ABSTRACT
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 (EC50) 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.
Subject(s)
Antiviral Agents , Classical Swine Fever Virus , Dihydroorotate Dehydrogenase , Viral Nonstructural Proteins , Virus Replication , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Cell Line , Classical Swine Fever/drug therapy , Classical Swine Fever/immunology , Classical Swine Fever/metabolism , Classical Swine Fever/virology , Classical Swine Fever Virus/drug effects , Classical Swine Fever Virus/growth & development , Classical Swine Fever Virus/immunology , Classical Swine Fever Virus/metabolism , Dihydroorotate Dehydrogenase/metabolism , Dose-Response Relationship, Drug , Immunity, Innate/drug effects , Immunity, Innate/immunology , Immunoprecipitation , Microscopy, Confocal , Mitochondria/enzymology , Mitochondria/metabolism , RNA, Viral/biosynthesis , Signal Transduction/drug effects , Swine/virology , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effectsABSTRACT
The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP53kD, NP47kD, and NP40kD. While both NP47kD and NP40kD have been previously shown to be products of caspase cleavage, here, we show that expression of the third isoform NP53kD 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, NP40kD plays the predominant role. In contrast to full-length NP (i.e., NP65kD), 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, NP53kD, NP47kD, and NP40kD 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 (NP47kD and NP40kD) are known to be produced by caspase cleavage, while, here, we show that NP53kD 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 NP40kD 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.
Subject(s)
Caspases , Cytoplasm , Hemorrhagic Fever, American , Host-Pathogen Interactions , Immunity, Innate , Junin virus , Nucleoproteins , Protein Biosynthesis , Humans , Apoptosis , Caspase Inhibitors/metabolism , Caspases/metabolism , Cytoplasm/metabolism , Cytoplasm/virology , Enzyme Activation , Hemorrhagic Fever, American/immunology , Hemorrhagic Fever, American/virology , Interferons/genetics , Interferons/immunology , Junin virus/genetics , Junin virus/metabolism , Junin virus/pathogenicity , Nucleoproteins/biosynthesis , Nucleoproteins/genetics , Nucleoproteins/metabolism , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , RNA, Viral/biosynthesis , RNA, Viral/genetics , Virus ReplicationABSTRACT
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 RNAs1. The RSV polymerase initiates RNA synthesis by binding to the conserved 3'-terminal RNA promoters of the genome or antigenome2. 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.
Subject(s)
Promoter Regions, Genetic , RNA-Dependent RNA Polymerase , Respiratory Syncytial Virus, Human , Promoter Regions, Genetic/genetics , Respiratory Syncytial Virus, Human/enzymology , Respiratory Syncytial Virus, Human/genetics , RNA, Viral/biosynthesis , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , RNA-Dependent RNA Polymerase/ultrastructure , Virus Replication/genetics , Cryoelectron Microscopy , Subgenomic RNA/biosynthesis , Subgenomic RNA/genetics , Subgenomic RNA/metabolismABSTRACT
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.
Subject(s)
Alphavirus , Gene Expression Regulation, Viral , Host Microbial Interactions , Replicon , Viral Proteins , Alphavirus/genetics , Alphavirus/metabolism , mRNA Vaccines/genetics , Replicon/genetics , Virus Replication/genetics , RNA, Viral/biosynthesis , RNA, Viral/genetics , Host Microbial Interactions/genetics , Viral Proteins/biosynthesis , Viral Proteins/geneticsABSTRACT
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 worldwide1. 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.
Subject(s)
Ebolavirus , RNA, Viral , RNA-Dependent RNA Polymerase , Virus Replication , Animals , Humans , Antiviral Agents/pharmacology , Ebolavirus/enzymology , Ebolavirus/genetics , Ebolavirus/growth & development , Hemorrhagic Fever, Ebola/virology , RNA, Viral/biosynthesis , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/metabolism , Genome, Viral , Nucleic Acid Conformation , Mutagenesis , RNA StabilityABSTRACT
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 remdesivir2. 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 triphosphate3,4. 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 propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.
Subject(s)
Coronavirus RNA-Dependent RNA Polymerase , Cryoelectron Microscopy , SARS-CoV-2 , Humans , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Coronavirus RNA-Dependent RNA Polymerase/ultrastructure , COVID-19/virology , Nucleosides/metabolism , Nucleosides/pharmacology , RNA, Viral/biosynthesis , RNA, Viral/chemistry , RNA, Viral/metabolism , SARS-CoV-2/enzymology , Substrate Specificity , Guanosine Triphosphate/metabolism , RNA CapsABSTRACT
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.
Subject(s)
Alphainfluenzavirus , Antiviral Agents , Betainfluenzavirus , Biological Products , Enzyme Inhibitors , Methyltransferases , RNA Caps , Tubercidin , Virus Replication , Animals , Humans , Mice , RNA Caps/metabolism , RNA, Messenger/metabolism , RNA, Viral/biosynthesis , Virus Replication/drug effects , Alphainfluenzavirus/drug effects , Betainfluenzavirus/drug effects , Biological Products/chemistry , Biological Products/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Tubercidin/analogs & derivatives , Tubercidin/pharmacology , Methyltransferases/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Streptomyces/chemistry , Computer Simulation , A549 CellsABSTRACT
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 disease1,2, 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 synthesis3. 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.
Subject(s)
Cryoelectron Microscopy , DNA-Directed RNA Polymerases , Ebolavirus , Viral Regulatory and Accessory Proteins , Antiviral Agents/pharmacology , Catalytic Domain , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/metabolism , DNA-Directed RNA Polymerases/ultrastructure , Ebolavirus/enzymology , Hemorrhagic Fever, Ebola/drug therapy , Hemorrhagic Fever, Ebola/virology , Humans , Protein Subunits/chemistry , Protein Subunits/metabolism , RNA, Viral/biosynthesis , Suramin/chemistry , Suramin/metabolism , Suramin/pharmacology , Suramin/therapeutic use , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism , Viral Regulatory and Accessory Proteins/ultrastructure , Virus ReplicationABSTRACT
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 3Cpro-interacting protein. 3Cpro 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 3Cpro-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, 3Cpro abolished DHX30 antiviral effects by inducing DHX30 cleavage. Thus, DHX30 is an intrinsic antiviral factor that inhibits SVV replication.
Subject(s)
3C Viral Proteases , Picornaviridae , Proteolysis , RNA Helicases , 3C Viral Proteases/metabolism , Animals , Chromatography, Liquid , Immunoprecipitation , Picornaviridae/enzymology , Picornaviridae/genetics , Picornaviridae/growth & development , Picornaviridae/physiology , Protein Binding , RNA Helicases/antagonists & inhibitors , RNA Helicases/metabolism , RNA, Double-Stranded/biosynthesis , RNA, Viral/biosynthesis , Swine/virology , Swine Vesicular Disease/virology , Tandem Mass Spectrometry , Virus ReplicationABSTRACT
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.
Subject(s)
Exoribonucleases , Genetic Fitness , Murine hepatitis virus , Proteolysis , RNA, Viral , Viral Nonstructural Proteins , Viral Replicase Complex Proteins , Animals , Exoribonucleases/genetics , Exoribonucleases/metabolism , Mice , Murine hepatitis virus/enzymology , Murine hepatitis virus/genetics , Murine hepatitis virus/growth & development , Murine hepatitis virus/physiology , Mutation , Polyproteins/chemistry , Polyproteins/genetics , Polyproteins/metabolism , RNA, Viral/biosynthesis , RNA, Viral/genetics , Recombination, Genetic , Transcription, Genetic , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Replicase Complex Proteins/chemistry , Viral Replicase Complex Proteins/genetics , Viral Replicase Complex Proteins/metabolism , Virus ReplicationABSTRACT
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.
Subject(s)
Amino Acid Substitution , Conserved Sequence , DNA, Circular , Hepatitis B Core Antigens , Hepatitis B virus , Hepatitis B , Lysine , Viral Core Proteins , Amino Acid Sequence , Conserved Sequence/genetics , DNA, Circular/biosynthesis , DNA, Circular/genetics , DNA, Circular/metabolism , DNA, Viral/genetics , DNA, Viral/metabolism , Hepatitis B/virology , Hepatitis B Core Antigens/chemistry , Hepatitis B Core Antigens/genetics , Hepatitis B Core Antigens/metabolism , Hepatitis B virus/chemistry , Hepatitis B virus/genetics , Hepatitis B virus/growth & development , Hepatitis B virus/metabolism , Humans , Lysine/genetics , Lysine/metabolism , Mutation , Nucleocapsid/metabolism , Polyadenylation/genetics , RNA, Viral/biosynthesis , RNA, Viral/genetics , Viral Core Proteins/chemistry , Viral Core Proteins/genetics , Viral Core Proteins/metabolism , Virion/growth & development , Virus Replication/geneticsABSTRACT
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 (IC50 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.
Subject(s)
Chromosomal Proteins, Non-Histone , DNA-Directed DNA Polymerase , Hepatitis A virus , Hepatitis A , Intrinsically Disordered Proteins , RNA Nucleotidyltransferases , RNA, Viral , Virus Replication , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Chromosomal Proteins, Non-Histone/metabolism , DNA-Directed DNA Polymerase/metabolism , Hepatitis A/drug therapy , Hepatitis A/metabolism , Hepatitis A/virology , Hepatitis A virus/drug effects , Hepatitis A virus/genetics , Hepatitis A virus/physiology , Humans , Intrinsically Disordered Proteins/metabolism , Mice , Mice, Mutant Strains , RNA Nucleotidyltransferases/metabolism , RNA, Viral/biosynthesis , RNA, Viral/genetics , Receptor, Interferon alpha-beta/genetics , Virus Replication/drug effectsABSTRACT
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).
Subject(s)
Antiviral Agents/pharmacology , COVID-19 Drug Treatment , Nucleic Acid Synthesis Inhibitors/pharmacology , SARS-CoV-2/drug effects , Tubercidin/analogs & derivatives , Cell Line , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/pharmacology , Drug Evaluation, Preclinical/methods , HEK293 Cells , Humans , Microbial Sensitivity Tests , RNA, Viral/biosynthesis , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/genetics , Thionucleosides/pharmacology , Tubercidin/pharmacology , Vidarabine/analogs & derivatives , Vidarabine/pharmacology , Vidarabine Phosphate/analogs & derivatives , Vidarabine Phosphate/pharmacologySubject(s)
COVID-19/pathology , Lung/pathology , SARS-CoV-2/pathogenicity , Turbinates/physiology , Animals , COVID-19/immunology , COVID-19/virology , Cricetulus , Female , Host Specificity , Humans , Lung/virology , Mice , Mice, Inbred BALB C , RNA, Viral/biosynthesis , SARS-CoV-2/growth & development , Severity of Illness Index , Turbinates/pathology , Turbinates/virology , Viral LoadABSTRACT
[Figure: see text].
Subject(s)
Antiviral Agents/pharmacology , Cytidine/analogs & derivatives , Hydroxylamines/pharmacology , Mutagenesis , RNA Viruses/drug effects , SARS-CoV-2/drug effects , Animals , Antiviral Agents/adverse effects , Antiviral Agents/therapeutic use , COVID-19/virology , Cytidine/adverse effects , Cytidine/metabolism , Cytidine/pharmacology , Cytidine/therapeutic use , Cytidine/toxicity , DNA/biosynthesis , Evolution, Molecular , Genome, Viral , Humans , Hydroxylamines/adverse effects , Hydroxylamines/metabolism , Hydroxylamines/therapeutic use , Mutagenicity Tests , Phosphorylation , RNA Virus Infections/drug therapy , RNA Virus Infections/virology , RNA Viruses/genetics , RNA, Viral/biosynthesis , RNA, Viral/genetics , Ribonucleosides/metabolism , SARS-CoV-2/genetics , COVID-19 Drug TreatmentABSTRACT
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.
Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , Exoribonucleases/metabolism , Genome, Viral/genetics , Genomic Instability , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Exoribonucleases/antagonists & inhibitors , Genome, Viral/drug effects , Genomic Instability/drug effects , Genomic Instability/genetics , HIV Integrase Inhibitors/pharmacology , Isoindoles/pharmacology , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/metabolism , Organoselenium Compounds/pharmacology , RNA, Viral/biosynthesis , RNA, Viral/genetics , Raltegravir Potassium/pharmacology , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Virus Replication/drug effects , Virus Replication/geneticsABSTRACT
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.
Subject(s)
Genome, Viral , Hepatitis Delta Virus/physiology , RNA, Viral/biosynthesis , Virus Replication , Cell Line , Guanosine , Hepatitis Delta Virus/genetics , Humans , RNA Caps , RNA Polymerase II/metabolism , RNA, Circular/biosynthesis , RNA, Circular/chemistry , RNA, Viral/chemistry , Templates, Genetic , TransfectionABSTRACT
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 ß (IFN-ß). The promotion of viral replication by HEV-miR-A6 further identified in vivo. Significant suppression of IFN-ß 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-ß expression.
Subject(s)
Gene Expression Regulation, Viral , Hepatitis E virus/physiology , Hepatitis E/metabolism , Interferon-beta/metabolism , MicroRNAs/biosynthesis , RNA, Viral/biosynthesis , Virus Replication , Female , Humans , Male , Organ SpecificityABSTRACT
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.