Viral RNA Replication Research Articles

Cellular fate of a plant virus immunotherapy candidate

Cowpea mosaic virus (CPMV) is a plant virus that is currently being developed for intratumoral immunotherapy. CPMV relieves the immune system from tumor-induced immunosuppression; reprograms the tumor microenvironment to an activated state whereby the treated and distant tumors are recognized and eradicated. Toward translational studies, we investigated the safety of CPMV, specifically addressing whether pathogenicity would be induced in mammalian cells. We show that murine macrophage immune cells recognize CPMV; however, there is no indication of de novo viral protein synthesis or RNA replication. Furthermore, we show that CPMV does not induce hemolysis, platelet aggregation and plasma coagulation amongst other assays in human blood and immune cells. Taken together, we anticipate that these results will reinforce the development of CPMV as an immunotherapeutic platform.

Replication capacity and susceptibility of nirmatrelvir-resistant mutants to next-generation Mpro inhibitors in a SARS-CoV-2 replicon system

There is an ongoing need to expand the anti-SARS-CoV-2 armamentarium to include agents capable of suppressing replication of drug-resistant mutants emerging during monotherapy with approved direct-acting antivirals. Using a subgenomic SARS-CoV-2 replicon system, we studied the RNA replication capacity of nirmatrelvir (NTV)-resistant mutants and their susceptibility to next-generation Mpro inhibitors, including ibuzatrelvir (ITV), ensitrelvir (ETV), and ML2006a4. Our findings revealed that E166V Mpro mutants reduced viral RNA replication, whereas other Mpro mutations retained or increased the replication capacity, suggesting the potential of the latter to dominate under NTV selective pressure. Except for having an advantage against E166A mutants, ITV largely showed the same mutational sensitivity as NTV. ETV was more effective than NTV against E166V mutants but less effective against S144A, E166A, and L167F mutants. ML2006a4 demonstrated the most effective suppression across most mutants (S144A, E166V, S144A + L50F, E166 A/V + L50F, L167F + L50F, and E166A + L167F + L50F). Thus, ML2006a4 represents an attractive investigational candidate against clinically relevant NTV-resistant SARS-CoV-2 mutants.

Coronavirus M protein promotes mitophagy over virophagy by recruiting PDPK1 to phosphorylate SQSTM1 at T138

Autophagy plays a dual role in coronavirus infection, facilitating the elimination of either proviral components (virophagy) or antiviral factors such as mitochondria (mitophagy), leading to complex mechanisms of immune evasion. Understanding the mechanisms that govern the switch between the autophagic degradation of deleterious or beneficial substrates in coronavirus infection is crucial for developing precise drug targets to treat virus-induced diseases. However, this switch remains largely unknown. Using a dual split-fluorescence assay, we identify PDPK1 as a negative regulator of innate immunity, directing the transition from virophagy to mitophagy through the phosphorylation of SQSTM1 at T138. Remarkably, a PDPK1-targeting peptide inhibits the replication of various RNA viruses by restoring innate immunity through enhanced virophagy and suppressed mitophagy, thereby protecting female mice from lethal infections. These findings underscore the detrimental role of PDPK1 in innate immunity by orchestrating the shift from virophagy to mitophagy, positioning PDPK1 as a promising pharmacological target for effectively combating a broad spectrum of virus infections.

Structure of the RNA-dependent RNA polymerase P2 from the cystovirus φ8

The replication of RNA viruses relies on the activity of RNA-dependent RNA polymerases (RdRps). Despite large variations in their genomic sequences, viral RdRps share a common architecture generally known as a closed right hand. The P2 polymerase of cystovirus φ6 is currently among the best characterized viral RdRps. This polymerase is responsible for carrying out both replication and transcription of the viral double-stranded RNA genome using de novo initiation. Despite the extensive biochemical and structural studies conducted on φ6 P2, further structural information on other cystoviral RdRps is crucial to elucidate the structural and functional diversity of viral RdRps. Here, we have determined the atomic X-ray structure of the RdRp P2 from the φ6-related cystovirus φ8 at 3Å resolution. This structure completes the existing set of structural information on the φ8 polymerase complex and sheds light on the difference and similarities with related cystoviral RdRps.

The hnRNP A2B1 is important for the replication of SFTSV and other RNA viruses.

The heterogeneous nuclear ribonucleoprotein (hnRNP A2B1) is a key component of the hnRNP complex involving RNA modulation in eukaryotic cells and it has also been reported to be involved in the replication of the hepatitis E virus, influenza A virus, and hepatitis B virus. However, it is not clear whether the role of the hnRNP A2B1 in viral replication is conserved among RNA viruses and what is the mechanism of hnRNP A2B1 in RNA virus replication. In this study, we first used severe fever with thrombocytopenia syndrome virus (SFTSV), a tick-borne RNA virus that causes a severe viral hemorrhagic fever as well as other RNA viruses including VSV-GFP, SeV, EV71, and ZIKV to demonstrate that knockout hnRNPA2B1 gene inhibited viral RNA replication and overexpression of hnRNP A2B1 could restore the RNA levels of all tested RNA viruses. These results suggest that hnRNPA2B1 upregulation of viral replication is conserved among RNA viruses. Next, we demonstrated that hnRNP A2B1 was translocated from the nucleus to the cytoplasm under RNA virus infection including SFTSV, VSV-GFP, SeV, EV71, and ZIKV, suggesting translocation of hnRNP A2B1 from the nucleus to the cytoplasm is crucial for RNA virus replication. We then used SFTSV as a model to demonstrate the mechanism of hnRNP A2B1 in the promotion of RNA virus replication. We found that overexpression of SFTSV nucleoprotein can also cause hnRNP A2B1 translocation from the nucleus to the cytoplasm and that the SFTSV NP interacted with the RNA recognition motif 1 domain of hnRNP A2B1. We further demonstrated that the hnRNP A2B1 interacted with the 5' UTR of SFTSV RNA. In conclusion, we revealed that the hnRNP A2B1 upregulation of viral RNA replication is conserved among RNA viruses; the mechanism of hnRNP A2B1 in promotion of SFTSV viral RNA replication is that SFTSV NP interacted with the hnRNPA2B1 to retain it in the cytoplasm where the hnRNP A2B1 interacted with the 5' UTR of SFTSV RNA to promote the viral RNA replication.IMPORTANCESevere fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne RNA virus with a high mortality rate of up to 30%. In this study, we first used SFTSV as a model to demonstrate that the role of hnRNPA2B1 in viral replication is conserved in SFTSV. Then we used other RNA viruses, including VSV-GFP, SeV, EV71, and ZIKV, to repeat the experiment and demonstrated the same results as SFTSV in all tested RNA viruses. By knocking out the hnRNPA2B1 gene, SFTSV RNA replication was inhibited, and overexpression of hnRNPA2B1 restored RNA levels of SFTSV and other tested RNA viruses. We revealed a novel mechanism where the SFTSV nucleoprotein interacts with hnRNPA2B1, retaining it in the cytoplasm. This interaction promotes viral RNA replication by binding to the 5' UTR of SFTSV RNA. The findings suggest that targeting hnRNPA2B1 could be a potential strategy for developing broad-spectrum antiviral therapies, given its conserved role across different RNA viruses. This research provides significant insights into the replication mechanisms of RNA viruses and highlights potential targets for antiviral interventions.

Molecular characterization and functional analysis of ZAP-like gene in common carp (Cyprinus carpio)

The zinc finger antiviral protein (ZAP) is a host antiviral factor that could restrict the replication of various RNA and DNA viruses. To date, the antiviral properties of ZAP gene have been demonstrated in multiple mammals and a few of bird species, while no data is available regarding the immune role of ZAP in fish. In this study, one ZAP-like gene (CcZAPL) was identified form common carp and its antiviral role was investigated. Expression analysis showed that CcZAPL was widely expressed in multiple fish tissues, with highest level in the head kidney, and confocal microscopy analysis showed the sublocation of CcZAPL mainly in the nucleus of Epithelioma papulosum cyprini (EPC) cells. After in vivo stimulation by Spring viraemia of carp virus (SVCV), CcZAPL was induced in gene expression, and in EPC cells overexpression of CcZAPL led to significantly decreased virus load of SVCV and diminished cytopathic effect (CPE). Moreover, after SVCV infection in vitro, expressions of cytokines including IFN, ISG15, PKR, Mx and TNF-α were observed to be up-regulated in CcZAPL-overexpressed EPC cells. Our findings indicated that CcZAPL played a positive role in the control of SVCV, which will allow us to gain new insights into the immune role of ZAP in fish antiviral immunity.

Drugs Targeting Sirtuin 2 Exhibit Broad-Spectrum Anti-Infective Activity.

Direct-acting anti-infective drugs target pathogen-coded gene products and are a highly successful therapeutic paradigm. However, they generally target a single pathogen or family of pathogens, and the targeted organisms can readily evolve resistance. Host-targeted agents can overcome these limitations. One family of host-targeted, anti-infective agents modulate human sirtuin 2 (SIRT2) enzyme activity. SIRT2 is one of seven human sirtuins, a family of NAD+-dependent protein deacylases. It is the only sirtuin that is found predominantly in the cytoplasm. Multiple, structurally distinct SIRT2-targeted, small molecules have been shown to inhibit the replication of both RNA and DNA viruses, as well as intracellular bacterial pathogens, in cell culture and in animal models of disease. Biochemical and X-ray structural studies indicate that most, and probably all, of these compounds act as allosteric modulators. These compounds appear to impact the replication cycles of intracellular pathogens at multiple levels to antagonize their replication and spread. Here, we review SIRT2 modulators reported to exhibit anti-infective activity, exploring their pharmacological action as anti-infectives and identifying questions in need of additional study as this family of anti-infective agents advances to the clinic.

Duck hepatitis A virus utilizes PCBP2 to facilitate viral translation and replication

Duck hepatitis A virus type 1 (DHAV-1) is an important member of the Picornaviridae family that causes highly fatal hepatitis in ducklings. Since picornaviruses have small genomes with limited coding capacity, they must utilize host proteins for viral cap-independent translation and RNA replication. Here, we report the role of duck poly(rC)-binding protein 2 (PCBP2) in regulating the replication and translation of DHAV-1. During DHAV-1 infection, PCBP2 expression was upregulated. A biotinylated RNA pull-down assay revealed that PCBP2 positively regulates DHAV-1 translation through specific interactions with structural domains II and III of the DHAV-1 internal ribosome entry site (IRES). Further studies revealed that PCBP2 promotes DHAV-1 replication via an interaction of its KH1 domain (aa 1–92) with DHAV-1 3Dpol. Thus, our studies demonstrated the specific role of PCBP2 in regulating DHAV-1 translation and replication, revealing a novel mechanism by which host‒virus interactions regulate viral translation and replication. These findings contribute to further understanding of the pathogenesis of picornavirus infections.

Small hydrophobic (SH) proteins of Pneumoviridae and Paramyxoviridae: small but mighty.

Small hydrophobic (SH) proteins are a class of viral accessory proteins expressed by many members of the negative-stranded RNA viral families Paramyxoviridae and Pneumoviridae. Identified SH proteins are type I or II transmembrane (TM) proteins with a single-pass TM domain. Little is known about the functions of SH proteins; however, several possess viroporin activity, enhancing membrane permeability of infected cells or those expressing SH protein. Moreover, several SH proteins inhibit apoptosis and immune signaling pathways within infected cells, including TNF and interferon signaling, or activate inflammasomes. SH proteins are generally nonessential for viral replication in vitro, but loss of SH is often associated with reduced replication in vivo, suggesting a role in enhancing viral replication or evading host immunity. Analogous proteins are expressed by a variety of pathogens of public health importance; thus, understanding the functional importance and mechanisms of SH proteins provides insight into the pathogenesis and replication of negative-sense RNA viruses.

Zinc-finger CCHC-type containing protein 8 promotes RNA virus replication by suppressing the type-I interferon responses.

Host cells have evolved an intricate regulatory network to fine tune the type-I interferon responses. However, the full picture of this regulatory network remains to be depicted. In this study, we found that knock out of zinc-finger CCHC-type containing protein 8 (ZCCHC8) impairs the replication of influenza A virus (IAV), Sendai virus (Sev), Japanese encephalitis virus (JEV), and vesicular stomatitis virus (VSV). Further investigation unveiled that ZCCHC8 suppresses the type-I interferon responses by targeting the interferon regulatory factor 3 (IRF3) signaling pathway. Mechanistically, ZCCHC8 associates with phosphorylated IRF3 and disrupts the interaction of IRF3 with the co-activator CREB-binding protein (CBP). Additionally, the direct binding of ZCCHC8 with the IFN-stimulated response element (ISRE) impairs the ISRE-binding of IRF3. Our study contributes to the comprehensive understanding for the negative regulatory network of the type-I interferon responses and provides valuable insights for the control of multiple viruses from a host-centric perspective.IMPORTANCEThe innate immune responses serve as the initial line of defense against invading pathogens and harmful substances. Negative regulation of the innate immune responses plays an essential role in avoiding auto-immune diseases and over-activated immune responses. Hence, the comprehensive understanding of the negative regulation network for innate immune responses could provide novel therapeutic insights for the control of viral infections and immune dysfunction. In this study, we report that ZCCHC8 negatively regulates the type-I interferon responses. We illustrate that ZCCHC8 impedes the IRF3-CBP association by interacting with phosphorylated IRF3 and competes with IRF3 for binding to ISRE. Our study demonstrates the role of ZCCHC8 in the replication of multiple RNA viruses and contributes to a deeper understanding of the negative regulation system for the type-I interferon responses.

Requirement of the N-terminal region of nonstructural protein 1 in cis for SARS-CoV-2 defective RNA replication.

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.

Independent repeated mutations within the alphaviruses Ross River virus and Barmah Forest virus indicates convergent evolution and past positive selection in ancestral populations despite ongoing purifying selection.

Ross River virus (RRV) and Barmah Forest virus (BFV) are arthritogenic arthropod-borne viruses (arboviruses) that exhibit generalist host associations and share distributions in Australia and Papua New Guinea (PNG). Using stochastic mapping and discrete-trait phylogenetic analyses, we profiled the independent evolution of RRV and BFV signature mutations. Analysis of 186 RRV and 88 BFV genomes demonstrated their viral evolution trajectories have involved repeated selection of mutations, particularly in the nonstructural protein 1 (nsP1) and envelope 3 (E3) genes suggesting convergent evolution. Convergent mutations in the nsP1 genes of RRV (residues 248 and 441) and BFV (residues 297 and 447) may be involved with catalytic enzyme mechanisms and host membrane interactions during viral RNA replication and capping. Convergent E3 mutations (RRV site 59 and BFV site 57) may be associated with enzymatic furin activity and cleavage of E3 from protein precursors assisting viral maturation and infectivity. Given their requirement to replicate in disparate insect and vertebrate hosts, convergent evolution in RRV and BFV may represent a dynamic link between their requirement to selectively 'fine-tune' intracellular host interactions and viral replicative enzymatic processes. Despite evidence of evolutionary convergence, selection pressure analyses did not reveal any RRV or BFV amino acid sites under strong positive selection and only weak positive selection for nonstructural protein sites. These findings may indicate that their alphavirus ancestors were subject to positive selection events which predisposed ongoing pervasive convergent evolution, and this largely supports continued purifying selection in RRV and BFV populations during their replication in mosquito and vertebrate hosts.

Bithiazole inhibitors of PI4KB show broad-spectrum antiviral activity against different viral families

Broad-spectrum antivirals can be extremely important for pandemic preparedness. Targeting host factors dispensable for the host but indispensable for the virus can result in high barrier to resistance and a large range of viruses targeted. PI4KB is a lipid kinase involved in the replication of several RNA viruses, but common inhibitors of this target are mainly active against members of the Picornaviridae family. Herein we describe the optimization of bithiazole PI4KB inhibitors as broad-spectrum antivirals (BSAs) active against different members of the Picornaviridae, Coronaviridae, Flaviviridae and Poxviridae families. Since some of these viruses are transmitted via respiratory route, the efficacy of one of the most promising compounds was evaluated in an airway model. The molecule showed complete viral inhibition and absence of toxicity. These results pave the road for the development of new BSAs.

Tanomastat exerts multi-targeted inhibitory effects on viral capsid dissociation and RNA replication in human enteroviruses

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. 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 invivo efficacy in an EV-A71-infected murine model. We demonstrated that Tanomastat exerts dose-dependent inhibition on EV-A71 replication, with comparable efficacy profiles in enterovirus species A, B, C, and D invitro. 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, 3Dpol 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. Our study establishes Tanomastat as a broad-spectrum anti-enterovirus candidate with promising pre-clinical efficacy, warranting further testing for potential therapeutic application. 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).

Bis-benzylisoquinoline alkaloids inhibit flavivirus entry and replication by compromising endolysosomal trafficking and autophagy

Flaviviruses, such as dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV), represent a substantial public health challenge as there are currently no approved treatments available. Here, we investigated the antiviral effects of bis-benzylisoquinoline alkaloids (BBAs) on flavivirus infections. We evaluated five specific BBAs—berbamine, tetrandrine, iso-tetrandrine, fangchinoline, and cepharanthine—and found that they effectively inhibited infections by ZIKV, DENV, or JEV by blocking virus entry and genome replication stages in the flavivirus life cycle. Furthermore, we synthesized a fluorophore-conjugated BBA and showed that BBAs targeted endolysosomes, causing lysosomal pH alkalization. Mechanistic studies on inhibiting ZIKV infection by BBAs revealed that these compounds blocked TRPML channels, leading to lysosomal dysfunction and reducing the expression of NCAM1, a key receptor for the entry of ZIKV into cells, thereby decreasing cells susceptibility to ZIKV infection. Additionally, BBAs inhibited the fusion of autophagosomes and lysosomes, significantly reducing viral RNA replication. Collectively, our results suggest that BBAs inhibit flavivirus entry and replication by compromising endolysosomal trafficking and autophagy, respectively, underscoring the potential of BBAs as therapeutic agents against flavivirus infections.

Positive-strand RNA virus replication organelles at a glance.

Membrane-bound replication organelles (ROs) are a unifying feature among diverse positive-strand RNA viruses. These compartments, formed as alterations of various host organelles, provide a protective niche for viral genome replication. Some ROs are characterised by a membrane-spanning pore formed by viral proteins. The RO membrane separates the interior from immune sensors in the cytoplasm. Recent advances in imaging techniques have revealed striking diversity in RO morphology and origin across virus families. Nevertheless, ROs share core features such as interactions with host proteins for their biogenesis and for lipid and energy transfer. The restructuring of host membranes for RO biogenesis and maintenance requires coordinated action of viral and host factors, including membrane-bending proteins, lipid-modifying enzymes and tethers for interorganellar contacts. In this Cell Science at a Glance article and the accompanying poster, we highlight ROs as a universal feature of positive-strand RNA viruses reliant on virus-host interplay, and we discuss ROs in the context of extensive research focusing on their potential as promising targets for antiviral therapies and their role as models for understanding fundamental principles of cell biology.

Intra-Host Citrus Tristeza Virus Populations during Prolonged Infection Initiated by a Well-Defined Sequence Variant in Nicotiana benthamiana.

Due to the error-prone nature of viral RNA-dependent RNA polymerases, the replication of RNA viruses results in a diversity of viral genomes harboring point mutations, deletions, insertions, and genome rearrangements. Citrus tristeza virus (CTV), a causal agent of diseases of economically important citrus species, shows intrinsic genetic stability. While the virus appears to have some mechanism that limits the accumulation of single-nucleotide variants, the production of defective viral genomes (DVGs) during virus infection has been reported for certain variants of CTV. The intra-host diversity generated during plant infection with variant T36 (CTV-T36) remains unclear. To address this, we analyzed the RNA species accumulated in the initially infected and systemic leaves of Nicotiana benthamiana plants inoculated with an infectious cDNA clone of CTV-T36, which warranted that infection was initiated by a known, well-defined sequence variant of the virus. CTV-T36 limited the accumulation of single-nucleotide mutants during infection. With that, four types of DVGs-deletions, insertions, and copy- and snap-backs-were found in all the samples, with deletions and insertions being the most common types. Hot-spots across the genome for DVG recombination and short direct sequence repeats suggest that sequence complementarity could mediate DVG formation. In conclusion, our study illustrates the formation of diverse DVGs during CTV-T36 infection. To the best of our knowledge, this is the first study that has analyzed the genetic variability and recombination of a well-defined sequence variant of CTV in an herbaceous host.

In Vitro Cleavage Assay to Characterize DENV NS2B3 Antagonism of cGAS.

cGAS is a key cytosolic dsDNA receptor that senses viral infection and elicits interferon production through the cGAS-cGAMP-STING axis. cGAS is activated by dsDNA from viral and bacterial origins as well as dsDNA leaked from damaged mitochondria and nucleus. Eventually, cGAS activation launches the cell into an antiviral state to restrict the replication of both DNA and RNA viruses. Throughout the long co-evolution, viruses devise many strategies to evade cGAS detection or suppress cGAS activation. We recently reported that the Dengue virus protease NS2B3 proteolytically cleaves human cGAS in its N-terminal region, effectively reducing cGAS binding to DNA and consequent production of the second messenger cGAMP. Several other RNA viruses likely adopt the cleavage strategy. Here, we describe a protocol for the purification of recombinant human cGAS and Dengue NS2B3 protease, as well as the in vitro cleavage assay.

Regulation viral RNA transcription and replication by higher-order RNA structures within the nsp1 coding region of MERS coronavirus

Coronavirus (CoV) possesses numerous functional cis-acting elements in its positive-strand genomic RNA. Although most of these RNA structures participate in viral replication, the functions of RNA structures in the genomic RNA of CoV in viral replication remain unclear. In this study, we investigated the functions of the higher-order RNA stem-loop (SL) structures SL5B, SL5C, and SL5D in the ORF1a coding region of Middle East respiratory syndrome coronavirus (MERS-CoV) in viral replication. Our approach, using reverse genetics of a bacterial artificial chromosome system, revealed that SL5B and SL5C play essential roles in the discontinuous transcription of MERS-CoV. In silico analyses predicted that SL5C interacts with a bulged stem-loop (BSL) in the 3′ untranslated region, suggesting that the RNA structure of SL5C is important for viral RNA transcription. Conversely, SL5D did not affect transcription, but mediated the synthesis of positive-strand genomic RNA. Additionally, the RNA secondary structure of SL5 in the revertant virus of the SL5D mutant was similar to that of the wild-type, indicating that the RNA structure of SL5D can finely tune RNA replication in MERS-CoV. Our data indicate novel regulatory mechanisms of viral RNA transcription and replication by higher-order RNA structures in the MERS-CoV genomic RNA.

Antiviral Activity of Selective Estrogen Receptor Modulators against Severe Fever with Thrombocytopenia Syndrome Virus In Vitro and In Vivo.

Severe fever with thrombocytopenia syndrome virus (SFTSV), also known as the Dabie Banda virus, is an emerging tick-borne Bunyavirus that causes severe fever with thrombocytopenia syndrome (SFTS). Currently, symptomatic treatment and antiviral therapy with ribavirin and favipiravir are used in clinical management. However, their therapeutical efficacy is hardly satisfactory in patients with high viral load. In this study, we explored the antiviral effects of selective estrogen receptor modulators (SERMs) on SFTSV infection and the antiviral mechanisms of a representative SERM, bazedoxifene acetate (BZA). Our data show that SERMs potently inhibited SFTSV-induced cytopathic effect (CPE), the proliferation of infectious viral particles, and viral RNA replication and that BZA effectively protected mice from lethal viral challenge. The mode of action analysis reveals that BZA exerts antiviral effects during the post-entry stage of SFTSV infection. The transcriptome analysis reveals that GRASLND and CYP1A1 were upregulated, while TMEM45B and TXNIP were downregulated. Our findings suggest that SERMs have the potential to be used in the treatment of SFTSV infection.