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Coronaviruses remodel the intracellular host membranes during replication, forming double-membrane vesicles (DMVs) to accommodate viral RNA synthesis and modifications 1,2 . SARS-CoV-2 non-structural protein 3 (nsp3) and nsp4 are the minimal viral components required to induce DMV formation and to form a double-membrane-spanning pore, essential for the transport of newly synthesized viral RNAs 3-5 . The mechanism of DMV pore complex formation remains unknown. Here we describe the molecular architecture of the SARS-CoV-2 nsp3-nsp4 pore complex, as resolved by cryogenic electron tomography and subtomogram averaging in isolated DMVs. The structures uncover an unexpected stoichiometry and topology of the nsp3-nsp4 pore complex comprising 12 copies each of nsp3 and nsp4, organized in 4 concentric stacking hexamer rings, mimicking a miniature nuclear pore complex. The transmembrane domains are interdigitated to create a high local curvature at the double-membrane junction, coupling double-membrane reorganization with pore formation. The ectodomains form extensive contacts in a pseudo-12-fold symmetry, belting the pore complex from the intermembrane space. A central positively charged ring of arginine residues coordinates the putative RNA translocation, essential for virus replication. Our work establishes a framework for understanding DMV pore formation and RNA translocation, providing a structural basis for the development of new antiviral strategies to combat coronavirus infection., (© 2024. The Author(s).)

Mitochondria are energy producers in cells, which can affect viral replication by regulating the host innate immune signaling pathways, and the changes in their biological functions are inextricably linked the viral life cycle. In this study, we screened a library of 382 mitochondria-targeted compounds and identified the antiviral inhibitors of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo synthesis pathway of pyrimidine ribonucleotides, against classical swine fever virus (CSFV). Our data showed that the inhibitors interfered with viral RNA synthesis in a dose-dependent manner, with half-maximal effective concentrations (EC 50 ) ranging from 0.975 to 26.635 nM. Remarkably, DHODH inhibitors obstructed CSFV replication by enhancing the innate immune response including the TBK1-IRF3-STAT1 and NF-κB signaling pathways. Furthermore, the data from a series of compound addition and supplementation trials indicated that DHODH inhibitors also inhibited CSFV replication by blocking the de novo pyrimidine synthesis. Remarkably, DHODH knockdown demonstrated that it was essential for CSFV replication. Mechanistically, confocal microscopy and immunoprecipitation assays showed that the non-structural protein 4A (NS4A) recruited and interacted with DHODH in the perinuclear. Notably, NS4A enhanced the DHODH activity and promoted the generation of UMP for efficient viral replication. Structurally, the amino acids 65-229 of DHODH and the amino acids 25-40 of NS4A were pivotal for this interaction. Taken together, our findings highlight the critical role of DHODH in the CSFV life cycle and offer a potential antiviral target for the development of novel therapeutics against CSF., Importance: Classical swine fever remains one of the most economically important viral diseases of domestic pigs and wild boar worldwide. dihydroorotate dehydrogenase (DHODH) inhibitors have been shown to suppress the replication of several viruses in vitro and in vivo , but the effects on Pestivirus remain unknown. In this study, three specific DHODH inhibitors, including DHODH-IN-16, BAY-2402234, and Brequinar were found to strongly suppress classical swine fever virus (CSFV) replication. These inhibitors target the host DHODH, depleting the pyrimidine nucleotide pool to exert their antiviral effects. Intriguingly, we observed that the non-structural protein 4A of CSFV induced DHODH to accumulate around the nucleus in conjunction with mitochondria. Moreover, NS4A exhibited a strong interaction with DHODH, enhancing its activity to promote efficient CSFV replication. In conclusion, our findings enhance the understanding of the pyrimidine synthesis in CSFV infection and expand the novel functions of CSFV NS4A in viral replication, providing a reference for further exploration of antiviral targets against CSFV., Competing Interests: The authors declare no conflict of interest.

The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP 53kD , NP 47kD , and NP 40kD . While both NP 47kD and NP 40kD have been previously shown to be products of caspase cleavage, here, we show that expression of the third isoform NP 53kD is due to alternative in-frame translation from M80. Based on this information, we were able to generate recombinant JUNVs lacking each of these isoforms. Infection with these mutants revealed that, while all three isoforms contribute to the efficient control of caspase activation, NP 40kD plays the predominant role. In contrast to full-length NP (i.e., NP 65kD ), which is localized to inclusion bodies, where viral RNA synthesis takes place, the loss of portions of the N-terminal coiled-coil region in these isoforms leads to a diffuse cytoplasmic distribution and a loss of function in viral RNA synthesis. Nonetheless, NP 53kD , NP 47kD , and NP 40kD all retain robust interferon antagonistic and 3'-5' exonuclease activities. We suggest that the altered localization of these NP isoforms allows them to be more efficiently targeted by activated caspases for cleavage as decoy substrates, and to be better positioned to degrade viral double-stranded (ds)RNA species that accumulate in the cytoplasm during virus infection and/or interact with cytosolic RNA sensors, thereby limiting dsRNA-mediated innate immune responses. Taken together, this work provides insight into the mechanism by which JUNV leverages apoptosis during infection to generate biologically distinct pools of NP and contributes to our understanding of the expression and biological relevance of alternative protein isoforms during virus infection.IMPORTANCEA limited coding capacity means that RNA viruses need strategies to diversify their proteome. The nucleoprotein (NP) of the highly pathogenic arenavirus Junín virus (JUNV) produces three N-terminally truncated isoforms: two (NP 47kD and NP 40kD ) are known to be produced by caspase cleavage, while, here, we show that NP 53kD is produced by alternative translation initiation. Recombinant JUNVs lacking individual NP isoforms revealed that all three isoforms contribute to inhibiting caspase activation during infection, but cleavage to generate NP 40kD makes the biggest contribution. Importantly, all three isoforms retain their ability to digest double-stranded (ds)RNA and inhibit interferon promoter activation but have a diffuse cytoplasmic distribution. Given the cytoplasmic localization of both aberrant viral dsRNAs, as well as dsRNA sensors and many other cellular components of innate immune activation pathways, we suggest that the generation of NP isoforms not only contributes to evasion of apoptosis but also robust control of the antiviral response., Competing Interests: The authors declare no conflict of interest.

The respiratory syncytial virus (RSV) polymerase is a multifunctional RNA-dependent RNA polymerase composed of the large (L) protein and the phosphoprotein (P). It transcribes the RNA genome into ten viral mRNAs and replicates full-length viral genomic and antigenomic RNAs 1 . The RSV polymerase initiates RNA synthesis by binding to the conserved 3'-terminal RNA promoters of the genome or antigenome 2 . However, the lack of a structure of the RSV polymerase bound to the RNA promoter has impeded the mechanistic understanding of RSV RNA synthesis. Here we report cryogenic electron microscopy structures of the RSV polymerase bound to its genomic and antigenomic viral RNA promoters, representing two of the first structures of an RNA-dependent RNA polymerase in complex with its RNA promoters in non-segmented negative-sense RNA viruses. The overall structures of the promoter-bound RSV polymerases are similar to that of the unbound (apo) polymerase. Our structures illustrate the interactions between the RSV polymerase and the RNA promoters and provide the structural basis for the initiation of RNA synthesis at positions 1 and 3 of the RSV promoters. These structures offer a deeper understanding of the pre-initiation state of the RSV polymerase and could aid in antiviral research against RSV., (© 2023. The Author(s).)

Importance: Alphavirus replicons are being developed as self-amplifying RNAs aimed at improving the efficacy of mRNA vaccines. These replicons are convenient for genetic manipulations and can express heterologous genetic information more efficiently and for a longer time than standard mRNAs. However, replicons mimic many aspects of viral replication in terms of induction of innate immune response, modification of cellular transcription and translation, and expression of nonstructural viral genes. Moreover, all replicons used in this study demonstrated expression of heterologous genes in cell- and replicon's origin-specific modes. Thus, many aspects of the interactions between replicons and the host remain insufficiently investigated, and further studies are needed to understand the biology of the replicons and their applicability for designing a new generation of mRNA vaccines. On the other hand, our data show that replicons are very flexible expression systems, and additional modifications may have strong positive impacts on protein expression., Competing Interests: The authors declare no conflict of interest.

Non-segmented negative-strand RNA viruses, including Ebola virus (EBOV), rabies virus, human respiratory syncytial virus and pneumoviruses, can cause respiratory infections, haemorrhagic fever and encephalitis in humans and animals, and are considered a substantial health and economic burden worldwide 1 . Replication and transcription of the viral genome are executed by the large (L) polymerase, which is a promising target for the development of antiviral drugs. Here, using the L polymerase of EBOV as a representative, we show that de novo replication of L polymerase is controlled by the specific 3' leader sequence of the EBOV genome in an enzymatic assay, and that formation of at least three base pairs can effectively drive the elongation process of RNA synthesis independent of the specific RNA sequence. We present the high-resolution structures of the EBOV L-VP35-RNA complex and show that the 3' leader RNA binds in the template entry channel with a distinctive stable bend conformation. Using mutagenesis assays, we confirm that the bend conformation of the RNA is required for the de novo replication activity and reveal the key residues of the L protein that stabilize the RNA conformation. These findings provide a new mechanistic understanding of RNA synthesis for polymerases of non-segmented negative-strand RNA viruses, and reveal important targets for the development of antiviral drugs., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Orthomyxo- and bunyaviruses steal the 5' cap portion of host RNAs to prime their own transcription in a process called "cap snatching." We report that RNA modification of the cap portion by host 2'-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces , called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S -adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC) 1 . Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir 2 . Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate 3,4 . Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation 5 . The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap 6 ., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Respiratory syncytial virus (RSV) is a significant threat to infants and elderly individuals globally. Currently, there are no effective therapies or treatments for RSV infection because of an insufficient understanding of the RSV viral machinery. In this study, we investigated the effects of the template variations on RNA synthesis by the RSV polymerase through in vitro RNA synthesis assays. We confirmed the previously reported back-priming activity of the RSV polymerase, which is likely due to the secondary structure of the RNA template. We found that the expansion of the hairpin loop size of the RNA template abolishes the RSV polymerase back-priming activity. At the same time, it seemingly does not affect the de novo RNA synthesis activities of the RSV polymerase. Interestingly, our results show that the RSV polymerase also has a new primer-based terminal extension activity that adds nucleotides to the template and primer in a nonspecific manner. We also mapped the impact of the RNA 5' chemical group on its mobility in a urea-denaturing RNA gel shift assay. Overall, these results enhance our knowledge about the RNA synthesis processes of the RSV polymerase and may guide future therapeutic efforts to develop effective antiviral drugs for RSV treatment.

Filoviruses, including Ebola virus, pose an increasing threat to the public health. Although two therapeutic monoclonal antibodies have been approved to treat the Ebola virus disease 1,2 , there are no approved broadly reactive drugs to control diverse filovirus infection. Filovirus has a large polymerase (L) protein and the cofactor viral protein 35 (VP35), which constitute the basic functional unit responsible for virus genome RNA synthesis 3 . Owing to its conservation, the L-VP35 polymerase complex is a promising target for broadly reactive antiviral drugs. Here we determined the structure of Ebola virus L protein in complex with tetrameric VP35 using cryo-electron microscopy (state 1). Structural analysis revealed that Ebola virus L possesses a filovirus-specific insertion element that is essential for RNA synthesis, and that VP35 interacts extensively with the N-terminal region of L by three protomers of the VP35 tetramer. Notably, we captured the complex structure in a second conformation with the unambiguous priming loop and supporting helix away from polymerase active site (state 2). Moreover, we demonstrated that the century-old drug suramin could inhibit the activity of the Ebola virus polymerase in an enzymatic assay. The structure of the L-VP35-suramin complex reveals that suramin can bind at the highly conserved NTP entry channel to prevent substrates from entering the active site. These findings reveal the mechanism of Ebola virus replication and may guide the development of more powerful anti-filovirus drugs., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Seneca Valley virus (SVV) is a new pathogen associated with porcine idiopathic vesicular disease (PIVD) in recent years. However, SVV-host interaction is still unclear. In this study, through LC-MS/MS analysis and coimmunoprecipitation analysis, DHX30 was identified as a 3C pro -interacting protein. 3C pro mediated the cleavage of DHX30 at a specific site, which depends on its protease activity. Further study showed that DHX30 was an intrinsic antiviral factor against SVV that was dependent on its helicase activity. DHX30 functioned as a viral-RNA binding protein that inhibited SVV replication at the early stage of viral infection. RIP-seq showed comparatively higher coverage depth at SVV 5'UTR, but the distribution across SVV RNA suggested that the interaction had low specificity. DHX30 expression strongly inhibited double-stranded RNA (dsRNA) production. Interestingly, DHX30 was determined to interact with 3D in an SVV RNA-dependent manner. Thus, DHX30 negatively regulated SVV propagation by blocking viral RNA synthesis, presumably by participating in the viral replication complex. IMPORTANCE DHX30, an RNA helicase, is identified as a 3C pro -interacting protein regulating Seneca Valley virus (SVV) replication dependent on its helicase activity. DHX30 functioned as a viral-RNA binding protein that inhibited SVV replication at the early stage of virus infection. DHX30 expression strongly inhibited double-stranded RNA (dsRNA) production. In addition, 3C pro abolished DHX30 antiviral effects by inducing DHX30 cleavage. Thus, DHX30 is an intrinsic antiviral factor that inhibits SVV replication.

Coronaviruses (CoVs) initiate replication by translation of the positive-sense RNA genome into the replicase polyproteins connecting 16 nonstructural protein domains (nsp1-16), which are subsequently processed by viral proteases to yield mature nsp. For the betacoronavirus murine hepatitis virus (MHV), total inhibition of translation or proteolytic processing of replicase polyproteins results in rapid cessation of RNA synthesis. The nsp5-3CLpro (Mpro) processes nsps7-16, which assemble into functional replication-transcription complexes (RTCs), including the enzymatic nsp12-RdRp and nsp14-exoribonuclease (ExoN)/N7-methyltransferase. The nsp14-ExoN activity mediates RNA-dependent RNA proofreading, high-fidelity RNA synthesis, and replication. To date, the solved partial RTC structures, biochemistry, and models use or assume completely processed, mature nsp. Here, we demonstrate that in MHV, engineered deletion of the cleavage sites between nsp13-14 and nsp14-15 allowed recovery of replication-competent virus. Compared to wild-type (WT) MHV, the nsp13-14 and nsp14-15 cleavage deletion mutants demonstrated delayed replication kinetics, impaired genome production, altered abundance and patterns of recombination, and impaired competitive fitness. Further, the nsp13-14 and nsp14-15 mutant viruses demonstrated mutation frequencies that were significantly higher than with the WT. The results demonstrate that cleavage of nsp13-14 or nsp14-15 is not required for MHV viability and that functions of the RTC/nsp14-ExoN are impaired when assembled with noncleaved intermediates. These data will inform future genetic, structural, biochemical, and modeling studies of coronavirus RTCs and nsp 13, 14, and 15 and may reveal new approaches for inhibition or attenuation of CoV infection. IMPORTANCE Coronavirus replication requires proteolytic maturation of the nonstructural replicase proteins to form the replication-transcription complex. Coronavirus replication-transcription complex models assume mature subunits; however, mechanisms of coronavirus maturation and replicase complex formation have yet to be defined. Here, we show that for the coronavirus murine hepatitis virus, cleavage between the nonstructural replicase proteins nsp13-14 and nsp14-15 is not required for replication but does alter RNA synthesis and recombination. These results shed new light on the requirements for coronavirus maturation and replication-transcription complex assembly, and they may reveal novel therapeutic targets and strategies for attenuation.

Hepatitis B virus (HBV) core protein (HBc), the building block of the viral capsid, plays a critical role throughout the HBV life cycle. There are two highly conserved lysine residues, namely, K7 and K96, on HBc, which have been proposed to function at various stages of viral replication, potentially through lysine-specific posttranslational modifications (PTMs). Here, we substituted K7 and K96 with alanine or arginine, which would also block potential PTMs on these two lysine residues, and tested the effects of these substitutions on HBV replication and infection. We found that the two lysine residues were dispensable for all intracellular steps of HBV replication. In particular, all mutants were competent to form the covalently closed circular DNA (cccDNA) via the intracellular amplification pathway, indicating that K7 and K96, or any PTMs of these residues, were not essential for nucleocapsid uncoating, a prerequisite for cccDNA formation. Furthermore, we found that K7A and K7R mutations did not affect de novo cccDNA formation and RNA transcription during infection, indicating that K7 or any PTMs of this residue were dispensable for HBV infection. In addition, we demonstrated that the HBc K7 coding sequence (AAA), as part of the HBV polyadenylation signal UAUAAA, was indispensable for viral RNA production, implicating this cis requirement at the RNA level, instead of any function of HBc-K7, likely constrains the identity of the 7th residue of HBc. In conclusion, our results provided novel insights regarding the roles of lysine residues on HBc, and their coding sequences, in the HBV life cycle. IMPORTANCE Hepatitis B virus (HBV) infection remains a public health burden that affects 296 million individuals worldwide. HBV core protein (HBc) is involved in almost all steps in the HBV life cycle. There are two conserved lysine residues on HBc. Here, we found that neither of them is essential for HBV intracellular replication, including the formation of covalently closed circular DNA (cccDNA), the molecular basis for establishing and sustaining the HBV infection. However, K96 is critical for virion morphogenesis, while the K7 coding sequence, but not HBc-K7 itself, is indispensable, as part of the RNA polyadenylation signal, for HBV RNA production from cccDNA. Our results provide novel insights regarding the role of the conserved lysine residues on HBc, and their coding sequences, in viral replication, and should facilitate the development of antiviral drugs against the HBV capsid protein.

Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5' untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3' poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3' poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC 50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1 -/- mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.

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.

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 negative-sense 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 5' 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 5' end of the genome RNA. The 5' 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 5' 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.

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

[Figure: see text].

Coronavirus disease 2019 (COVID-19) is a newly emerged infectious disease caused by a novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid global emergence of SARS-CoV-2 highlights the importance and urgency for potential drugs to control the pandemic. The functional importance of RNA-dependent RNA polymerase (RdRp) in the viral life cycle, combined with structural conservation and absence of closely related homologs in humans, makes it an attractive target for designing antiviral drugs. Nucleos(t)ide analogs (NAs) are still the most promising broad-spectrum class of viral RdRp inhibitors. In this study, using our previously developed cell-based SARS-CoV-2 RdRp report system, we screened 134 compounds in the Selleckchemicals NAs library. Four candidate compounds, Fludarabine Phosphate, Fludarabine, 6-Thio-20-Deoxyguanosine (6-Thio-dG), and 5-Iodotubercidin, exhibit remarkable potency in inhibiting SARS-CoV-2 RdRp. Among these four compounds, 5-Iodotubercidin exhibited the strongest inhibition upon SARS-CoV-2 RdRp, and was resistant to viral exoribonuclease activity, thus presenting the best antiviral activity against coronavirus from a different genus. Further study showed that the RdRp inhibitory activity of 5-Iodotubercidin is closely related to its capacity to inhibit adenosine kinase (ADK)., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The coronavirus nucleocapsid (N) protein comprises two RNA-binding domains connected by a central spacer, which contains a serine- and arginine-rich (SR) region. The SR region engages the largest subunit of the viral replicase-transcriptase, nonstructural protein 3 (nsp3), in an interaction that is essential for efficient initiation of infection by genomic RNA. We carried out an extensive genetic analysis of the SR region of the N protein of mouse hepatitis virus in order to more precisely define its role in RNA synthesis. We further examined the N-nsp3 interaction through construction of nsp3 mutants and by creation of an interspecies N protein chimera. Our results indicate a role for the central spacer as an interaction hub of the N molecule that is partially regulated by phosphorylation. These findings are discussed in relation to the recent discovery that nsp3 forms a molecular pore in the double-membrane vesicles that sequester the coronavirus replicase-transcriptase., (Copyright © 2021 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs), the non-coding RNAs of ~22 nucleotides (nt) in length, play a vital role in regulating viral replication. Hepatitis E virus (HEV), a single-stranded RNA virus, is a predominant pathogen of acute hepatitis worldwide. Virus-encoded miRNAs regulate the viral life cycle and escape from the host innate immune system. However, it is rarely known about HEV-encoded miRNA (HEV-miR-A6). In the present study, HEV-miR-A6 was screened by microarray, and further identified in vivo and in vitro. HEV-miR-A6 originated from the methylase (MeT) of HEV open reading frame 1 (ORF1) and was highly conserved in eight HEV genotypes. HEV-miR-A6 expression was growing during HEV replication, and significantly increased in acute hepatitis E patients than convalescence patients. Furthermore, HEV-miR-A6 was specifically detected in liver, spleen, kidney and colon by in situ hybridization. To identify the specificity of HEV-miR-A6, its mutants (HEV-miR-A6M1 and HEV-miR-A6M2) were constructed to change the stem-loop structure. Interestingly, over-expression of HEV-miR-A6 or HEV-miR-A6M1 significantly facilitated viral replication, while HEV-miR-A6M2, another mutant completely changed the stem-loop structure was invalid. SIRP-α, a candidate target gene of HEV-miR-A6, was activated when HEV-miR-A6 over-expressed to inhibit the phosphorylation of IRF3, and subsequently suppressed the expression of type I interferon β (IFN-β). The promotion of viral replication by HEV-miR-A6 further identified in vivo. Significant suppression of IFN-β production in the serum of HEV-infected mice pre-treated with HEV-miR-A6 was observed. In summary, HEV-miR-A6 activates SIRP-α to promote viral replication by inhibition of IFN-β expression., (© 2021 Federation of American Societies for Experimental Biology.)

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them.

SARS-CoV-2 is a positive-sense RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors involved in RNA proofreading and 5' capping of viral RNAs. The formation of the 5' 7-methylguanosine (m7G) cap structure is known to require a guanylyltransferase (GTase) as well as a 5' triphosphatase and methyltransferases; however, the mechanism of SARS-CoV-2 RNA capping remains poorly understood. Here we find that SARS-CoV-2 nsp12 is involved in viral RNA capping as a GTase, carrying out the addition of a GTP nucleotide to the 5' end of viral RNA via a 5' to 5' triphosphate linkage. We further show that the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase) domain performs this reaction, and can be inhibited by remdesivir triphosphate, the active form of the antiviral drug remdesivir. These findings improve understanding of coronavirus RNA synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: There is an urgent need for new antivirals with powerful therapeutic potential and tolerable side effects., Methods: Here, we tested the antiviral properties of interferons (IFNs), alone and with other drugs in vitro., Results: While IFNs alone were insufficient to completely abolish replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), IFNα, in combination with remdesivir, EIDD-2801, camostat, cycloheximide, or convalescent serum, proved to be more effective. Transcriptome and metabolomic analyses revealed that the IFNα-remdesivir combination suppressed SARS-CoV-2-mediated changes in Calu-3 cells and lung organoids, although it altered the homeostasis of uninfected cells and organoids. We also demonstrated that IFNα combinations with sofosbuvir, telaprevir, NITD008, ribavirin, pimodivir, or lamivudine were effective against HCV, HEV, FLuAV, or HIV at lower concentrations, compared to monotherapies., Conclusions: Altogether, our results indicated that IFNα can be combined with drugs that affect viral RNA transcription, protein synthesis, and processing to make synergistic combinations that can be attractive targets for further pre-clinical and clinical development against emerging and re-emerging viral infections.

Lassa virus is endemic in West Africa and can cause severe hemorrhagic fever. The viral L protein transcribes and replicates the RNA genome via its RNA-dependent RNA polymerase activity. Here, we present nine cryo-EM structures of the L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved 3' and 5' promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and early elongation states, the endonuclease is inhibited by two distinct L protein peptides, whereas in the pre-initiation state it is uninhibited. In the early elongation state, a template-product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide and the full C-terminal region of the L protein, including the putative cap-binding domain, is well-ordered. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated., (© 2021. The Author(s).)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of Coronavirus Disease 2019 (COVID-19) pandemic. Despite intensive and global efforts to discover and develop novel antiviral therapies, only Remdesivir has been approved as a treatment for COVID-19. Therefore, effective antiviral therapeutics are still urgently needed to combat and halt the pandemic. Viral RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 demonstrates high potential as a reliable target for the development of antivirals. We previously developed a cell-based assay to assess the efficiency of compounds that target SARS-CoV-2 RdRp, as well as their tolerance to viral exoribonuclease-mediated proof-reading. In our previous study, we discovered that 2-((1H-indol-3-yl)thio)-N-phenyl-acetamides specifically targets the RdRp of both respiratory syncytial virus (RSV) and influenza A virus. Thus, we hypothesize that 2-((1H-indol-3-yl)thio)-N-phenyl-acetamides may also have the ability to inhibit SARS-CoV-2 replication by targeting its RdRp activity. In this research, we test a compound library containing 103 of 2-((1H-indol-3-yl)thio)-N-phenyl-acetamides against SARS-CoV-2 RdRp, using our cell-based assay. Among these compounds, the top five candidates strongly inhibit SARS-CoV-2 RdRp activity while exhibiting low cytotoxicity and resistance to viral exoribonuclease. Compound 6-72-2a is the most promising candidate with the lowest EC 50 value of 1.41 μM and highest selectivity index (CC 50 /EC 50 ) (above 70.92). Furthermore, our data suggests that 4-46b and 6-72-2a also inhibit the replication of HCoV-OC43 and HCoV-NL63 virus in a dose-dependent manner. Compounds 4-46b and 6-72-2a exhibit EC 50 values of 1.13 μM and 0.94 μM, respectively, on HCoV-OC43 viral replication. However, higher concentrations of these compounds are needed to effectively block HCoV-NL63 replication. Together, our findings successfully identified 4-46b and 6-72-2a as promising inhibitors against SARS-CoV-2 RdRp., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Positive-strand RNA viruses replicate in close association with rearranged intracellular membranes. For hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), these rearrangements comprise endoplasmic reticulum (ER)-derived double membrane vesicles (DMVs) serving as RNA replication sites. Cellular factors involved in DMV biogenesis are poorly defined. Here, we show that despite structural similarity of viral DMVs with autophagosomes, conventional macroautophagy is dispensable for HCV and SARS-CoV-2 replication. However, both viruses exploit factors involved in autophagosome formation, most notably class III phosphatidylinositol 3-kinase (PI3K). As revealed with a biosensor, PI3K is activated in cells infected with either virus to produce phosphatidylinositol 3-phosphate (PI3P) while kinase complex inhibition or depletion profoundly reduces replication and viral DMV formation. The PI3P-binding protein DFCP1, recruited to omegasomes in early steps of autophagosome formation, participates in replication and DMV formation of both viruses. These results indicate that phylogenetically unrelated HCV and SARS-CoV-2 exploit similar components of the autophagy machinery to create their replication organelles., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The large (L) polymerase proteins of most nonsegmented, negative-stranded (NNS) RNA viruses have conserved methyltransferase motifs, (G)-G-G-D and K-D-K-E, which are important for the stabilization and translation of mRNA. However, the function of the (G)-G-G-D and K-D-K-E motifs in the NNS RNA virus Newcastle disease virus (NDV) remains unclear. We observed G-G-D and K-D-K-E motifs in all NDV genotypes. By using the infection cloning system of NDV rSG10 strain, recombinant NDVs with a single amino acid mutated to alanine in one motif (G-G-D or K-D-K-E) were rescued. The intracerebral pathogenicity index and mean death time assay results revealed that the G-G-D motif and K-D-K-E motif attenuate the virulence of NDV to various degrees. The replication, transcription, and translation levels of the K-D-K-E motif-mutant strains were significantly higher than those of wild-type virus owing to their altered regulation of the affinity between nucleocapsid protein and eukaryotic translation initiation factor 4E. When the infection dose was changed from a multiplicity of infection (MOI) of 10 to an MOI of 0.01, the cell-to-cell spread abilities of G-G-D- and K-D-K-E-mutant strains were reduced, according to plaque assay and dynamic indirect immunofluorescence assay results. Finally, we found that NDV strains with G-G-D or K-D-K-E motif mutations had less pathogenicity in 3-week-old specific-pathogen-free chickens than wild-type NDV. Therefore, these methyltransferase motifs can affect virulence by regulating the translation and cell-to-cell spread abilities of NDV. This work provides a feasible approach for generating vaccine candidates for viruses with methyltransferase motifs. IMPORTANCE Newcastle disease virus (NDV) is an important pathogen that is widespread globally. Research on its pathogenic mechanism is an important means of improving prevention and control efforts. Our study found that a deficiency in its methyltransferase motifs (G-G-D and K-D-K-E motifs) can attenuate NDV and revealed the molecular mechanism by which these motifs affect pathogenicity, which provides a new direction for the development of NDV vaccines. In addition to the (G)-G-G-D and K-D-K-E motifs of many nonsegmented, negative-stranded RNA viruses, similar motifs have been found in dengue virus, Zika virus, Japanese encephalitis virus (JEV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This suggests that such motifs may be present in more viruses. Our finding also provides a molecular basis for the discovery and functional study of (G)-G-G-D and K-D-K-E motifs of other viruses.

Human coronavirus (HCoV) similar to other viruses rely on host cell machinery for both replication and to spread. The p97/VCP ATPase is associated with diverse pathways that may favor HCoV replication. In this study, we assessed the role of p97 and associated host responses in human lung cell line H1299 after HCoV-229E or HCoV-OC43 infection. Inhibition of p97 function by small molecule inhibitors shows antiviral activity, particularly at early stages of the virus life cycle, during virus uncoating and viral RNA replication. Importantly, p97 activity inhibition protects human cells against HCoV-induced cytopathic effects. The p97 knockdown also inhibits viral production in infected cells. Unbiased quantitative proteomics analyses reveal that HCoV-OC43 infection resulted in proteome changes enriched in cellular senescence and DNA repair during virus replication. Further analysis of protein changes between infected cells with control and p97 shRNA identifies cell cycle pathways for both HCoV-229E and HCoV-OC43 infection. Together, our data indicate a role for the essential host protein p97 in supporting HCoV replication, suggesting that p97 is a therapeutic target to treat HCoV infection.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiologic agent that causes Coronavirus Disease 2019 (COVID-19) pandemic, is a newly emerging respiratory RNA virus with exceptional transmissibility and pathogenicity. Numerous COVID-19 related studies have been fast-tracked, with the ultimate goal to end the pandemic. Here we review the major stages of SARS-CoV-2 infection cycle in cells, with specific emphasis on essential host factors. Insights into the cell biology of SARS-CoV-2 infection have accelerated the development of host-directed therapeutics, as shown by dozens of clinical trials evaluating COVID-19 treatments using host-targeting compounds., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The norovirus genome consists of a single positive-stranded RNA. The mechanism by which this single-stranded RNA genome is replicated is not well understood. To reveal the mechanism underlying the initiation of the norovirus genomic RNA synthesis by its RNA-dependent RNA polymerase (RdRp), we used an in vitro assay to detect the complementary RNA synthesis activity. Results showed that the purified recombinant RdRp was able to synthesize the complementary positive-sense RNA from a 100-nt template corresponding to the 3'-end of the viral antisense genome sequence, but that the RdRp could not synthesize the antisense genomic RNA from the template corresponding to the 5'-end of the positive-sense genome sequence. We also predicted that the 31 nt region at the 3'-end of the RNA antisense template forms a stem-loop structure. Deletion of this sequence resulted in the loss of complementary RNA synthesis by the RdRp, and connection of the 31 nt to the 3'-end of the inactive positive-sense RNA template resulted in the gain of complementary RNA synthesis by the RdRp. Similarly, an electrophoretic mobility shift assay further revealed that the RdRp bound to the antisense RNA specifically, but was dependent on the 31 nt at the 3'-end. Therefore, based on this observation and further deletion and mutation analyses, we concluded that the predicted stem-loop structure in the 31 nt end and the region close to the antisense viral genomic stem sequences are both important for initiating the positive-sense human norovirus genomic RNA synthesis by its RdRp., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Several viruses have been proven to inhibit the formation of RNA processing bodies (P-bodies); however, knowledge regarding whether enterovirus blocks P-body formation remains unclear, and the detailed molecular mechanisms and functions of picornavirus regulation of P-bodies are limited. Here, we show the crucial role of 2A protease in inhibiting P-bodies to promote viral replication during enterovirus 71 infection. Moreover, we found that the activity of 2A protease is essential to inhibit P-body formation, which was proven by the result that infection with EV71-2A C110S , a 2A protease activity-inactivated recombinant virus, failed to block the formation of P-bodies. Furthermore, we show that DDX6, a scaffolding protein of P-bodies, interacted with viral RNA to facilitate viral replication rather than viral translation, by using a Renilla luciferase mRNA reporter system and nascent RNA capture assay. Altogether, our data first demonstrate that the 2A protease of enterovirus inhibits P-body formation to facilitate viral RNA synthesis by recruiting the P-body components to viral RNA. IMPORTANCE Processing bodies (P-bodies) are constitutively present in eukaryotic cells and play an important role in the mRNA cycle, including regulation of gene expression and mRNA degradation. The P-body is the structure that viruses manipulate to facilitate their survival. Here, we show that the 2A protease alone was efficient to block P-body formation during enterovirus 71 infection, and its activity is essential. When the assembly of P-bodies was blocked by 2A protease, DDX6 and 4E-T, which were required for P-body formation, bound to viral RNA to facilitate viral RNA synthesis. We propose a model revealing that EV71 manipulates P-body formation to generate an environment that is conducive to viral replication by facilitating viral RNA synthesis: 2A protease blocked P-body assembly to make it possible for virus to take advantage of P-body components.

Based on WHO reports the new SARS-CoV-2 coronavirus is currently widespread all over the world. So far > 162 million cases have been confirmed, including > 3 million deaths. Because of the pandemic still spreading across the globe the accomplishment of computational methods to find new potential mechanisms of virus inhibitions is necessary. According to the fact that C 60 fullerene (a sphere-shaped molecule consisting of carbon) has shown inhibitory activity against various protein targets, here the analysis of the potential binding mechanism between SARS-CoV-2 proteins 3CLpro and RdRp with C 60 fullerene was done; it has resulted in one and two possible binding mechanisms, respectively. In the case of 3CLpro, C 60 fullerene interacts in the catalytic binding pocket. And for RdRp in the first model C 60 fullerene blocks RNA synthesis pore and in the second one it prevents binding with Nsp8 co-factor (without this complex formation, RdRp can't perform its initial functions). Then the molecular dynamics simulation confirmed the stability of created complexes. The obtained results might be a basis for other computational studies of 3CLPro and RdRp potential inhibition ways as well as the potential usage of C 60 fullerene in the fight against COVID-19 disease., (© 2021. The Author(s).)

Coronavirus 3′-to-5′ exoribonuclease (ExoN), residing in the nonstructural protein (nsp) 10–nsp14 complex, boosts replication fidelity by proofreading RNA synthesis and is critical for the virus life cycle. ExoN also recognizes and excises nucleotide analog inhibitors incorporated into the nascent RNA, undermining the effectiveness of nucleotide analog–based antivirals. Here we present cryo–electron microscopy structures of both wild-type and mutant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp10-nsp14 in complex with an RNA substrate bearing a 3′-end mismatch at resolutions ranging from 2.5 to 3.9 angstroms. The structures reveal the molecular determinants of ExoN substrate specificity and offer insight into the molecular mechanisms of mismatch correction during coronavirus RNA synthesis. Our findings provide guidance for rational design of improved anticoronavirus therapies.

Although understanding the diversity of HIV-1 reservoirs is key to achieving a cure, their study at the single-cell level in primary samples remains challenging. We combine flow cytometric multiplexed fluorescent in situ RNA hybridization for different viral genes with HIV-1 p24 protein detection, cell phenotyping, and downstream near-full-length single-cell vDNA sequencing. Stimulation-induced viral RNA-positive (vRNA + ) cells from viremic and antiretroviral-therapy (ART)-suppressed individuals differ in their ability to produce p24. In participants on ART, latency-reversing agents (LRAs) induce a wide variety of viral gene transcription and translation patterns with LRA class-specific differences in reactivation potency. Reactivated proviruses, including in p24 + cells, are mostly defective. Although LRAs efficiently induce transcription in all memory cell subsets, we observe induction of translation mostly in effector memory cells, rather than in the long-lived central memory pool. We identify HIV-1 clones with diverse transcriptional and translational patterns between individual cells, and this finding suggests that cell-intrinsic factors influence reservoir persistence and heterogeneity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We use a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide evidence that the RNA-dependent RNA polymerase (RdRp) uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enable the direct observation of coronavirus polymerase deep and long-lived backtracking that is strongly stimulated by secondary structures in the template. The framework we present here elucidates one of the most important structure-dynamics-function relationships in human health today and will form the grounds for understanding the regulation of this complex., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The nucleotide analogue prodrug remdesivir remains the only FDA-approved antiviral small molecule for the treatment of infection with SARS-CoV-2. Biochemical studies revealed that the active form of the drug targets the viral RNA-dependent RNA polymerase and causes delayed chain-termination. Delayed chain-termination is incomplete, but the continuation of RNA synthesis enables a partial escape from viral proofreading. Remdesivir becomes embedded in the copy of the RNA genome that later serves as a template. Incorporation of an incoming nucleotide triphosphate is now inhibited by the modified template. Knowledge on the mechanism of action matters. Enzymatic inhibition links to antiviral effects in cell cultures, animal models and viral load reduction in patients, which provides the logical chain that is expected for a direct acting antiviral. Hence, remdesivir also serves as a benchmark in current drug development efforts that will hopefully lead to orally available treatments to the benefit of a broader population., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

Snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus isolated from diseased hybrid snakehead fish, has caused great economic losses in snakehead fish culture in China. The large (L) protein, together with its cofactor phosphoprotein (P), forms a P/L polymerase complex and catalyzes the transcription and replication of viral genomic RNA. In this study, the cellular heat shock protein 90 (Hsp90) was identified as an interacting partner of SHVV L protein. Hsp90 activity was required for the stability of SHVV L because Hsp90 dysfunction caused by using its inhibitor destabilized SHVV L and thereby suppressed SHVV replication via reducing viral RNA synthesis. SHVV L expressed alone was detected mainly in the insoluble fraction, and the insoluble L was degraded by Hsp90 dysfunction through the proteasomal pathway, while the presence of SHVV P promoted the solubility of SHVV L and the soluble L was degraded by Hsp90 dysfunction through the autophagy pathway. Collectively, our data suggest that Hsp90 contributes to the maturation of SHVV L and ensures the effective replication of SHVV, which exhibits an important anti-SHVV target. This study will help us to understand the role of Hsp90 in stabilizing the L protein and regulating the replication of negative-stranded RNA viruses. IMPORTANCE It has long been proposed that cellular proteins are involved in viral RNA synthesis via interacting with the viral polymerase protein. This study focused on identifying cellular proteins interacting with the SHVV L protein, studying the effects of their interactions on SHVV replication, and revealing the underlying mechanisms. We identified Hsp90 as an interacting partner of SHVV L and found that Hsp90 activity was required for SHVV replication. Hsp90 functioned in maintaining the stability of SHVV L. Inhibition of Hsp90 activity with its inhibitor degraded SHVV L through different pathways based on the solubility of SHVV L due to the presence or absence of SHVV P. Our data provide important insights into the role of Hsp90 in SHVV polymerase maturation, which will help us to understand the polymerase function of negative-stranded RNA viruses.

Cyclin-dependent kinase 9 (CDK9) is a key regulator of RNA-polymerase II and a candidate therapeutic target for various virus infections such as respiratory syncytial virus, herpes simplex virus, human adenovirus, human cytomegalovirus, hepatitis virus B, and human papillomavirus. We employed CDK9-IN-1, a selective CDK9 inhibitor, to investigate the role of CDK9 in porcine reproductive and respiratory syndrome virus (PRRSV) infection. CDK9-IN-1 dose-dependently reduced PRRSV replication without cytotoxicity in the infected cells. The antiviral activity of CDK9-IN-1 was further confirmed by evaluating the effects of lentivirus-mediated CDK9 knockdown or CDK9 overexpression on PRRSV infection. Briefly, the depletion of CDK9 significantly inhibited viral replication, while the overexpression of CDK9 promoted viral replication. PRRSV infection also enhanced the nuclear export of CDK9 without affecting CDK9 protein expression. Viral replication cycle analyses further revealed that functionally active CDK9 in the cytosol advanced viral subgenomic RNA synthesis. Collectively, our data illustrated that CDK9 was a new host factor that was involved in PRRSV subgenomic RNA synthesis, and CDK9 inhibitor, CDK9-IN-1 was a promising antiviral candidate for PRRSV infection., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Frog Virus 3 (FV3) is a large dsDNA virus belonging to Ranaviruses of family Iridoviridae . Ranaviruses infect cold-blood vertebrates including amphibians, fish and reptiles, and contribute to catastrophic amphibian declines. FV3 has a genome at ~105 kb that contains nearly 100 coding genes and 50 intergenic regions as annotated in its reference genome. Previous studies have mainly focused on coding genes and rarely addressed potential non-coding regulatory role of intergenic regions., Results: Using a whole transcriptomic analysis of total RNA samples containing both the viral and cellular transcripts from FV3-infected frog tissues, we detected virus-specific reads mapping in non-coding intergenic regions, in addition to reads from coding genes. Further analyses identified multiple cis -regulatory elements ( CREs ) in intergenic regions neighboring highly transcribed coding genes. These CREs include not only a virus TATA-Box present in FV3 core promoters as in eukaryotic genes, but also viral mimics of CREs interacting with several transcription factors including CEBPs, CREBs, IRFs, NF-κB, and STATs, which are critical for regulation of cellular immunity and cytokine responses. Our study suggests that intergenic regions immediately upstream of highly expressed FV3 genes have evolved to bind IRFs, NF-κB, and STATs more efficiently. Moreover, we found an enrichment of putative microRNA (miRNA) sequences in more than five intergenic regions of the FV3 genome. Our sequence analysis indicates that a fraction of these viral miRNAs is targeting the 3'-UTR regions of Xenopus genes involved in interferon (IFN)-dependent responses, including particularly those encoding IFN receptor subunits and IFN-regulatory factors (IRFs)., Conclusions: Using the FV3 model, this study provides a first genome-wide analysis of non-coding regulatory mechanisms adopted by ranaviruses to epigenetically regulate both viral and host gene expressions, which have co-evolved to interact especially with the host IFN response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tian, Khwatenge, Li, De Jesus Andino, Robert and Sang.)

Coronavirus disease 2019 (COVID-19) is a fatal respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The identification of potential drugs is urgently needed to control the pandemic. RNA dependent RNA polymerase (RdRp) is a conserved protein within RNA viruses and plays a crucial role in the viral life cycle, thus making it an attractive target for development of antiviral drugs. In this study, 101 quinoline and quinazoline derivatives were screened against SARS-CoV-2 RdRp using a cell-based assay. Three compounds I-13e , I-13h , and I-13i exhibit remarkable potency in inhibiting RNA synthesis driven by SARS-CoV-2 RdRp and relatively low cytotoxicity. Among these three compounds, I-13e showed the strongest inhibition upon RNA synthesis driven by SARS-CoV-2 RdRp, the resistance to viral exoribonuclease activity and the inhibitory effect on the replication of CoV, thus holding potential of being drug candidate for treatment of SARS-CoV-2.

Dengue virus (DENV) is spread from human to human through the bite of the female Aedes aegypti mosquito and leads to about 100 million clinical infections yearly. Treatment options and vaccine availability for DENV are limited. Defective interfering particles (DIPs) are considered a promising antiviral approach but infectious virus contamination has limited their development. Here, a DENV-derived DIP production cell line was developed that continuously produced DENV-free DIPs. The DIPs contained and could deliver to cells a DENV serotype 2 subgenomic defective-interfering RNA, which was originally discovered in DENV infected patients. The DIPs released into cell culture supernatant were purified and could potently inhibit replication of all DENV serotypes in cells. Antiviral therapeutics are limited for many viral infection. The DIP system described could be re-purposed to make antiviral DIPs for many other RNA viruses such as SARS-CoV-2, yellow fever, West Nile and Zika viruses.

Infection with influenza A and B viruses results in a mild to severe respiratory tract infection. It is widely accepted that many factors affect the severity of influenza disease, including viral replication, host adaptation, innate immune signalling, pre-existing immunity, and secondary infections. In this review, we will focus on the interplay between influenza virus RNA synthesis and the detection of influenza virus RNA by our innate immune system. Specifically, we will discuss the generation of various RNA species, host pathogen receptors, and host shut-off. In addition, we will also address outstanding questions that currently limit our knowledge of influenza virus replication and host adaption. Understanding the molecular mechanisms underlying these factors is essential for assessing the pandemic potential of future influenza virus outbreaks.

Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5'-viral genome-linked protein (VPg) and have a 3' polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3' untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5' regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5' region and 3' UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5' region and 3' UTR of RNA2 are required for efficient cap-independent translation of these transcripts. We identify translation-enhancing elements in the 5' proximal coding region of the RNA2 polyprotein and in the RNA2 3' UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3' UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3' UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5' region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773-972) of the 1547 nt long 3' UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion virus, another nepovirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two viruses., Competing Interests: The authors have declared that no competing interests exist.

Positive-strand RNA virus genome replication takes place on intracellular membranes that separate the reduced cytosol from the oxidized extracellular/luminal milieu. Ongoing studies of these membrane-bounded genome replication complexes have revealed underlying common principles in their structure, assembly and functionalization, including transmembrane features and redox dependencies. Among these, members of the alphavirus, flavivirus, and picornavirus supergroups all encode membrane-permeabilizing viroporins required for efficient RNA replication. For flaviviruses and particularly alphavirus supergroup members, these viroporins are linked to activating viral RNA capping and potentially other later-stage RNA replication functions, and to local transmembrane release of oxidizing potential to trigger these changes in cytoplasmic RNA replication complexes. Further exploration of these emerging shared principles could spur development of broad-spectrum antivirals., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Baloxavir marboxil has been used for influenza treatment since March 2018 in Japan. After baloxavir treatment, the most frequently detected substitution is Ile38Thr in polymerase acidic protein (PA/I38T), and this substitution reduces baloxavir susceptibility in influenza A viruses. To rapidly investigate the frequency of PA/I38T in influenza A (H1N1)pdm09 and A (H3N2) viruses in clinical samples, we established a rapid real-time system to detect single nucleotide polymorphisms in PA, using cycling probe real-time PCR. We designed two sets of probes that were labeled with either 6-carboxyfluorescein (FAM) or 6-carboxy-X-rhodamine (ROX) to identify PA/I38 (wild type strain) or PA/I38T, respectively. The established cycling probe real-time PCR system showed a dynamic linear range of 10 1 to 10 6 copies with high sensitivity in plasmid DNA controls. This real-time PCR system discriminated between PA/I38T and wild type viruses well. During the 2018/19 season, 377 influenza A-positive clinical samples were collected in Japan before antiviral treatment. Using our cycling probe real-time PCR system, we detected no (0/129, 0.0%) influenza A (H1N1)pdm09 viruses with PA/I38T substitutions and four A (H3N2) (4/229, 1.7%) with PA/I38T substitution prior to treatment. In addition, we found PA/I38T variant in siblings who did not received baloxavir treatment during an infection caused by A (H3N2) that afflicted the entire family. Although human-to-human transmission of PA/I38T variant may have occurred in a closed environment, the prevalence of this variant in influenza A viruses was still limited. Our cycling probe-PCR system is thus useful for antiviral surveillance of influenza A viruses possessing PA/I38T., (Copyright © 2021 Elsevier B.V. All rights reserved.)

SARS-CoV-2 is thought to have originated in the human population from a zoonotic spillover event. Infection in humans results in a variety of outcomes ranging from asymptomatic cases to the disease COVID-19, which can have significant morbidity and mortality, with over two million confirmed deaths worldwide as of January 2021. Over a year into the pandemic, sequencing analysis has shown that variants of SARS-CoV-2 are being selected as the virus continues to circulate widely within the human population. The predominant drivers of genetic variation within SARS-CoV-2 are single nucleotide polymorphisms (SNPs) caused by polymerase error, potential host factor driven RNA modification, and insertion/deletions (indels) resulting from the discontinuous nature of viral RNA synthesis. While many mutations represent neutral 'genetic drift' or have quickly died out, a subset may be affecting viral traits such as transmissibility, pathogenicity, host range, and antigenicity of the virus. In this review, we summarise the current extent of genetic change in SARS-CoV-2, particularly recently emerging variants of concern, and consider the phenotypic consequences of this viral evolution that may impact the future trajectory of the pandemic.