA for an enterovirus infection to proceed efficiently.

Influenza virus contains a single-stranded negative-sense RNA genome. Replication of the genome is carried out by the viral RNA-dependent RNA polymerase in the context of the viral ribonucleoprotein (RNP) complex, through a positive-sense complementary RNA intermediate. Genome replication is tightly controlled through interactions with accessory viral and host factors. Propelled by developments in recombinant protein expression, and technical improvements in X-ray crystallography and cryo-electron microscopy, snapshots of the replication process have been captured. Here, we review how recent structural data shed light on the molecular mechanisms of influenza virus genome replication, in particular, encapsidation of nascent RNA, de novo RNP assembly, and regulation of replication initiation through interactions with host and viral cues. Influenza virus RNA genome replication is carried out by a dimeric form of the influenza virus RNA polymerase with one of the polymerases acting as a replicating polymerase, synthesizing product RNA, and the other acting as an encapsidating polymerase, capturing the nascent RNA product and initiating its assembly into a ribonucleoprotein (RNP) complex. Proteins of the acidic nuclear phosphoprotein 32 (ANP32) family, essential host factors for influenza virus RNA genome replication, bridge the replicating and encapsidating polymerase. The disordered highly acidic C-terminal tail of ANP32 proteins acts as a platform for binding viral nucleoprotein, increasing nucleoprotein density around the site of RNA genome replication, and thus facilitating its recruitment to nascent viral RNA. Using a dimeric form of the viral RNA polymerase to replicate viral genomic RNA could be a general feature of negative-sense RNA viruses. [ABSTRACT FROM AUTHOR]

Tacaribe virus (TCRV) is the prototype of New World mammarenaviruses, a group that includes several members that cause hemorrhagic fevers in humans. The TCRV genome comprises two RNA segments, named S (small) and L (large). Both genomic segments contain noncoding regions (NCRs) at their 59 and 39 ends. While the 59- and 39-terminal 19-nucleotide sequences are known to be essential for promoter function, the role of their neighboring internal noncoding region (iNCR) sequences remains poorly understood. To analyze the relevance of the 59 and 39 iNCRs in TCRV S RNA synthesis, mutant S-like minigenomes and miniantigenomes were generated. Using a minireplicon assay, Northern blotting, and reverse transcription-quantitative PCR, we demonstrated that the genomic 59 iNCR is specifically engaged in minigenome replication yet is not directly involved in minigenome transcription, and we showed that the S genome 39 iNCR is barely engaged in this process. Analysis of partial deletions and point mutations, as well as total or partial substitution of the 59 iNCR sequence, led us to conclude that the integrity of the whole genomic 59 iNCR is essential and that a local predicted secondary structure or RNA-RNA interactions between the 59 and 39 iNCRs are not strictly required for viral S RNA synthesis. Furthermore, we employed a TCRV reverse genetic approach to ask whether manipulation of the S genomic 59 iNCR sequence may be suitable for viral attenuation. We found that mutagenesis of the 59 promoter-proximal subregion slightly impacted recombinant TCRV virulence in vivo. IMPORTANCE The Mammarenavirus genus of the Arenaviridae family includes several members that cause severe hemorrhagic fevers associated with high morbidity and mortality rates, for which no FDA-approved vaccines and limited therapeutic resources are available. We provide evidence demonstrating the specific involvement of the TCRV S 59 noncoding sequence adjacent to the viral promoter in replication. In addition, we examined the relevance of this region in the context of an in vivo infection. Our findings provide insight into the mechanism through which this 59 viral RNA noncoding region assists the L polymerase for efficient viral S RNA synthesis. Also, these findings expand our understanding of the effect of genetic manipulation of New World mammarenavirus sequences aimed at the rational design of attenuated recombinant virus vaccine platforms. [ABSTRACT FROM AUTHOR]

Crimean-Congo hemorrhagic fever virus (CCHFV) infection can result in a severe hemorrhagic syndrome for which there are no antiviral interventions available to date. Certain RNA viruses, such as CCHFV, encode cysteine proteases of the ovarian tumor (OTU) family that antagonize interferon (IFN) production by deconjugating ubiquitin (Ub). The OTU of CCHFV, a negative-strand RNA virus, is dispensable for replication of the viral genome, despite being part of the large viral RNA polymerase. Here, we show that mutations that prevent binding of the OTU to cellular ubiquitin are required for the generation of recombinant CCHFV containing a mutated catalytic cysteine. Similarly, the high-affinity binding of a synthetic ubiquitin variant (UbV-CC4) to CCHFV OTU strongly inhibits viral growth. UbV-CC4 inhibits CCHFV infection even in the absence of intact IFN signaling, suggesting that its antiviral activity is not due to blocking the OTUs immunosuppressive function. Instead, the prolonged occupancy of the OTU with UbV-CC4 directly targets viral replication by interfering with CCHFV RNA synthesis. Together, our data provide mechanistic details supporting the development of antivirals targeting viral OTUs.IMPORTANCE Crimean-Congo hemorrhagic fever virus is an important human pathogen with a wide global distribution for which no therapeutic interventions are available. CCHFV encodes a cysteine protease belonging to the ovarian tumor (OTU) family which is involved in host immune suppression. Here we demonstrate that artificially prolonged binding of the OTU to a substrate inhibits virus infection. This provides novel insights into CCHFV OTU function during the viral replicative cycle and highlights the OTU as a potential antiviral target.

Eastern equine encephalitis virus (EEEV) usually cycles between Culiseta melanura mosquitoes and birds; however, it can also infect humans. EEEV has a positive-sense RNA genome that, in infected cells, serves as an mRNA for the P1234 polyprotein. P1234 undergoes a series of precise cleavage events producing four nonstructural proteins (nsP1–4) representing subunits of the RNA replicase. Here, we report the construction and properties of a trans-replicase for EEEV. The template RNA of EEEV was shown to be replicated by replicases of diverse alphaviruses. The EEEV replicase, on the other hand, demonstrated limited ability in replicating template RNAs originating from alphaviruses of the Semliki Forest virus complex. The replicase of EEEV was also successfully reconstructed from P123 and nsP4 components. The ability of EEEV P123 to form functional RNA replicases with heterologous nsP4s was more efficient using EEEV template RNA than heterologous alphavirus template RNA. This finding indicates that unlike with previously studied Semliki Forest complex alphaviruses, P123 and/or its processing products have a leading role in EEEV template RNA recognition. Infection of HEK293T cells harboring the EEEV template RNA with EEEV or Western equine encephalitis virus prominently activated expression of a reporter encoded in the template RNA; the effect was much smaller for infection with other alphaviruses and not detectable upon flavivirus infection. At the same time, EEEV infection resulted only in a limited activation of the template RNA of chikungunya virus. Thus, cells harboring reporter-carrying template RNAs can be used as sensitive and selective biosensors for different alphaviruses. IMPORTANCE Infection of EEEV in humans can cause serious neurologic disease with an approximately 30% fatality rate. Although human infections are rare, a record-breaking number was documented in 2019. The replication of EEEV has a unique requirement for host factors but is poorly studied, partly because the virus requires biosafety level 3 facilities which can limit the scope of experiments; at the same time, these studies are crucial for developing antiviral approaches. The EEEV trans-replicase developed here contributes significantly to research on EEEV, providing a safe and versatile tool for studying the virus RNA replication. Using this system, the compatibility of EEEV replicase components with counterparts from other alphaviruses was analyzed. The obtained data can be used to develop unique biosensors that provide alternative methods for detection, identification, quantitation, and neutralization of viable alphaviruses that are compatible with high throughput, semiautomated approaches. [ABSTRACT FROM AUTHOR]

The cellular mRNA decay protein AUF1 acts as a restriction factor during infection by picornaviruses, including poliovirus, coxsackievirus, and human rhinovirus. AUF1 relocalizes from the nucleus to the cytoplasm during infection by these viruses due to the disruption of nucleocytoplasmic trafficking by viral proteinases. Previous studies have demonstrated that AUF1 binds to poliovirus and coxsackievirus B3 (CVB3) RNA during infection, with binding shown to occur within the internal ribosome entry site (IRES) of the 5' noncoding region (NCR) or the 3' NCR, respectively. Binding to different sites within the viral RNA suggests that AUF1 may negatively regulate infection by these viruses using different mechanisms. The work presented here addresses the mechanism of AUF1 inhibition of the replication of poliovirus and CVB3. We demonstrate that AUF1 knockdown in human cells results in increased viral translation, RNA synthesis, and virus production. AUF1 is shown to negatively regulate translation of a poliovirus and CVB3 IRES reporter RNA during infection but not in uninfected cells. We found that this inhibitory activity is not mediated through destabilization of viral genomic RNA; however, it does require virus-induced relocalization of AUF1 from the nucleus to the cytoplasm during the early phases of infection. Our findings suggest that AUF1 restriction of poliovirus and CVB3 replication uses a common mechanism through the viral IRES, which is distinct from the canonical role that AUF1 plays in regulated mRNA decay in uninfected host cells.IMPORTANCE Picornaviruses primarily infect the gastrointestinal or upper respiratory tracts of humans and animals and may disseminate to tissues of the central nervous system, heart, skin, liver, or pancreas. Many common human pathogens belong to the Picornaviridae family, which includes viruses known to cause paralytic poliomyelitis (poliovirus); myocarditis (coxsackievirus B3 [CVB3]); the common cold (human rhinovirus [HRV]); and hand, foot, and mouth disease (enterovirus 71 [EV71]), among other illnesses. There are no specific treatments for infection, and vaccines exist for only two picornaviruses: poliovirus and hepatitis A virus. Given the worldwide distribution and prevalence of picornaviruses, it is important to gain insight into the host mechanisms used to restrict infection. Other than proteins involved in the innate immune response, few host factors have been identified that restrict picornavirus replication. The work presented here seeks to define the mechanism of action for the host restriction factor AUF1 during infection by poliovirus and CVB3.

Retrozymes are nonautonomous retrotransposons with hammerhead ribozymes in their long terminal repeats (LTRs). Retrozyme transcripts can be self-cleaved by the LTR ribozyme, circularized, and can undergo RNA-to-RNA replication. Here, we demonstrate that the Nicotiana benthamiana genome contains hundreds of retrozyme loci, of which nine represent full-length retrozymes. The LTR contains a promoter directing retrozyme transcription. Although retrozyme RNA is easily detected in plants, the LTR region is heavily methylated, pointing to its transcriptional silencing, which can be mediated by 24 nucleotide-long retrozyme-specific RNAs identified in N. benthamiana. A transcriptome analysis revealed that half of the retrozyme-specific RNAs in plant leaves have no exact matches to genomic retrozyme loci, containing up to 13% mismatches with the closest genomic sequences, and could arise as a result of many rounds of RNA-to-RNA replication leading to error accumulation. Using a cloned retrozyme copy, we show that retrozyme RNA is capable of replication and systemic transport in plants. The presented data suggest that retrozyme loci in the N. benthamiana genome are transcriptionally inactive, and that circular retrozyme RNA can persist in cells due to its RNA-to-RNA replication and be transported systemically, emphasizing functional and, possibly, evolutionary links of retrozymes to viroids—noncoding circular RNAs that infect plants. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus. In infected cells, its positive-sense RNA genome is translated into polyproteins that are subsequently processed into four nonstructural proteins (nsP1 to 4), the virus-encoded subunits of the RNA replicase. However, for RNA replication, interactions between nsPs and host proteins are also needed. These interactions are mostly mediated through the intrinsically disordered C-terminal hypervariable domain (HVD) in nsP3. Duplicate FGDF motifs in the HVD are required for interaction with mammalian RasGAP SH3-binding proteins (G3BPs) and their mosquito homolog Rin; these interactions are crucial for CHIKV RNA replication. In this study, we inactivated G3BP/Rin-binding motifs in the HVD and inserted peptides containing either native or inactivated G3BP/Rin-binding motifs into flexible regions of nsP1, nsP2, or nsP4. Insertion of native motifs into nsP1 or nsP2 but not into the C terminus of nsP4 activated CHIKV RNA replication in human cells in a G3BP-dependent manner. In mosquito cells, activation also resulted from the insertion of inactive motifs after residue 8 or 466 in nsP2; however, the effect was significantly larger when the inserted sequence contained native motifs. Nonetheless, CHIKV mutants harboring mutations in the HVD and containing insertions of native motifs in nsP2 were not viable in mosquito cells. In contrast, mutant genomes containing native motifs after residue 466 or 618 in nsP2 replicated in BHK-21 cells, with the latter mutant forming infectious progeny. Thus, the binding of G3BPs to nsP2 can support CHIKV RNA replication and restore the infectivity of viruses lacking G3BP-binding motifs in the HVD of nsP3. [ABSTRACT FROM AUTHOR]

New variations of SARS-CoV-2 continue to emerge in the global pandemic, which may be resistant to at least some vaccines in COVID-19, indicating that drug and vaccine development must be continuously strengthened. NSP10 plays an essential role in SARS-CoV-2 viral life cycle. It stimulates the enzymatic activities of NSP14-ExoN and NSP16–O-MTase by the formation of NSP10/NSP14 and NSP10/NSP16 complexes. Inhibiting NSP10 can block the binding of NSP10 to NSP14 and NSP16. This study has identified potential natural NSP10 inhibitors from ZINC database. The protein druggable pocket was identified for screening candidates. Molecular docking of the selected compounds was performed and MM-GBSA binding energy was calculated. After ADMET assessment, 4 hits were obtained for favorable druggability. The analysis of site interactions suggested that the hits all had excellent binding. Molecular dynamics studies revealed that selected natural compounds stably bind to NSP10. These compounds were identified as potential leads against NSP10 for the development of strategies to combat SARS-CoV-2 replication and could serve as the basis for further studies. • NSP10 as an attractive drug target offers the possibility of COVID-19 treatment. • Binding modes and binding energies of the candidates were revealed using molecular docking and binding energy calculations. • Candidate compounds with good pharmacokinetic properties were evaluated by ADMET analysis. • Four hits as potential NSP10 inhibitors exhibited binding stability in MD simulations. [ABSTRACT FROM AUTHOR]

Replication proteins of tobacco mosaic virus (TMV), a positive-sense RNA virus, co-translationally bind to a 5′-proximal ~70-nucleotide (nt) region of the genomic RNA, referred to as the nuclease-resistant (NR) region for replication template selection. Therefore, disruption of the interaction between the viral replication proteins and viral genomic RNA is expected to inhibit the replication of TMV. In this study, we demonstrate that the addition of small RNA fragments (18–33 nts in length) derived from different regions within the NR region inhibit the binding of TMV replication proteins to viral RNA and TMV RNA replication in a cell-free system. Intriguingly, some of the small RNA fragments also inhibited the translation of mRNA in a sequence-nonspecific manner. These results highlight the pleiotropic roles of the 5′-proximal region of the TMV genome. [ABSTRACT FROM AUTHOR]

An RNA ligase ribozyme that catalyzes the joining of RNA molecules of the opposite chiral handedness was optimized for the ability to synthesize its own enantiomer from two component fragments. The mirror-image D- and L-ligases operate in concert to provide a system for cross-chiral replication, whereby they catalyze each other's synthesis and undergo mutual amplification at constant temperature, with apparent exponential growth and a doubling time of about 1 h. Neither the D- nor the L-RNA components alone can achieve autocatalytic self-replication. Cross-chiral exponential amplification can be continued indefinitely through a serial-transfer process that provides an ongoing supply of the component D- and L-substrates. Unlike the familiar paradigm of semiconservative nucleic acid replication that relies on Watson-Crick pairing between complementary strands, cross-chiral replication relies on tertiary interactions between structured nucleic acids "across the mirror." There are few examples, outside of biology, of autocatalytic self-replication systems that undergo exponential amplification and there are no prior examples, in either biological or chemical systems, of cross-chiral replication enabling exponential amplification., Competing Interests: Competing interests statement:The authors declare no competing interest.

RNA viruses have evolved sophisticated strategies to exploit the limited encoded information within their typically compact genomes. One of them, named transcriptional slippage (TS), is characterized by the appearance of indels in nascent viral RNAs, leading to changes in the open reading frame (ORF). Although members of unrelated viral families express key proteins via TS, the available information about this phenomenon is still limited. In potyvirids (members of the Potyviridae family), TS has been defined by the insertion of an additional A at A n motifs ( n ≥ 6) in newly synthesized transcripts at a low frequency, modulated by nucleotides flanking the A-rich motif. Here, by using diverse experimental approaches and a collection of plant/virus combinations, we discover cases not following this definition. In summary, we observe (i) a high rate of single-nucleotide deletions at slippage motifs, (ii) overlapping ORFs acceded by slippage at an U 8 stretch, and (iii) changes in slippage rates induced by factors not related to cognate viruses. Moreover, a survey of whole-genome sequences from potyvirids shows a widespread occurrence of species-specific A n /U n ( n ≥ 6) motifs. Even though many of them, but not all, lead to the production of truncated proteins rather than access to overlapping ORFs, these results suggest that slippage motifs appear more frequently than expected and play relevant roles during virus evolution. Considering the potential of this phenomenon to expand the viral proteome by acceding to overlapping ORFs and/or producing truncated proteins, a re-evaluation of TS significance during infections of RNA viruses is required.IMPORTANCETranscriptional slippage (TS) is used by RNA viruses as another strategy to maximize the coding information in their genomes. This phenomenon is based on a peculiar feature of viral replicases: they may produce indels in a small fraction of newly synthesized viral RNAs when transcribing certain motifs and then produce alternative proteins due to a change of the reading frame or truncated products by premature termination. Here, using plant-infecting RNA viruses as models, we discover cases expanding on previously established features of plant virus TS, prompting us to reconsider and redefine this expression strategy. An interesting conclusion from our study is that TS might be more relevant during RNA virus evolution and infection processes than previously assumed., Competing Interests: The authors declare no conflict of interest.

SARS-CoV-2 belongs to the family Coronaviridae and carries a single-stranded positive-sense RNA genome. During coronavirus (CoV) replication, defective or defective interfering RNAs that lack a large portion of the genome often emerge. These defective RNAs typically carry the necessary RNA elements that are required for replication and packaging. We identified the minimum requirement of the 5' proximal region necessary for viral RNA replication by using artificially generated SARS-CoV-2 minigenomes. The minigenomes consist of the 5'-proximal region, an open reading frame (ORF) that encodes a fusion protein consisting of the N-terminal of viral NSP1 and a reporter gene, and the 3' untranslated region of the SARS-CoV-2 genome. We used a modified SARS-CoV-2 variant to support replication of the minigenomes. A minigenome carrying the 5' proximal 634 nucleotides replicated, whereas those carrying shorter than 634 nucleotides did not, demonstrating that the entire 265 nt-long 5' untranslated region and N-terminal portion of the NSP1 coding region are required for the minigenome replication. Minigenome RNAs carrying a specific amino acid substitution or frame shift insertions in the partial NSP1 coding sequence abrogated minigenome replication. Introduction of synonymous mutations in the minigenome RNAs also affected the replication efficiency of the minigenomes. These data suggest that the expression of the N-terminal portion of NSP1 and the primary sequence of the 5' proximal 634 nucleotides are important for minigenome replication.IMPORTANCESARS-CoV-2, the causative agent of COVID-19, is highly transmissible and continues to have a significant impact on public health and the global economy. While several vaccines mitigate the severe consequences of SARS-CoV-2 infection, mutant viruses with reduced reactivity to current vaccines continue to emerge and circulate. This study aimed to identify the minimal 5' proximal region of SARS-CoV-2 genomic RNA required for SARS-CoV-2 defective RNA replication and investigate the importance of an ORF encoded in these defective RNAs. Identifying cis-acting replication signals of SARS-CoV-2 genomic RNA is critical for the development of antivirals that target these signals. Additionally, replication-competent defective RNAs can serve as therapeutic reagents to interfere with SARS-CoV-2 replication. Our findings provide valuable insights into the mechanisms of SARS-CoV-2 RNA replication and the development of reagents that suppress SARS-CoV-2 replication., Competing Interests: The authors declare no conflict of interest.

Background: Global cyclical outbreaks of human enterovirus infections has positioned human enterovirus A71 (EV-A71) as a neurotropic virus of clinical importance. However, there remains a scarcity of internationally approved antivirals and vaccines., Methods: In pursuit of repurposing drugs for combating human enteroviruses, we employed a comprehensive pharmacophore- and molecular docking-based virtual screen targeting EV-A71 capsid protein VP1-4, 3C protease, and 3D polymerase proteins. Among 15 shortlisted ligand candidates, we dissected the inhibitory mechanism of Tanomastat in cell-based studies and evaluated its in vivo efficacy in an EV-A71-infected murine model., Findings: We demonstrated that Tanomastat exerts dose-dependent inhibition on EV-A71 replication, with comparable efficacy profiles in enterovirus species A, B, C, and D in vitro. Time-course studies suggested that Tanomastat predominantly disrupts early process(es) of the EV-A71 replication cycle. Mechanistically, live virus particle tracking and docking predictions revealed that Tanomastat specifically impedes viral capsid dissociation, potentially via VP1 hydrophobic pocket binding. Bypassing its inhibition on entry stages, we utilized EV-A71 replication-competent, 3D pol replication-defective, and bicistronic IRES reporter replicons to show that Tanomastat also inhibits viral RNA replication, but not viral IRES translation. We further showed that orally administered Tanomastat achieved 85% protective therapeutic effect and alleviated clinical symptoms in EV-A71-infected neonatal mice., Interpretation: Our study establishes Tanomastat as a broad-spectrum anti-enterovirus candidate with promising pre-clinical efficacy, warranting further testing for potential therapeutic application., Funding: MOE Tier 2 grants (MOE-T2EP30221-0005, R571-000-068-592, R571-000-076-515, R571-000-074-733) and A∗STARBiomedical Research Council (BMRC)., Competing Interests: Declaration of interests HF received grants from Synthetic Biology Translational Research Program, School of Medicine, National University of Singapore., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

ABSTRACT Marburg virus (MARV) is an enveloped, negative-sense RNA virus from the filovirus family that causes outbreaks of severe, frequently fatal illness in humans. Of the seven MARV proteins, the VP30 protein stands out because it is essential for viral growth but lacks a definitive function. Here, we used model MARV genome RNAs for one or two reporter genes and the MARV VP40, glycoprotein (GP), and VP24 genes to demonstrate that VP30 is dispensable for the transcription of some genes but critical for transcription reinitiation at the GP gene. This results in the loss of the expression of GP and downstream genes and the impaired production of infectious particles when VP30 is absent. Bicistronic minigenome assays demonstrate that the VP40 gene end/GP gene start junction specifically confers VP30 dependence. A region at the GP gene start site predicted to form a stem-loop contributes to VP30 dependence because the replacement of the GP stem-loop with corresponding sequences from the MARV VP35 gene relieves VP30 dependence. Finally, a Cys3-His zinc binding motif characteristic of filovirus VP30 proteins was demonstrated to be critical for reinitiation at GP. These findings address a long-standing gap in our understanding of MARV biology by defining a critical role for VP30 in MARV transcription. IMPORTANCE Marburg virus and Ebola virus encode VP30 proteins. While the role of VP30 in Ebola virus transcription has been well studied, the role of VP30 in the Marburg virus life cycle is not well understood. The work here demonstrates that different gene start sites within the Marburg viral genome have variable levels of dependence on Marburg virus VP30, with its expression being critical for transcription reinitiation at the GP gene start site. These findings address a long-standing question regarding Marburg virus VP30 function and further our understanding of how Marburg virus gene expression is regulated.

Alphaviruses are positive-strand RNA viruses, mostly being mosquito-transmitted. Cells infected by an alphavirus become resistant to superinfection due to a block that occurs at the level of RNA replication. Alphavirus replication proteins, called nsP1-4, are produced from nonstructural polyprotein precursors, processed by the protease activity of nsP2. Trans-replicase systems and replicon vectors were used to study effects of nsP2 of chikungunya virus and Sindbis virus on alphavirus RNA replication in mosquito cells. Co-expressed wild-type nsP2 reduced RNA replicase activity of homologous virus; this effect was reduced but typically not abolished by mutation in the protease active site of nsP2. Mutations in the replicase polyprotein that blocked its cleavage by nsP2 reduced the negative effect of nsP2 co-expression, confirming that nsP2-mediated inhibition of RNA replicase activity is largely due to nsP2-mediated processing of the nonstructural polyprotein. Co-expression of nsP2 also suppressed the activity of replicases of heterologous alphaviruses. Thus, the presence of nsP2 inhibits formation and activity of alphavirus RNA replicase in protease activity-dependent and -independent manners. This knowledge improves our understanding about mechanisms of superinfection exclusion for alphaviruses and may aid the development of anti-alphavirus approaches. [ABSTRACT FROM AUTHOR]

Group B coxsackieviruses are responsible for chronic cardiac infections. However, the molecular mechanisms by which the virus can persist in the human heart long after the signs of acute myocarditis have abated are still not completely understood. Recently, coxsackievirus B3 strains with 5'-terminal deletions in genomic RNAs were isolated from a patient suffering from idiopathic dilated cardiomyopathy, suggesting that such mutant viruses may be the forms responsible for persistent infection. These deletions lacked portions of 5' stem-loop I, which is an RNA secondary structure required for viral RNA replication. In this study, we assessed the consequences of the genomic deletions observed in vivo for coxsackievirus B3 biology. Using cell extracts from HeLa cells, as well as transfection of luciferase replicons in two types of cardiomyocytes, we demonstrated that coxsackievirus RNAs harboring 5' deletions ranging from 7 to 49 nucleotides in length can be translated nearly as efficiently as those of wild-type virus. However, these 5' deletions greatly reduced the synthesis of viral RNA in vitro, which was detected only for the 7- and 21-nucleotide deletions. Since 5' stem-loop I RNA forms a ribonucleoprotein complex with cellular and viral proteins involved in viral RNA replication, we investigated the binding of the host cell protein PCBP2, as well as viral protein 3CDpro, to deleted positive-strand RNAs corresponding to the 5' end. We found that binding of these proteins was conserved but that ribonucleoprotein complex formation required higher PCBP2 and 3CDpro concentrations, depending on the size of the deletion. Overall, this study confirmed the characteristics of persistent CVB3 infection observed in heart tissues and provided a possible explanation for the low level of RNA replication observed for the 5'-deleted viral genomes-a less stable ribonucleoprotein complex formed with proteins involved in viral RNA replication.IMPORTANCE Dilated cardiomyopathy is the most common indication for heart transplantation worldwide, and coxsackie B viruses are detected in about one-third of idiopathic dilated cardiomyopathies. Terminal deletions at the 5' end of the viral genome involving an RNA secondary structure required for RNA replication have been recently reported as a possible mechanism of virus persistence in the human heart. These mutations are likely to disrupt the correct folding of an RNA secondary structure required for viral RNA replication. In this report, we demonstrate that transfected RNAs harboring 5'-terminal sequence deletions are able to direct the synthesis of viral proteins, but not genomic RNAs, in human and murine cardiomyocytes. Moreover, we show that the binding of cellular and viral replication factors to viral RNA is conserved despite genomic deletions but that the impaired RNA synthesis associated with terminally deleted viruses could be due to destabilization of the ribonucleoprotein complexes formed.

Zika virus is a mosquito-borne flavivirus known to cause severe birth defects and neuroimmunological disorders. We have previously demonstrated that mosquito transmission of Zika virus decreases with temperature. While transmission was optimized at 29°C, it was limited at cool temperatures (,22°C) due to poor virus establishment in the mosquitoes. Temperature is one of the strongest drivers of vector-borne disease transmission due to its profound effect on ectothermic mosquito vectors, viruses, and their interaction. Although there is substantial evidence of temperature effects on arbovirus replication and dissemination inside mosquitoes, little is known about whether temperature affects virus replication directly or indirectly through mosquito physiology. In order to determine the mechanisms behind temperature-induced changes in Zika virus transmission potential, we investigated different steps of the virus replication cycle in mosquito cells (C6/36) at optimal (28°C) and cool (20°C) temperatures. We found that the cool temperature did not alter Zika virus entry or translation, but it affected genome replication and reduced the amount of double-stranded RNA replication intermediates. If replication complexes were first formed at 28°C and the cells were subsequently shifted to 20°C, the late steps in the virus replication cycle were efficiently completed. These data suggest that cool temperature decreases the efficiency of Zika virus genome replication in mosquito cells. This phenotype was observed in the Asian lineage of Zika virus, while the African lineage Zika virus was less restricted at 20°C. [ABSTRACT FROM AUTHOR]

As the most abundant biological entities with incredible diversity, bacteriophages (also known as phages) have been recognized as an important source of molecular machines for the development of genetic-engineering tools. At the same time, phages are crucial for establishing and improving basic theories of molecular biology. Studies on phages provide rich sources of essential elements for synthetic circuit design as well as powerful support for the improvement of directed evolution platforms. Therefore, phages play a vital role in the development of new technologies and central scientific concepts. After the RNA world hypothesis was proposed and developed, novel biological functions of RNA continue to be discovered. RNA and its related elements are widely used in many fields such as metabolic engineering and medical diagnosis, and their versatility led to a major role of RNA in synthetic biology. Further development of RNA-based technologies will advance synthetic biological tools as well as provide verification of the RNA world hypothesis. Most synthetic biology efforts are based on reconstructing existing biological systems, understanding fundamental biological processes, and developing new technologies. RNA-based technologies derived from phages will offer abundant sources for synthetic biological components. Moreover, phages as well as RNA have high impact on biological evolution, which is pivotal for understanding the origin of life, building artificial life-forms, and precisely reprogramming biological systems. This review discusses phage-derived RNA-based technologies terms of phage components, the phage lifecycle, and interactions between phages and bacteria. The significance of RNA-based technology derived from phages for synthetic biology and for understanding the earliest stages of biological evolution will be highlighted.

Influenza A viruses are negative-sense RNA viruses that rely on their own viral replication machinery to replicate and transcribe their segmented single-stranded RNA genome. The viral ribonucleoprotein complexes in which viral RNA is replicated consist of a nucleoprotein scaffold around which the RNA genome is wound, and a heterotrimeric RNA-dependent RNA polymerase that catalyzes viral replication. The RNA polymerase copies the viral RNA (vRNA) via a replicative intermediate, called the cRNA, and subsequently uses this cRNA to make more vRNA copies. To ensure that new cRNA and vRNA molecules are associated with ribonucleoproteins in which they can be amplified, the active RNA polymerase recruits a second polymerase to encapsidate the cRNA or vRNA. Host factor ANP32A has been shown to be essential for viral replication and to facilitate the formation of a dimer between viral RNA polymerases. Differences between mammalian and avian ANP32A proteins are sufficient to restrict viral replication. It has been proposed that ANP32A is only required for the synthesis of vRNA molecules from cRNA but not vice versa. However, this view does not match recent molecular evidence. Here we use minigenome assays, virus infections, and viral promoter mutations to demonstrate that ANP32A is essential for both vRNA and cRNA synthesis. Moreover, we show that ANP32A is not only needed for the actively replicating polymerase, but not for the polymerase that is encapsidating nascent viral RNA products. Overall, these results provide new insights into influenza A virus replication and host adaptation. IMPORTANCE Zoonotic avian influenza A viruses pose a constant threat to global health, and they have the potential to cause pandemics. Species variations in host factor ANP32A play a key role in supporting the activity of avian influenza A virus RNA polymerases in mammalian hosts. Here we show that ANP32A acts at two stages in the influenza A virus replication cycle, supporting recent structural experiments, in line with its essential role. Understanding how ANP32A supports viral RNA polymerase activity and how it supports avian polymerase function in mammalian hosts is important for understanding influenza A virus replication and the development of antiviral strategies against influenza A viruses. [ABSTRACT FROM AUTHOR]

Hepatitis delta virus (HDV) is a significant human pathogen that causes acute and chronic liver disease; there is no licensed therapy. HDV is a circular negativesense single-stranded RNA (ssRNA) virus that produces three RNAs in infected cells, genome, antigenome, and mRNA; the latter encodes hepatitis delta antigen (HDAg), the viral protein. These RNAs are synthesized by host DNA-dependent RNA polymerase acting as an RNA-dependent RNA polymerase. Although HDV genome RNA accumulates to high levels in infected cells, the mechanism by which this process occurs remains poorly understood. For example, the nature of the 59 end of the genome, including the synthesis start site and its chemical composition, is not known. Analysis of this process has been challenging because the initiation site is part of an unstable precursor in the rolling-circle mechanism by which HDV genome RNA is synthesized. In this study, circular HDV antigenome RNAs synthesized in vitro were used to directly initiate HDV genome RNA synthesis in transfected cells, thus enabling the detection of the 59 end of the genome RNA. The 59 end of this RNA is capped, as expected for a polymerase II product. Initiation begins at position 1646 on the genome, which is located near the loop end proximal to the start site for HDAg mRNA synthesis. Unexpectedly, synthesis begins with a guanosine that is not conventionally templated by the HDV RNA. IMPORTANCE Hepatitis delta virus (HDV) is a unique virus that causes severe liver disease. It uses host RNA polymerase II to copy its circular RNA genome in a unique and poorly understood process. Although the virus RNA accumulates to high levels within infected cells, it is not known how the synthesis of the viral RNA begins or even where on the genome synthesis starts. Here, we identify the start site for the initiation of HDV genome RNA synthesis as position 1646, which is at one end of the closed-hairpin-like structure of the viral RNA. The 59 end of the RNA is capped, as expected for polymerase II products. However, RNA synthesis begins with a guanosine that is not present in the genome. Thus, although HDV uses polymerase II to synthesize the viral genome, some details of the initiation process are different. These differences could be important for successfully targeting virus replication. [ABSTRACT FROM AUTHOR]

Infectious pancreatic necrosis virus (IPNV) is an important pathogen that is threatening the worldwide salmon and trout industry. But there is no therapeutic drug available for now. In this study, we demonstrate that MK-0608 is highly efficient against IPNV and low cytotoxic, with a 50 % effective concentration (EC 50 ) of 0.20 μM and selectivity index (SI) of about 268. Time of addition assay illustrated that MK-0608 targeted the early stage of IPNV life cycle. Furthermore, we found that MK-0608 blocked IPNV attachment on the premise of sufficient pre-incubation time but MK-0608 did not influence viral internalization and release. MK-0608 could inhibit IPNV genome synthesis, and combination with ribavirin enhanced the inhibition effect, which might be functional via binding to IPNV RNA dependent RNA polymerase (RdRp), which was predicted by using molecular docking methods. In vivo test showed that IPNV was extremely suppressed in the rainbow trout (Oncorhynchus mykiss) with one single dose of MK-0608, and the higher dosage of 50 mg/kg could cause 3 log decrease of IPNV loads in fish tissues., Competing Interests: Declaration of competing interest All authors declare that no conflicts of interest exist., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hepatitis C virus (HCV) is a positive-stranded RNA virus that mainly causes chronic hepatitis, cirrhosis and hepatocellular carcinoma. Recently we confirmed m5C modifications within NS5A gene of HCV RNA genome. However, the roles of the m5C modification and its interaction with host proteins in regulating HCV's life cycle, remain unexplored. Here, we demonstrate that HCV infection enhances the expression of the host m5C reader YBX1 through the transcription factor MAX. YBX1 acts as an m5C reader, recognizing the m5C-modified NS5A C7525 site in the HCV RNA genome and significantly enhancing HCV RNA stability. This m5C-modification is also required for YBX1 colocalization with lipid droplets and HCV Core protein. Moreover, YBX1 facilitates HCV RNA replication, as well as viral assembly/budding. The tryptophan residue at position 65 (W65) of YBX1 is critical for these functions. Knockout of YBX1 or the application of YBX1 inhibitor SU056 suppresses HCV RNA replication and viral protein translation. To our knowledge, this is the first report demonstrating that the interaction between host m5C reader YBX1 and HCV RNA m5C methylation facilitates viral replication. Therefore, hepatic-YBX1 knockdown holds promise as a potential host-directed strategy for HCV therapy.

Self-replicating RNAs (srRNAs) are synthetic molecules designed to mimic the self-replicating ability of viral RNAs. srRNAs hold significant promise for a range of applications, including enhancing protein expression, reprogramming cells into pluripotent stem cells, and creating cell-free systems for experimental evolution. However, the development of srRNAs for use in bacterial systems remains limited. Here, we demonstrate how a srRNA scaffold from Emesvirus zinderi can be engineered into a self-encoding srRNA by incorporating the coding region of the catalytically active replicase subunit. With the help of in vitro replication assays, including an in vitro translation-coupled replication approach, we show that the resulting system enables complete replication cycles of RNA both in cis and trans, including long cargo RNAs such as tethered 5S, 16S, and 23S rRNAs. In summary, our findings suggest that these srRNAs have significant potential for fundamental research, synthetic biology, and general in vitro evolution.

Chikungunya virus (CHIKV) is an enveloped, positive-sense RNA virus that has re-emerged to cause millions of human infections worldwide. In humans, acute CHIKV infection causes fever and severe muscle and joint pain. Chronic and debilitating arthritis and joint pain can persist for months to years. To date, there are no approved antivirals against CHIKV. Recently, the ribonucleoside analog 4'-fluorouridine (4'-FlU) was reported as a highly potent orally available inhibitor of SARS-CoV-2, respiratory syncytial virus, and influenza virus replication. In this study, we assessed 4'-FlU's potency and breadth of inhibition against a panel of alphaviruses including CHIKV, and found that it broadly suppressed alphavirus production in cell culture. 4'-FlU acted on the viral RNA replication step, and the first 4 hours post-infection were the critical time for its antiviral effect. In vitro replication assays identified nsP4 as the target of inhibition. In vivo , treatment with 4'-FlU reduced disease signs, inflammatory responses, and viral tissue burden in mouse models of CHIKV and Mayaro virus infection. Treatment initiated at 2 hours post-infection was most effective; however, treatment initiated as late as 24-48 hours post-infection produced measurable antiviral effects in the CHIKV mouse model. 4'-FlU showed effective oral delivery in our mouse model and resulted in the accumulation of both 4'-FlU and its bioactive triphosphate form in tissues relevant to arthritogenic alphavirus pathogenesis. Together, our data indicate that 4'-FlU inhibits CHIKV infection in vitro and in vivo and is a promising oral therapeutic candidate against CHIKV infection.IMPORTANCEAlphaviruses including chikungunya virus (CHIKV) are mosquito-borne positive-strand RNA viruses that can cause various diseases in humans. Although compounds that inhibit CHIKV and other alphaviruses have been identified in vitro , there are no licensed antivirals against CHIKV. Here, we investigated a ribonucleoside analog, 4'-fluorouridine (4'-FlU), and demonstrated that it inhibited infectious virus production by several alphaviruses in vitro and reduced virus burden in mouse models of CHIKV and Mayaro virus infection. Our studies also indicated that 4'-FlU treatment reduced CHIKV-induced footpad swelling and reduced the production of pro-inflammatory cytokines. Inhibition in the mouse model correlated with effective oral delivery of 4'-FlU and accumulation of both 4'-FlU and its bioactive form in relevant tissues. In summary, 4'-FlU exhibits potential as a novel anti-alphavirus agent targeting the replication of viral RNA., Competing Interests: G.R.P. is a coinventor on patent WO 2019/1736002 covering the composition of matter and the use of EIDD-2749 and its analogs as an antiviral treatment. This study could affect his personal financial status. The other authors declare no conflict of interest.

Coxsackievirus B3 (CVB3) is an enterovirus belonging to the family Picornaviridae. Its 5' untranslated region (UTR) contains a cloverleaf structure followed by an internal ribosome entry site (IRES). The cloverleaf forms an RNA-protein complex known to regulate virus replication, translation, and stability of the genome, and the IRES regulates virus RNA translation. For positive-strand RNA-containing viruses, such as members of the flaviviruses or enteroviruses, the genomic RNA is used for translation, replication, and encapsidation. Only a few regulatory mechanisms which govern the accessibility of genomic RNA templates for translation or replication have been reported. Here, we report the role of human antigen R (HuR) in regulating the fate of CVB3 positive-strand RNA into the replication cycle or translation cycle. We have observed that synthesis of HuR is induced during CVB3 infection, and it suppresses viral replication by displacing PCBP-2 (a positive regulator of virus replication) at the cloverleaf RNA. Silencing of HuR increases viral RNA replication and consequently reduces viral RNA translation in a replication-dependent manner. Furthermore, we have shown that HuR level is upregulated upon CVB3 infection. Moreover, HuR limits virus replication and can coordinate the availability of genomic RNA templates for translation, replication, or encapsidation. Our study highlights the fact that the relative abundance of translation factors and replication factors in the cell decides the outcome of viral infection. IMPORTANCE A positive-strand RNA virus must balance the availability of its genomic template for different viral processes at different stages of its life cycle. A few host proteins are shown to be important to help the virus in switching the usage of a template between these processes. These proteins inhibit translation either by displacing a stimulator of translation or by binding to an alternative site. Both mechanisms lead to ribosome clearance and availability of the genomic strand for replication. We have shown that HuR also helps in maintaining this balance by inhibiting replication and subsequently promoting translation and packaging. [ABSTRACT FROM AUTHOR]

Alphaviruses (family Togaviridae) include both human pathogens such as chikungunya virus (CHIKV) and Sindbis virus (SINV) and model viruses such as Semliki Forest virus (SFV). The alphavirus positive-strand RNA genome is translated into nonstructural (ns) polyprotein(s) that are precursors for four nonstructural proteins (nsPs). The three-dimensional structures of nsP2 and the N-terminal 2/3 of nsP3 reveal that these proteins consist of several domains. Cleavage of the ns-polyprotein is performed by the strictly regulated protease activity of the nsP2 region. Processing results in the formation of a replicase complex that can be considered a network of functional modules. These modules work cooperatively and should perform the same task for each alphavirus. To investigate functional interactions between replicase components, we generated chimeras using the SFV genome as a backbone. The functional modules corresponding to different parts of nsP2 and nsP3 were swapped with their counterparts from CHIKV and SINV. Although some chimeras were nonfunctional, viruses harboring the CHIKV N-terminal domain of nsP2 or any domain of nsP3 were viable. Viruses harboring the protease part of nsP2, the full-length nsP2 of CHIKV, or the nsP3 macrodomain of SINV required adaptive mutations for functionality. Seven mutations that considerably improved the infectivity of the corresponding chimeric genomes affected functionally important hot spots recurrently highlighted in previous alphavirus studies. These data indicate that alphaviruses utilize a rather limited set of strategies to survive and adapt. Furthermore, functional analysis revealed that the disturbance of processing was the main defect resulting from chimeric alterations within the ns-polyprotein. [ABSTRACT FROM AUTHOR]

Alphaviruses have positive-strand RNA genomes containing two open reading frames (ORFs). The first ORF encodes the nonstructural (ns) polyproteins P123 and P1234 that act as precursors for the subunits of the viral RNA replicase (nsP1 to nsP4). Processing of P1234 leads to the formation of a negative-strand replicase consisting of nsP4 (RNA polymerase) and P123 components. Subsequent processing of P123 results in a positive-strand replicase. The second ORF encoding the structural proteins is expressed via the synthesis of a subgenomic RNA. Alphavirus replicase is capable of using template RNAs that contain essential cis-active sequences. Here, we demonstrate that the replicases of nine alphaviruses, expressed in the form of separate P123 and nsP4 components, are active. Their activity depends on the abundance of nsP4. The match of nsP4 to its template strongly influences efficient subgenomic RNA synthesis. nsP4 of Barmah Forest virus (BFV) formed a functional replicase only with matching P123, while nsP4s of other alphaviruses were compatible also with several heterologous P123s. The P123 components of Venezuelan equine encephalitis virus and Sindbis virus (SINV) required matching nsP4s, while P123 of other viruses could form active replicases with different nsP4s. Chimeras of Semliki Forest virus, harboring the nsP4 of chikungunya virus, Ross River virus, BFV, or SINV were viable. In contrast, chimeras of SINV, harboring an nsP4 from different alphaviruses, exhibited a temperature-sensitive phenotype. These findings highlight the possibility for formation of new alphaviruses via recombination events and provide a novel approach for the development of attenuated chimeric viruses for vaccination strategies. [ABSTRACT FROM AUTHOR]

The template‐directed synthesis of RNA played an important role in the transition from prebiotic chemistry to the beginnings of RNA based life, but the mechanism of RNA copying chemistry is incompletely understood. We measured the kinetics of template copying with a set of primers with modified 3′‐nucleotides and determined the crystal structures of these modified nucleotides in the context of a primer/template/substrate‐analog complex. pH‐rate profiles and solvent isotope effects show that deprotonation of the primer 3′‐hydroxyl occurs prior to the rate limiting step, the attack of the alkoxide on the activated phosphate of the incoming nucleotide. The analogs with a 3E ribose conformation show the fastest formation of 3′–5′ phosphodiester bonds. Among those derivatives, the reaction rate is strongly correlated with the electronegativity of the 2′‐substituent. We interpret our results in terms of differences in steric bulk and charge distribution in the ground vs. transition states. [ABSTRACT FROM AUTHOR]

Life history theory posits that the sequence and timing of events in an organism's lifespan are fine-tuned by evolution to maximize the production of viable offspring. In a virus, a life history strategy is largely manifested in its replication mode. Here, we develop a stochastic mathematical model to infer the replication mode shaping the structure and mutation distribution of a poliovirus population in an intact single infected cell. We measure production of RNA and poliovirus particles through the infection cycle, and use these data to infer the parameters of our model. We find that on average the viral progeny produced from each cell are approximately five generations removed from the infecting virus. Multiple generations within a single cell infection provide opportunities for significant accumulation of mutations per viral genome and for intracellular selection.

Replication proteins of tobacco mosaic virus (TMV), a positive-sense RNA virus, co-translationally bind to a 5′-proximal ~70-nucleotide (nt) region of the genomic RNA, referred to as the nuclease-resistant (NR) region for replication template selection. Therefore, disruption of the interaction between the viral replication proteins and viral genomic RNA is expected to inhibit the replication of TMV. In this study, we demonstrate that the addition of small RNA fragments (18–33 nts in length) derived from different regions within the NR region inhibit the binding of TMV replication proteins to viral RNA and TMV RNA replication in a cell-free system. Intriguingly, some of the small RNA fragments also inhibited the translation of mRNA in a sequence-nonspecific manner. These results highlight the pleiotropic roles of the 5′-proximal region of the TMV genome.

The emergence of new viruses is a matter of significant concern in the current decade. It has dazed the indigenous healthcare systems in various parts of the world. Consequently, the resources for fighting the rapid spread of the DENV are ineffectual, insufficient, and incompetent. Several environmental factors have been allied with dengue fever transmission. High documentation of neuropathogenesis in dengue-infected individuals in recent years reflects the significance of our study, and the information discussed in this review can be used to enhance clinical awareness along with advances in diagnostic approaches. The replication of the DENV genome is usually engaged in a membrane-linked replication complex; the virion budding and morphogenesis have been found to take place in the modified endoplasmic reticulum membranes. Most of these non-structural proteins are believed to be responsible for RNA replication and polyprotein processing. The nucleic acid-based strategies have provided high sensitivity and progressively replacing conventional techniques. Advanced technologies like PCR have offered timely serotyping of dengue viruses, which illustrates the early warning of dengue epidemics. The molecular methods help to characterize the key factors responsible for the rapid spread of viruses and thereby update the vector control programmes targeted at extenuating their adverse impacts on public health. The vast diversity of dengue virus strains worldwide with special inference on their clinical manifestations and genetic characteristics was also analyzed in this study. As dengue threatens one third of the world's population, practical applications of advanced molecular strategies from an environmental and phylogenetic perspective are critical for disease control. [ABSTRACT FROM AUTHOR]

The origin of genetic systems is the central problem in the study of the origin of life for which various explanatory hypotheses have been presented. One model suggests that both ancestral transfer ribonucleic acid (tRNA) molecules and primitive ribosomes were originally involved in RNA replication (Campbell 1991). According to this model the early tRNA molecules catalyzed their own self-loading with a trinucleotide complementary to their anticodon triplet, while the primordial ribosome (protoribosome) catalyzed the transfer of these terminal trinucleotides from one tRNA to another tRNA harboring the growing RNA polymer at the 3´-end. Here we present the notion that the anticodon-codon-like pairs presumably located in the acceptor stem of primordial tRNAs (Rodin et al. 1996) (thus being and remaining, after the code and translation origins, the major contributor to the RNA operational code (Schimmel et al. 1993)) might have originally been used for RNA replication rather than translation; these anticodon and acceptor stem triplets would have been involved in accurately loading the 3'-end of tRNAs with a trinucleotide complementary to their anticodon triplet, thus allowing the accurate repair of tRNAs for their use by the protoribosome during RNA replication. We propose that tRNAs could have catalyzed their own trinucleotide self-loading by forming catalytic tRNA dimers which would have had polymerase activity. Therefore, the loading mechanism and its evolution may have been a basic step in the emergence of new genetic mechanisms such as genetic translation. The evolutionary implications of this proposed loading mechanism are also discussed. [ABSTRACT FROM AUTHOR]

Alphaviruses are positive-strand RNA viruses, mostly being mosquito-transmitted. Cells infected by an alphavirus become resistant to superinfection due to a block that occurs at the level of RNA replication. Alphavirus replication proteins, called nsP1-4, are produced from nonstructural polyprotein precursors, processed by the protease activity of nsP2. Trans-replicase systems and replicon vectors were used to study effects of nsP2 of chikungunya virus and Sindbis virus on alphavirus RNA replication in mosquito cells. Co-expressed wild-type nsP2 reduced RNA replicase activity of homologous virus; this effect was reduced but typically not abolished by mutation in the protease active site of nsP2. Mutations in the replicase polyprotein that blocked its cleavage by nsP2 reduced the negative effect of nsP2 co-expression, confirming that nsP2-mediated inhibition of RNA replicase activity is largely due to nsP2-mediated processing of the nonstructural polyprotein. Co-expression of nsP2 also suppressed the activity of replicases of heterologous alphaviruses. Thus, the presence of nsP2 inhibits formation and activity of alphavirus RNA replicase in protease activity-dependent and -independent manners. This knowledge improves our understanding about mechanisms of superinfection exclusion for alphaviruses and may aid the development of anti-alphavirus approaches.

Flavivirus replication is intimately associated with re-organized cellular membranes. These virus-induced changes in membrane architecture form three distinct membranous “organelles” that have specific functions during the flavivirus life cycle. One of these structures is the replication complex in which the flaviviral RNA is replicated to produce progeny genomes. We have previously observed that this process is strictly dependent on cellular cholesterol. In this study we have identified a putative cholesterol recognition/interaction amino acid consensus (CRAC) motif within the West Nile virus strain Kunjin virus (WNVKUN) NS4A protein. Site-directed mutagenesis of this motif within a WNVKUN infectious clone severely attenuated virus replication and the capacity of the mutant viruses to form the replication complex. Replication of the mutant viruses also displayed reduced co-localization with cellular markers recruited to replication sites during wild-type virus replication. In addition, we observed that the mutant viruses were significantly impaired in their ability to remodel cytoplasmic membranes. However, after extensive analysis we are unable to conclusively reveal a role for the CRAC motif in direct cholesterol binding to NS4A, suggesting additional complex lipid-protein and protein-protein interactions. We believe this study highlights the crucial role for this region within NS4A protein in recruitment of cellular and viral proteins to specialized subdomains on membrane platforms to promote efficient virus replication.

Chikungunya virus (CHIKV, family Togaviridae) is a mosquito-transmitted alphavirus. The positive-sense RNA genome of CHIKV encodes four nonstructural proteins (nsP1 to nsP4) that are virus-specific subunits of the RNA replicase. Among nsP functions, those of nsP3 are the least understood. The C-terminal hypervariable domain (HVD) in nsP3 is disordered and serves as a platform for interactions with multiple host proteins. For Sindbis virus (SINV) and Semliki Forest virus (SFV), the nsP3 HVD has been shown to be phosphorylated. Deletion of phosphorylated regions has a mild effect on the growth of SFV and SINV in vertebrate cells. Using radiolabeling, we demonstrated that nsP3 in CHIKV and o'nyong-nyong virus is also phosphorylated. We showed that the phosphorylated residues in CHIKV nsP3 are not clustered at the beginning of the HVD. The substitution of 20 Ser/Thr residues located in the N-terminal half of the HVD or 26 Ser/Thr residues located in its C-terminal half with Ala residues reduced the activity of the CHIKV replicase and the infectivity of CHIKV in mammalian cells. Furthermore, the substitution of all 46 potentially phosphorylated residues resulted in the complete loss of viral RNA synthesis and infectivity. The mutations did not affect the interaction of the HVD in nsP3 with the host G3BP1 protein; interactions with CD2AP, BIN1, and FHL1 proteins were significantly reduced but not abolished. Thus, CHIKV differs from SFV and SINV both in the location of the phosphorylated residues in the HVD in nsP3 and, significantly, in their effect on replicase activity and virus infectivity. [ABSTRACT FROM AUTHOR]

The capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements using chaperone activity. However, the role of DENV2C during the interaction of RNA elements in the conserved 5' untranslated region (5'UTR) to the 3' untranslated region (3'UTR) is still unclear. Thus, we investigated the effect of DENV2C on the annealing mechanism of two RNA hairpin elements from the 5'UTR to their complementary sequences during (+)/(-) ds-RNAformation and (+) RNA circularization. DENV2C was found to switch the annealing pathway for RNA elements involved in (+)/(-) ds-RNA formation, but not for RNA elements related to (+) RNA circularization. In addition, we also determined that DENV2C modulates intrinsic dynamics and reduces kinetically trapped unfavourable conformations of the 5'UTR sequence. Thus, our results provide mechanistic insights by which DENV2C chaperones the interactions between RNA elements at the 5' and 3' ends during genome recombination, a prerequisite for DENV replication. [ABSTRACT FROM AUTHOR]

Enterovirus replication requires the cellular protein GBF1, a guanine nucleotide exchange factor for small Arf GTPases. When activated, Arfs associate with membranes, where they regulate numerous steps of membrane homeostasis. The requirement for GBF1 implies that Arfs are important for replication, but which of the different Arfs function(s) during replication remains poorly understood. Here, we established cell lines expressing each of the human Arfs fused to a fluorescent tag and investigated their behavior during enterovirus infection. Arf1 was the first to be recruited to the replication organelles, where it strongly colocalized with the viral antigen 2B and mature virions but not double-stranded RNA. By the end of the infectious cycle, Arf3, Arf4, Arf5, and Arf6 were also concentrated on the replication organelles. Once on the replication membranes, all Arfs except Arf3 were no longer sensitive to inhibition of GBF1, suggesting that in infected cells they do not actively cycle between GTP- and GDP-bound states. Only the depletion of Arf1, but not other class 1 and 2 Arfs, significantly increased the sensitivity of replication to GBF1 inhibition. Surprisingly, depletion of Arf6, a class 3 Arf, normally implicated in plasma membrane events, also increased the sensitivity to GBF1 inhibition. Together, our results suggest that GBF1-dependent Arf1 activation directly supports the development and/or functioning of the replication complexes and that Arf6 plays a previously unappreciated role in viral replication. Our data reveal a complex pattern of Arf activation in enterovirus-infected cells that may contribute to the resilience of viral replication in different cellular environments. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) has gained notoriety in recent years because there are no targeted therapies or vaccines available so far. Caveolin-1 (Cav-1) in host cells plays crucial functions in the invasion of many viruses. However, its specific involvement in ZIKV infection has remained unclear. Here, we reveal that depleting Cav-1 leads to a substantial reduction in ZIKV RNA levels, protein expression and viral particle production, indicating that ZIKV exploits Cav-1 for its infection. By dissecting each stage of the viral life cycle, we unveil that, unlike its invasion role in many other viruses, Cav-1 depletion selectively impairs ZIKV replication, resulting in altered replication dynamics and reduced strand-specific RNA levels, but does not affect viral entry, maturation and release. These results reveal an unforeseen function of Cav-1 in facilitating ZIKV replication, which provides new insights into the intricate interaction between Cav-1 and ZIKV and underscores Cav-1 as a potential candidate for anti-ZIKV approaches., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The nonstructural protein 12, known as RNA-dependent RNA polymerase (RdRp), is essential for both replication and repair of the viral genome. The RdRp of SARS-CoV-2 has been used as a promising candidate for drug development since the inception of the COVID-19 spread. In this work, we performed an in silico investigation on the insertion of the naturally modified pyrimidine nucleobase ddhCTP into the SARS-CoV-2 RdRp active site, in a comparative analysis with the natural one (CTP). The modification in ddhCTP involves the removal of the 3'-hydroxyl group that prevents the addition of subsequent nucleotides into the nascent strand, acting as an RNA chain terminator inhibitor. Quantum mechanical investigations helped to shed light on the mechanistic source of RdRp activity on the selected nucleobases, and comprehensive all-atom simulations provided insights about the structural rearrangements occurring in the active-site region when inorganic pyrophosphate (PPi) is formed. Subsequently, the intricate pathways for the release of PPi, the catalytic product of RdRp, were investigated using Umbrella Sampling simulations. The results are in line with the available experimental data and contribute to a more comprehensive point of view on such an important viral enzyme.