PARP12 is required to repress the replication of a Mac1 mutant coronavirus in a cell- and tissue-specific manner.

ADP-ribosyltransferases (ARTs) mediate the transfer of ADP-ribose from NAD+ to protein or nucleic acid substrates. This modification can be removed by several different types of proteins, including macrodomains. Several ARTs, also known as PARPs, are stimulated by interferon indicating ADP-ribosylation is an important aspect of the innate immune response. All coronaviruses (CoVs) encode for a highly conserved macrodomain (Mac1) that is critical for CoVs to replicate and cause disease, indicating that ADP-ribosylation can effectively control coronavirus infection. Our siRNA screen indicated that PARP12 might inhibit the replication of a murine hepatitis virus (MHV) Mac1 mutant virus in bone-marrow-derived macrophages (BMDMs). To conclusively demonstrate that PARP12 is a key mediator of the antiviral response to CoVs both in cell culture and in vivo, we produced PARP12-/-mice and tested the ability of MHV A59 (hepatotropic/neurotropic) and JHM (neurotropic) Mac1 mutant viruses to replicate and cause disease in these mice. Notably, in the absence of PARP12, Mac1 mutant replication was increased in BMDMs and mice. In addition, liver pathology was also increased in A59-infected mice. However, the PARP12 knockout did not restore Mac1 mutant virus replication to WT virus levels in all cell or tissue types and did not significantly increase the lethality of Mac1 mutant viruses. These results demonstrate that while PARP12 inhibits MHV Mac1 mutant virus infection, additional PARPs or innate immune factors must contribute to the extreme attenuation of this virus in mice. IMPORTANCE Over the last decade, the importance of ADP-ribosyltransferases (ARTs), also known as PARPs, in the antiviral response has gained increased significance as several were shown to either restrict virus replication or impact innate immune responses. However, there are few studies showing ART-mediated inhibition of virus replication or pathogenesis in animal models. We found that the CoV macrodomain (Mac1) was required to prevent ART-mediated inhibition of virus replication in cell culture. Using knockout mice, we found that PARP12, an interferon-stimulated ART, was required to repress the replication of a Mac1 mutant CoV both in cell culture and in mice, demonstrating that PARP12 represses coronavirus replication. However, the deletion of PARP12 did not fully rescue Mac1 mutant virus replication or pathogenesis, indicating that multiple PARPs function to counter coronavirus infection.

Identification of a 1.4-kb-Long Sequence Located in the nsp12 and nsp13 Coding Regions of SARS-CoV-2 Genomic RNA That Mediates Efficient Viral RNA Packaging.

The specific packaging of the viral RNA genome into virus particles is an essential step in the replication cycle of coronaviruses (CoVs). Using a single-cycle, replicable severe acute respiratory syndrome CoV-2 (SARS-CoV-2) mutant, we demonstrated the preferential packaging of the SARS-CoV-2 genomic RNA into purified virus particles. Furthermore, based on the sequence of an efficiently packaged defective interfering RNA of SARS-CoV, a closely related CoV, that was generated after serial passages of SARS-CoV in cell culture, we designed a series of replication-competent SARS-CoV-2 minigenome RNAs to identify the specific viral RNA region that is important for SARS-CoV-2 RNA packaging into virus particles. We showed that a 1.4-kb-long sequence, derived from the nsp12 and nsp13 coding regions of the SARS-CoV-2 genomic RNA, is required for the efficient packaging of SARS-CoV-2 minigenome RNA into SARS-CoV-2 particles. In addition, we also showed that the presence of possibly the entire 1.4-kb-long sequence is important for the efficient packaging of SARS-CoV-2 RNA. Our findings highlight the differences between the RNA packaging sequence identified in SARS-CoV-2, a Sarbecovirus, and the packaging signal of mouse hepatitis virus (MHV), an Embecovirus, which is a 95-nt-long sequence located at the nsp15 coding region of MHV genomic RNA. Collectively, our data imply that both the location and the sequence/structural features of the RNA element(s) that drives the selective and efficient packaging of viral genomic RNA are not conserved among the subgenera Embecovirus and Sarbecovirus within the Betacoronavirus genus. IMPORTANCE Elucidating the mechanism of SARS-CoV-2 RNA packaging into virus particles is important for the rational design of antiviral drugs that inhibit this vital step in the replication cycle of CoVs. However, our knowledge about the RNA packaging mechanism in SARS-CoV-2, including the identification of the viral RNA region important for SARS-CoV-2 RNA packaging, is limited, primarily due to the logistical challenges of handing SARS-CoV-2 in biosafety level 3 (BSL3) facilities. Our study, using a single-cycle, replicable SARS-CoV-2 mutant, which can be handled in a BSL2 lab, demonstrated the preferential packaging of full-length SARS-CoV-2 genomic RNA into virus particles and identified a specific 1.4-kb-long RNA region in SARS-CoV-2 genomic RNA that is required for the efficient packaging of SARS-CoV-2 RNA into virus particles. The information generated in our study could be valuable for clarifying the mechanisms of SARS-CoV-2 RNA packaging and for the development of targeted therapeutics against SARS-CoV-2 and other related CoVs.

Obesity fosters severe disease outcomes in a mouse model of coronavirus infection associated with transcriptomic abnormalities.

Obesity has been identified as an independent risk factor for severe outcomes in humans with coronavirus disease 2019 (COVID-19) and other infectious diseases. Here, we established a mouse model of COVID-19 using the murine betacoronavirus, mouse hepatitis virus 1 (MHV-1). C57BL/6 and C3H/HeJ mice exposed to MHV-1 developed mild and severe disease, respectively. Obese C57BL/6 mice developed clinical manifestations similar to those of lean controls. In contrast, all obese C3H/HeJ mice succumbed by 8 days postinfection, compared to a 50% mortality rate in lean controls. Notably, both lean and obese C3H/HeJ mice exposed to MHV-1 developed lung lesions consistent with severe human COVID-19, with marked evidence of diffuse alveolar damage (DAD). To identify early predictive biomarkers of worsened disease outcomes in obese C3H/HeJ mice, we sequenced RNA from whole blood 2 days postinfection and assessed changes in gene and pathway expression. Many pathways uniquely altered in obese C3H/HeJ mice postinfection aligned with those found in humans with severe COVID-19. Furthermore, we observed altered gene expression related to the unfolded protein response and lipid metabolism in infected obese mice compared to their lean counterparts, suggesting a role in the severity of disease outcomes. This study presents a novel model for studying COVID-19 and elucidating the mechanisms underlying severe disease outcomes in obese and other hosts.

Development of two novel on-site detection visualization methods for murine hepatitis virus based on the multienzyme isothermal rapid amplification.

Murine hepatitis virus (MHV) infection is one of the most prevalent types of mice infection in laboratory. MHV could cause death in mice and even interfere with the results in animal experiments. Herein, we developed two isothermal approaches based on the Multienzyme Isothermal Rapid Amplification (MIRA), for rapid detection of MHV in conserved M gene. We designed and screened several pairs of primers and probes and the isothermal fluorescence detector was applied for the exonuclease Ⅲ reverse transcription MIRA (exo-RT-MIRA) assay. To further simplify the workflow, the portable fluorescence visualization instrument, also as a palm-sized handheld system, was used for the naked-eye exo-RT-MIRA assay. The amplification temperature and time were optimized. The assay could be processed well at 42 °C 20 min for the exo-RT-MIRA and the naked-eye exo-RT-MIRA assay. The limit of detection (LoD) of the exo-RT-MIRA assay was 43.4 copies/µL. The LoD of the naked-eye exo-RT-MIRA assay was 68.2 copies/µL. No nonspecific amplifications were observed in the two assays. A total of 107 specimens were examined by qPCR and two assays developed. The experimental results statistical analysis demonstrated that the exo-RT-MIRA assay with the qPCR yielded sufficient agreement with a kappa value of 1.000 (p < 0.0001). The results also exhibited a good agreement (kappa value, 0.961) (p < 0.0001) between the naked-eye exo-RT-MIRA assay and the qPCR assay. In our study, the exo-RT-MIRA assay and the naked-eye exo-RT-MIRA assay presented the possibility of new methods in MHV point-of-testing diagnosis.

Proteolytic Processing of the Coronavirus Replicase Nonstructural Protein 14 Exonuclease Is Not Required for Virus Replication but Alters RNA Synthesis and Viral Fitness.

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.

Targeting the conserved coronavirus octamer motif GGAAGAGC is a strategy for the development of coronavirus vaccine.

BACKGROUND: Coronaviruses are pathogens of humans and animals that cause widespread and costly diseases. The development of effective strategies to combat the threat of coronaviruses is therefore a top priority. The conserved coronavirus octamer motif 5'GGAAGAGC3' exists in the 3' untranslated region of all identified coronaviruses. In the current study, we aimed to examine whether targeting the coronavirus octamer motif GGAAGAGC is a promising approach to develop coronavirus vaccine. METHODS: Plaque assays were used to determine the titers of mouse hepatitis virus (MHV)-A59 octamer mutant (MHVoctm) and wild-type (wt) MHV-A59 (MHVwt). Western blotting was used for the determination of translation efficiency of MHVoctm and MHVwt. Plaque assays and RT-qPCR were employed to examine whether MHVoctm was more sensitive to interferon treatment than MHVwt. Weight loss, clinical signs, survival rate, viral RNA detection and histopathological examination were used to evaluate whether MHVoctm was a vaccine candidate against MHVwt infection in BALB/c mice. RESULTS: In this study, we showed that (i) the MHVoctm with mutation of coronavirus octamer was able to grow to high titers but attenuated in mice, (ii) with the reduced multiplicity of infection (MOI), the difference in gene expression between MHVoctm and MHVwt became more evident in cultured cells, (iii) MHVoctm was more sensitive to interferon treatment than MHVwt and (iv) mice inoculated with MHVoctm were protected from MHVwt infection. CONCLUSIONS: Based on the results obtained from cultured cells, it was suggested that the synergistic effects of octamer mutation, multiplicity of infection and immune response may be a mechanism explaining the distinct phenotypes of octamer-mutated coronavirus in cell culture and mice. In addition, targeting the conserved coronavirus octamer motif is a strategy for development of coronavirus vaccine. Since the conserved octamer exists in all coronaviruses, this strategy of targeting the conserved octamer motif can also be applied to other human and animal coronaviruses for the development of coronavirus vaccines, especially the emergence of novel coronaviruses such as SARS-CoV-2, saving time and cost for vaccine development and disease control.

Biological characterization of coronavirus noncanonical transcripts in vitro and in vivo.

BACKGROUND: In addition to the well-known coronavirus genomes and subgenomic mRNAs, the existence of other coronavirus RNA species, which are collectively referred to as noncanonical transcripts, has been suggested; however, their biological characteristics have not yet been experimentally validated in vitro and in vivo. METHODS: To comprehensively determine the amounts, species and structures of noncanonical transcripts for bovine coronavirus in HRT-18 cells and mouse hepatitis virus A59, a mouse coronavirus, in mouse L cells and mice, nanopore direct RNA sequencing was employed. To experimentally validate the synthesis of noncanonical transcripts under regular infection, Northern blotting was performed. Both Northern blotting and nanopore direct RNA sequencing were also applied to examine the reproducibility of noncanonical transcripts. In addition, Northern blotting was also employed to determine the regulatory features of noncanonical transcripts under different infection conditions, including different cells, multiplicities of infection (MOIs) and coronavirus strains. RESULTS: In the current study, we (i) experimentally determined that coronavirus noncanonical transcripts were abundantly synthesized, (ii) classified the noncanonical transcripts into seven populations based on their structures and potential synthesis mechanisms, (iii) showed that the species and amounts of the noncanonical transcripts were reproducible during regular infection but regulated in altered infection environments, (iv) revealed that coronaviruses may employ various mechanisms to synthesize noncanonical transcripts, and (v) found that the biological characteristics of coronavirus noncanonical transcripts were similar between in vitro and in vivo conditions. CONCLUSIONS: The biological characteristics of noncanonical coronavirus transcripts were experimentally validated for the first time. The identified features of noncanonical transcripts in terms of abundance, reproducibility and variety extend the current model for coronavirus gene expression. The capability of coronaviruses to regulate the species and amounts of noncanonical transcripts may contribute to the pathogenesis of coronaviruses during infection, posing potential challenges in disease control. Thus, the biology of noncanonical transcripts both in vitro and in vivo revealed here can provide a database for biological research, contributing to the development of antiviral strategies.

Unveiling the biology of defective viral genomes in vitro and in vivo: implications for gene expression and pathogenesis of coronavirus.

BACKGROUND: Defective viral genome (DVG) is a truncated version of the full-length virus genome identified in most RNA viruses during infection. The synthesis of DVGs in coronavirus has been suggested; however, the fundamental characteristics of coronavirus DVGs in gene expression and pathogenesis have not been systematically analyzed. METHODS: Nanopore direct RNA sequencing was used to investigate the characteristics of coronavirus DVGs in gene expression including reproducibility, abundance, species and genome structures for bovine coronavirus in cells, and for mouse hepatitis virus (MHV)-A59 (a mouse coronavirus) in cells and in mice. The MHV-A59 full-length genomic cDNAs (~ 31 kilobases) were in vitro constructed to experimentally validate the origin of coronavirus DVG. The synthesis of DVGs was also experimentally identified by RT-PCR followed by sequencing. In addition, the alterations of DVGs in amounts and species under different infection environments and selection pressures including the treatment of antiviral remdesivir and interferon were evaluated based on the banding patterns by RT-PCR. RESULTS: The results are as follows: (i) the structures of DVGs are with diversity, (ii) DVGs are overall synthesized with moderate (MHV-A59 in cells) to high (BCoV in cells and MHV-A59 in mice) reproducibility under regular infection with the same virus inoculum, (iii) DVGs can be synthesized from the full-length coronavirus genome, (iv) the sequences flanking the recombination point of DVGs are AU-rich and thus may contribute to the recombination events during gene expression, (v) the species and amounts of DVG are altered under different infection environments, and (vi) the biological nature of DVGs between in vitro and in vivo is similar. CONCLUSIONS: The identified biological characteristics of coronavirus DVGs in terms of abundance, reproducibility, and variety extend the current model for coronavirus gene expression. In addition, the biological features of alterations in amounts and species of coronavirus DVGs under different infection environments may assist the coronavirus to adapt to the altered environments for virus fitness and may contribute to the coronavirus pathogenesis. Consequently, the unveiled biological features may assist the community to study the gene expression mechanisms of DVGs and their roles in pathogenesis, contributing to the development of antiviral strategy and public health.

Control of B Cell Lymphoma by Gammaherpesvirus-Induced Memory CD8 T Cells.

Persistent infection with gammaherpesviruses (γHV) can cause lymphomagenesis in immunocompromised patients. Murine γHV-68 (MHV-68) is an important tool for understanding immune factors contributing to γHV control; however, modeling control of γHV-associated lymphomagenesis has been challenging. Current model systems require very long incubation times or severe immune suppression, and tumor penetrance is low. In this report, we describe the generation of a B cell lymphoma on the C57BL/6 background, which is driven by the Myc oncogene and expresses an immunodominant CD8 T cell epitope from MHV-68. We determined MHV-68-specific CD8 T cells in latently infected mice use either IFN-γ or perforin/granzyme to control γHV-associated lymphoma, but perforin/granzyme is a more potent effector mechanism for lymphoma control than IFN-γ. Consistent with previous reports, CD4-depleted mice lost control of virus replication in persistently infected mice. However, control of lymphoma remained intact in the absence of CD4 T cells. Collectively, these data show the mechanisms of T cell control of B cell lymphoma in γHV-infected mice overlap with those necessary for control of virus replication, but there are also important differences. This study establishes a tool for further dissecting immune surveillance against, and optimizing adoptive T cell therapies for, γHV-associated lymphomas.

One proline deletion in the fusion peptide of neurotropic mouse hepatitis virus (MHV) restricts retrograde axonal transport and neurodegeneration.

Mouse hepatitis virus (MHV; murine coronavirus) causes meningoencephalitis, myelitis, and optic neuritis followed by axonal loss and demyelination. This murine virus is used as a common model to study acute and chronic virus-induced demyelination in the central nervous system. Studies with recombinant MHV strains that differ in the gene encoding the spike protein have demonstrated that the spike has a role in MHV pathogenesis and retrograde axonal transport. Fusion peptides (FPs) in the spike protein play a key role in MHV pathogenesis. In a previous study of the effect of deleting a single proline residue in the FP of a demyelinating MHV strain, we found that two central, consecutive prolines are important for cell-cell fusion and pathogenesis. The dihedral fluctuation of the FP was shown to be repressed whenever two consecutive prolines were present, in contrast to the presence of a single proline in the chain. Using this proline-deleted MHV strain, here we investigated whether intracranial injection of this strain can induce optic neuritis by retrograde axonal transport from the brain to the retina through the optic nerve. We observed that the proline-deleted recombinant MHV strain is restricted to the optic nerve, is unable to translocate to the retina, and causes only minimal demyelination and no neuronal death. We conclude that an intact proline dyad in the FP of the recombinant demyelinating MHV strain plays a crucial role in translocation of the virus through axons and subsequent neurodegeneration.

A proline insertion-deletion in the spike glycoprotein fusion peptide of mouse hepatitis virus strongly alters neuropathology.

Fusion peptides (FPs) in spike proteins are key players mediating early events in cell-to-cell fusion, vital for intercellular viral spread. A proline residue located at the central FP region has often been suggested to have a distinctive role in this fusion event. The spike glycoprotein from strain RSA59 (PP) of mouse hepatitis virus (MHV) contains two central, consecutive prolines in the FP. Here, we report that deletion of one of these proline residues, resulting in RSA59 (P), significantly affected neural cell syncytia formation and viral titers postinfection in vitro Transcranial inoculation of C57Bl/6 mice with RSA59 (PP) or RSA59 (P) yielded similar degrees of necrotizing hepatitis and meningitis, but only RSA59 (PP) produced widespread encephalitis that extended deeply into the brain parenchyma. By day 6 postinfection, both virus variants were mostly cleared from the brain. Interestingly, inoculation with the RSA59 (P)-carrying MHV significantly reduced demyelination at the chronic stage. We also found that the presence of two consecutive prolines in FP promotes a more ordered, compact, and rigid structure in the spike protein. These effects on FP structure were due to proline's unique stereochemical properties intrinsic to its secondary amino acid structure, revealed by molecular dynamics and NMR experiments. We therefore propose that the differences in the severity of encephalitis and demyelination between RSA59 (PP) and RSA59 (P) arise from the presence or absence, respectively, of the two consecutive prolines in FP. Our studies define a structural determinant of MHV entry in the brain parenchyma important for altered neuropathogenesis.

Analysis of Coronavirus Temperature-Sensitive Mutants Reveals an Interplay between the Macrodomain and Papain-Like Protease Impacting Replication and Pathogenesis.

Analysis of temperature-sensitive (ts) mutant viruses is a classic method allowing researchers to identify genetic loci involved in viral replication and pathogenesis. Here, we report genetic analysis of a ts strain of mouse hepatitis virus (MHV), tsNC11, focusing on the role of mutations in the macrodomain (MAC) and the papain-like protease 2 (PLP2) domain of nonstructural protein 3 (nsp3), a component of the viral replication complex. Using MHV reverse genetics, we generated a series of mutant viruses to define the contributions of macrodomain- and PLP2-specific mutations to the ts phenotype. Viral replication kinetics and efficiency-of-plating analysis performed at permissive and nonpermissive temperatures revealed that changes in the macrodomain alone were both necessary and sufficient for the ts phenotype. Interestingly, mutations in the PLP2 domain were not responsible for the temperature sensitivity but did reduce the frequency of reversion of macrodomain mutants. Coimmunoprecipitation studies are consistent with an interaction between the macrodomain and PLP2. Expression studies of the macrodomain-PLP2 portion of nsp3 indicate that the ts mutations enhance proteasome-mediated degradation of the protein. Furthermore, we found that during virus infection, the replicase proteins containing the MAC and PLP2 mutations were more rapidly degraded at the nonpermissive temperature than were the wild-type proteins. Importantly, we show that the macrodomain and PLP2 mutant viruses trigger production of type I interferon in vitro and are attenuated in mice, further highlighting the importance of the macrodomain-PLP2 interplay in viral pathogenesis.IMPORTANCE Coronaviruses (CoVs) are emerging human and veterinary pathogens with pandemic potential. Despite the established and predicted threat these viruses pose to human health, there are currently no approved countermeasures to control infections with these viruses in humans. Viral macrodomains, enzymes that remove posttranslational ADP-ribosylation of proteins, and viral multifunctional papain-like proteases, enzymes that cleave polyproteins and remove polyubiquitin chains via deubiquitinating activity, are two important virulence factors. Here, we reveal an unanticipated interplay between the macrodomain and the PLP2 domain that is important for replication and antagonizing the host innate immune response. Targeting the interaction of these enzymes may provide new therapeutic opportunities to treat CoV disease.

Small-Molecule Antiviral ß-d-N4-Hydroxycytidine Inhibits a Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance.

Coronaviruses (CoVs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no FDA-approved antivirals to treat the infections. One class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication. While these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and 5-fluorouracil, have been ineffective at inhibiting CoVs. This has been attributed to the proofreading activity of the viral 3'-5' exoribonuclease (ExoN). ß-d-N4-Hydroxycytidine (NHC) (EIDD-1931; Emory Institute for Drug Development) has recently been reported to inhibit multiple viruses. Here, we demonstrate that NHC inhibits both murine hepatitis virus (MHV) (50% effective concentration [EC50] = 0.17 µM) and Middle East respiratory syndrome CoV (MERS-CoV) (EC50 = 0.56 µM) with minimal cytotoxicity. NHC inhibited MHV lacking ExoN proofreading activity similarly to wild-type (WT) MHV, suggesting an ability to evade or overcome ExoN activity. NHC inhibited MHV only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of G:A and C:U transition mutations present after a single infection. Low-level NHC resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both MHV and MERS-CoV. These results point to a virus-mutagenic mechanism of NHC inhibition in CoVs and indicate a high genetic barrier to NHC resistance. Together, the data support further development of NHC for treatment of CoVs and suggest a novel mechanism of NHC interaction with the CoV replication complex that may shed light on critical aspects of replication.IMPORTANCE The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. However, no antivirals have been approved to treat these infections. Here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, ß-d-N4-hydroxycytidine (NHC), against two divergent CoVs. Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase. Further, passage in the presence of NHC generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections.

An in vivo cell-based assay for investigating the specific interaction between the SARS-CoV N-protein and its viral RNA packaging sequence.

The SARS-CoV nucleocapsid (N) protein serves multiple functions in viral replication, transcription, and assembly of the viral genome complex. Coronaviruses specifically package genomic RNA into assembled virions, and in SARS-CoV, it is reported that this process is driven by an interaction between the N-protein and a packaging signal encoded within the viral RNA. While recent studies have uncovered the sequence of this packaging signal, little is known about the specific interaction between the N-protein and the packaging signal sequence, and the mechanisms by which this interaction drives viral genome packaging. In this study, we developed a novel in vivo cell-based assay for examining this interaction between the N-protein and packaging signal RNA for SARS-CoV, as well as other viruses within the coronaviridae family. Our results demonstrate that the N-protein specifically recognizes the SARS-CoV packaging signal with greater affinity compared to signals from other coronaviruses or non-coronavirus species. We also use deletion mapping to identify a 151-nt region within the packaging signal sequence that is critical for N-protein-RNA binding, and conversely, we show that both the N-terminal and C-terminal domains of the N protein are necessary for recognizing the packaging RNA. These results describe, for the first time, in vivo evidence for an interaction between the SARS-CoV N-protein and its packaging signal RNA, and demonstrate the feasibility of using this cell-based assay to further probe viral RNA-protein interactions in future studies.

Identification of H209 as Essential for pH 8-Triggered Receptor-Independent Syncytium Formation by S Protein of Mouse Hepatitis Virus A59.

The spike glycoprotein (S) of murine coronavirus mouse hepatitis virus (MHV) strain A59 uses murine carcinoembryonic antigen-related cell adhesion molecule 1a as its receptor for cell entry, but S protein can also be triggered in the absence of receptor by pH 8.0 alone at 37°C. The mechanism by which conformational changes of this S glycoprotein can be triggered by pH 8.0 has not yet been determined. Here, we show that MHV-A59 S protein is triggered by pH 8.0 at 37°C to induce receptor-independent syncytium (RIS) formation on 293T cells, and that the conformational changes in S proteins triggered by pH 8.0 are very similar to those triggered by receptor binding. We systemically mutated each of 15 histidine residues in S protein and found that H209 is essential for pH 8.0-triggered RIS formation, while H179, H441, H643, and H759 also play important roles in this process. Replacement of H209 with Ala had no effect on receptor binding, but in murine 17Cl.1 cells mutant H209A MHV-A59 showed delayed growth kinetics and was readily outcompeted by wild-type virus when mixed together, indicating that the H209A mutation caused a defect in virus fitness. Finally, the H209A mutation significantly increased the thermostability of S protein in its prefusion conformation, which may raise the energy barrier for conformational change of S protein required for membrane fusion and lead to a decrease in virus fitness in cell culture. Thus, MHV-A59 may have evolved to lower the stability of its S protein in order to increase virus fitness.IMPORTANCE Enveloped viruses enter cells through fusion of viral and cellular membranes, and the process is mediated by interactions between viral envelope proteins and their host receptors. In the prefusion conformation, viral envelope proteins are metastable, and activation to the fusion conformation is tightly regulated, since premature activation would lead to loss of viral infectivity. The stability of viral envelope proteins greatly influences their activation and virus fitness. Here, we report that, similar to the A82V mutation in Ebola glycoprotein, in the S glycoprotein of murine coronavirus MHV-A59, the histidine residue at position of 209 significantly affects the thermal stability of the S protein, determines whether S protein can be activated at 37°C by either pH 8.0 alone or by receptor binding, and affects viral fitness in cell culture. Thus, the spike glycoprotein of MHV-A59 has evolved to retain histidine at position 209 to optimize virus fitness.

Murine Hepatitis Virus nsp14 Exoribonuclease Activity Is Required for Resistance to Innate Immunity.

Coronaviruses (CoVs) are positive-sense RNA viruses that infect numerous mammalian and avian species and are capable of causing severe and lethal disease in humans. CoVs encode several innate immune antagonists that counteract the host innate immune response to facilitate efficient viral replication. CoV nonstructural protein 14 (nsp14) encodes 3'-to-5' exoribonuclease activity (ExoN), which performs a proofreading function and is required for high-fidelity replication. Outside of the order Nidovirales, arenaviruses are the only RNA viruses that encode an ExoN, which functions to degrade double-stranded RNA (dsRNA) replication intermediates. In this study, we tested the hypothesis that CoV ExoN also functions to antagonize the innate immune response. We demonstrate that viruses lacking ExoN activity [ExoN(-)] are sensitive to cellular pretreatment with interferon beta (IFN-ß) in a dose-dependent manner. In addition, ExoN(-) virus replication was attenuated in wild-type bone marrow-derived macrophages (BMMs) and partially restored in interferon alpha/beta receptor-deficient (IFNAR-/-) BMMs. ExoN(-) virus replication did not result in IFN-ß gene expression, and in the presence of an IFN-ß-mediated antiviral state, ExoN(-) viral RNA levels were not substantially reduced relative to those of untreated samples. However, ExoN(-) virus generated from IFN-ß-pretreated cells had reduced specific infectivity and decreased relative fitness, suggesting that ExoN(-) virus generated during an antiviral state is less viable to establish a subsequent infection. Overall, our data suggest murine hepatitis virus (MHV) ExoN activity is required for resistance to the innate immune response, and antiviral mechanisms affecting the viral RNA sequence and/or an RNA modification act on viruses lacking ExoN activity.IMPORTANCE CoVs encode multiple antagonists that prevent or disrupt an efficient innate immune response. Additionally, no specific antiviral therapies or vaccines currently exist for human CoV infections. Therefore, the study of CoV innate immune antagonists is essential for understanding how CoVs overcome host defenses and to maximize potential therapeutic interventions. Here, we sought to determine the contributions of nsp14 ExoN activity in the induction of and resistance to the innate immune response. We show that viruses lacking nsp14 ExoN activity are more sensitive than wild-type MHV to restriction by exogenous IFN-ß and that viruses produced in the presence of an antiviral state are less capable of establishing a subsequent viral infection. Our results support the hypothesis that murine hepatitis virus ExoN activity is required for resistance to the innate immune response.

Identification of the Fusion Peptide-Containing Region in Betacoronavirus Spike Glycoproteins.

UNLABELLED: The fusion peptides (FP) play an essential role in fusion of viral envelope with cellular membranes. The location and properties of the FPs in the spike (S) glycoproteins of different coronaviruses (CoV) have not yet been determined. Through amino acid sequence analysis of S proteins of representative CoVs, we identified a common region as a possible FP (pFP) that shares the characteristics of FPs of class I viral fusion proteins, including high Ala/Gly content, intermediate hydrophobicity, and few charged residues. To test the hypothesis that this region contains the CoV FP, we systemically mutated every residue in the pFP of Middle East respiratory syndrome betacoronavirus (MERS-CoV) and found that 11 of the 22 residues in the pFP (from G953 to L964, except for A956) were essential for S protein-mediated cell-cell fusion and virus entry. The synthetic MERS-CoV pFP core peptide (955IAGVGWTAGL964) induced extensive fusion of liposome membranes, while mutant peptide failed to induce any lipid mixing. We also selectively mutated residues in pFPs of two other ß-CoVs, severe acute respiratory syndrome coronavirus (SARS-CoV) and mouse hepatitis virus (MHV). Although the amino acid sequences of these two pFPs differed significantly from that of MERS-CoV and each other, most of the pFP mutants of SARS-CoV and MHV also failed to mediate membrane fusion, suggesting that these pFPs are also the functional FPs. Thus, the FPs of 3 different lineages of ß-CoVs are conserved in location within the S glycoproteins and in their functions, although their amino acid sequences have diverged significantly during CoV evolution. IMPORTANCE: Within the class I viral fusion proteins of many enveloped viruses, the FP is the critical mediator of fusion of the viral envelope with host cell membranes leading to virus infection. FPs from within a virus family, like influenza viruses or human immunodeficiency viruses (HIV), tend to share high amino acid sequence identity. In this study, we determined the location and amino acid sequences of the FPs of S glycoproteins of 3 ß-CoVs, MERS-CoV, SARS-CoV, and MHV, and demonstrated that they were essential for mediating cell-cell fusion and virus entry. Interestingly, in marked contrast to the FPs of influenza and HIV, the primary amino acid sequences of the FPs of ß-CoVs in 3 different lineages differed significantly. Thus, during evolution the FPs of ß-CoVs have diverged significantly in their primary sequences while maintaining the same essential biological functions. Our findings identify a potential new target for development of drugs against CoVs.

Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens.

UNLABELLED: Positive-sense RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than-10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it on solved picornaviral RdRp structures. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-RdRp model structure and then engineered into the MHV genome with [nsp14-ExoN(+)] or without [nsp14-ExoN(-)] ExoN activity. Using this method, we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I, we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and a decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses. The results also indicate that, with regard to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-RdRp, consistent with its proposed role in a multiprotein replicase-proofreading complex. IMPORTANCE: RNA viruses have evolutionarily fine-tuned replication fidelity to balance requirements for genetic stability and diversity. Responsibility for replication fidelity in RNA viruses has been attributed to the RNA-dependent RNA polymerases, with mutations in RdRps for multiple RNA viruses shown to alter fidelity and attenuate virus replication and virulence. Coronaviruses (CoVs) are the only known RNA viruses to encode a proofreading exonuclease (nsp14-ExoN), as well as other replicase proteins involved in regulation of fidelity. This report shows that the CoV RdRp (nsp12) likely functions in replication fidelity; that residue determinants of CoV RdRp nucleotide selectivity map to similar structural regions of other, unrelated RNA viral polymerases; and that for CoVs, the proofreading activity of the nsp14-ExoN is epistatic to the function of the RdRp in fidelity.

Dissection of amino-terminal functional domains of murine coronavirus nonstructural protein 3.

UNLABELLED: Coronaviruses, the largest RNA viruses, have a complex program of RNA synthesis that entails genome replication and transcription of subgenomic mRNAs. RNA synthesis by the prototype coronavirus mouse hepatitis virus (MHV) is carried out by a replicase-transcriptase composed of 16 nonstructural protein (nsp) subunits. Among these, nsp3 is the largest and the first to be inserted into the endoplasmic reticulum. nsp3 comprises multiple structural domains, including two papain-like proteases (PLPs) and a highly conserved ADP-ribose-1″-phosphatase (ADRP) macrodomain. We have previously shown that the ubiquitin-like domain at the amino terminus of nsp3 is essential and participates in a critical interaction with the viral nucleocapsid protein early in infection. In the current study, we exploited atypical expression schemes to uncouple PLP1 from the processing of nsp1 and nsp2 in order to investigate the requirements of nsp3 domains for viral RNA synthesis. In the first strategy, a mutant was created in which replicase polyprotein translation initiated with nsp3, thereby establishing that complete elimination of nsp1 and nsp2 does not abolish MHV viability. In the second strategy, a picornavirus autoprocessing element was used to separate a truncated nsp1 from nsp3. This provided a platform for further dissection of amino-terminal domains of nsp3. From this, we found that catalytic mutation of PLP1 or complete deletion of PLP1 and the adjacent ADRP domain was tolerated by the virus. These results showed that neither the PLP1 domain nor the ADRP domain of nsp3 provides integral activities essential for coronavirus genomic or subgenomic RNA synthesis. IMPORTANCE: The largest component of the coronavirus replicase-transcriptase complex, nsp3, contains multiple modules, many of which do not have clearly defined functions in genome replication or transcription. These domains may play direct roles in RNA synthesis, or they may have evolved for other purposes, such as to combat host innate immunity. We initiated a dissection of MHV nsp3 aimed at identifying those activities or structures in this huge molecule that are essential to replicase activity. We found that both PLP1 and ADRP could be entirely deleted, provided that the requirement for proteolytic processing by PLP1 was offset by an alternative mechanism. This demonstrated that neither PLP1 nor ADRP plays an essential role in coronavirus RNA synthesis.

Mutations across murine hepatitis virus nsp4 alter virus fitness and membrane modifications.

UNLABELLED: A common feature of infection by positive-sense RNA virus is the modification of host cell cytoplasmic membranes that serve as sites of viral RNA synthesis. Coronaviruses induce double-membrane vesicles (DMVs), but the role of DMVs in replication and virus fitness remains unclear. Coronaviruses encode 16 nonstructural proteins (nsps), three of which, nsp3, nsp4, and nsp6, are necessary and sufficient for DMV formation. It has been shown previously that mutations in murine hepatitis virus (MHV) nsp4 loop 1 that alter nsp4 glycosylation are associated with disrupted DMV formation and result in changes in virus replication and RNA synthesis. However, it is not known whether DMV morphology or another function of nsp4 glycosylation is responsible for effects on virus replication. In this study, we tested whether mutations across nsp4, both alone and in combination with mutations that abolish nsp4 glycosylation, affected DMV formation, replication, and fitness. Residues in nsp4 distinct from glycosylation sites, particularly in the endoplasmic reticulum (ER) luminal loop 1, independently disrupted both the number and morphology of DMVs and exacerbated DMV changes associated with loss of glycosylation. Mutations that altered DMV morphology but not glycosylation did not affect virus fitness while viruses lacking nsp4 glycosylation exhibited a loss in fitness. The results support the hypothesis that DMV morphology and numbers are not key determinants of virus fitness. The results also suggest that nsp4 glycosylation serves roles in replication in addition to the organization and stability of MHV-induced double-membrane vesicles. IMPORTANCE: All positive-sense RNA viruses modify host cytoplasmic membranes for viral replication complex formation. Thus, defining the mechanisms of virus-induced membrane modifications is essential for both understanding virus replication and development of novel approaches to virus inhibition. Coronavirus-induced membrane changes include double-membrane vesicles (DMVs) and convoluted membranes. Three viral nonstructural proteins (nsps), nsp3, nsp4, and nsp6, are known to be required for DMV formation. It is unknown how these proteins induce membrane modification or which regions of the proteins are involved in DMV formation and stability. In this study, we show that mutations across nsp4 delay virus replication and disrupt DMV formation and that loss of nsp4 glycosylation is associated with a substantial fitness cost. These results support a critical role for nsp4 in DMV formation and virus fitness.