Nidovirus Replication Research Articles

Transcriptional and Translational Landscape of Equine Torovirus.

The genus Torovirus (subfamily Torovirinae, family Coronaviridae, order Nidovirales) encompasses a range of species that infect domestic ungulates, including cattle, sheep, goats, pigs, and horses, causing an acute self-limiting gastroenteritis. Using the prototype species equine torovirus (EToV), we performed parallel RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) to analyze the relative expression levels of the known torovirus proteins and transcripts, chimeric sequences produced via discontinuous RNA synthesis (a characteristic of the nidovirus replication cycle), and changes in host transcription and translation as a result of EToV infection. RNA sequencing confirmed that EToV utilizes a unique combination of discontinuous and nondiscontinuous RNA synthesis to produce its subgenomic RNAs (sgRNAs); indeed, we identified transcripts arising from both mechanisms that would result in sgRNAs encoding the nucleocapsid. Our ribosome profiling analysis revealed that ribosomes efficiently translate two novel CUG-initiated open reading frames (ORFs), located within the so-called 5' untranslated region. We have termed the resulting proteins U1 and U2. Comparative genomic analysis confirmed that these ORFs are conserved across all available torovirus sequences, and the inferred amino acid sequences are subject to purifying selection, indicating that U1 and U2 are functionally relevant. This study provides the first high-resolution analysis of transcription and translation in this neglected group of livestock pathogens.IMPORTANCE Toroviruses infect cattle, goats, pigs, and horses worldwide and can cause gastrointestinal disease. There is no treatment or vaccine, and their ability to spill over into humans has not been assessed. These viruses are related to important human pathogens, including severe acute respiratory syndrome (SARS) coronavirus, and they share some common features; however, the mechanism that they use to produce sgRNA molecules differs. Here, we performed deep sequencing to determine how equine torovirus produces sgRNAs. In doing so, we also identified two previously unknown open reading frames "hidden" within the genome. Together these results highlight the similarities and differences between this domestic animal virus and related pathogens of humans and livestock.

Antiviral activity of K22 against members of the order Nidovirales

Recently, a novel antiviral compound (K22) that inhibits replication of a broad range of animal and human coronaviruses was reported to interfere with viral RNA synthesis by impairing double-membrane vesicle (DMV) formation (Lundin et al., 2014). Here we assessed potential antiviral activities of K22 against a range of viruses representing two (sub)families of the order Nidovirales, the Arteriviridae (porcine reproductive and respiratory syndrome virus [PRRSV], equine arteritis virus [EAV] and simian hemorrhagic fever virus [SHFV]), and the Torovirinae (equine torovirus [EToV] and White Bream virus [WBV]).Possible effects of K22 on nidovirus replication were studied in suitable cell lines. K22 concentrations significantly decreasing infectious titres of the viruses included in this study ranged from 25 to 50 μM. Reduction of double-stranded RNA intermediates of viral replication in nidovirus-infected cells treated with K22 confirmed the anti-viral potential of K22. Collectively, the data show that K22 has antiviral activity against diverse lineages of nidoviruses, suggesting that the inhibitor targets a critical and conserved step during nidovirus replication.

What we know but do not understand about nidovirus helicases

Helicases are versatile NTP-dependent motor proteins of monophyletic origin that are found in all kingdoms of life. Their functions range from nucleic acid duplex unwinding to protein displacement and double-strand translocation. This explains their participation in virtually every metabolic process that involves nucleic acids, including DNA replication, recombination and repair, transcription, translation, as well as RNA processing. Helicases are encoded by all plant and animal viruses with a positive-sense RNA genome that is larger than 7kb, indicating a link to genome size evolution in this virus class. Viral helicases belong to three out of the six currently recognized superfamilies, SF1, SF2, and SF3. Despite being omnipresent, highly conserved and essential, only a few viral helicases, mostly from SF2, have been studied extensively. In general, their specific roles in the viral replication cycle remain poorly understood at present. The SF1 helicase protein of viruses classified in the order Nidovirales is encoded in replicase open reading frame 1b (ORF1b), which is translated to give rise to a large polyprotein following a ribosomal frameshift from the upstream ORF1a. Proteolytic processing of the replicase polyprotein yields a dozen or so mature proteins, one of which includes a helicase. Its hallmark is the presence of an N-terminal multi-nuclear zinc-binding domain, the nidoviral genetic marker and one of the most conserved domains across members of the order. This review summarizes biochemical, structural, and genetic data, including drug development studies, obtained using helicases originating from several mammalian nidoviruses, along with the results of the genomics characterization of a much larger number of (putative) helicases of vertebrate and invertebrate nidoviruses. In the context of our knowledge of related helicases of cellular and viral origin, it discusses the implications of these results for the protein's emerging critical function(s) in nidovirus evolution, genome replication and expression, virion biogenesis, and possibly also post-transcriptional processing of viral RNAs. Using our accumulated knowledge and highlighting gaps in our data, concepts and approaches, it concludes with a perspective on future research aimed at elucidating the role of helicases in the nidovirus replication cycle.

Ribavirin efficiently suppresses porcine nidovirus replication

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) are porcine nidoviruses that represent emerging viral pathogens causing heavy economic impacts on the swine industry. Although ribavirin is a well-known antiviral drug against a broad range of both DNA and RNA viruses in vitro, its inhibitory effect and mechanism of action on porcine nidovirus replication remains to be elucidated. Therefore, the present study was conducted to determine whether ribavirin suppresses porcine nidovirus infection. Our results demonstrated that ribavirin treatment dose-dependently inhibited the replication of both nidoviruses. The antiviral activity of ribavirin on porcine nidovirus replication was found to be primarily exerted at early times post-infection. Treatment with ribavirin resulted in marked reduction of viral genomic and subgenomic RNA synthesis, viral protein expression, and progeny virus production in a dose-dependent manner. Investigations into the mechanism of action of ribavirin against PRRSV and PEDV revealed that the addition of guanosine to the ribavirin treatment significantly reversed the antiviral effects, suggesting that depletion of the intracellular GTP pool by inhibiting IMP dehydrogenase may be essential for ribavirin activity. Further sequencing analysis showed that the mutation frequency in ribavirin-treated cells was similar to that in untreated cells, indicating that ribavirin did not induce error-prone replication. Taken together, our data indicate that ribavirin might not only be a good therapeutic agent against porcine nidovirus, but also a potential candidate to be evaluated against other human and animal coronaviruses.

Zn2+ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture

Increasing the intracellular Zn2+ concentration with zinc-ionophores like pyrithione (PT) can efficiently impair the replication of a variety of RNA viruses, including poliovirus and influenza virus. For some viruses this effect has been attributed to interference with viral polyprotein processing. In this study we demonstrate that the combination of Zn2+ and PT at low concentrations (2 µM Zn2+ and 2 µM PT) inhibits the replication of SARS-coronavirus (SARS-CoV) and equine arteritis virus (EAV) in cell culture. The RNA synthesis of these two distantly related nidoviruses is catalyzed by an RNA-dependent RNA polymerase (RdRp), which is the core enzyme of their multiprotein replication and transcription complex (RTC). Using an activity assay for RTCs isolated from cells infected with SARS-CoV or EAV—thus eliminating the need for PT to transport Zn2+ across the plasma membrane—we show that Zn2+ efficiently inhibits the RNA-synthesizing activity of the RTCs of both viruses. Enzymatic studies using recombinant RdRps (SARS-CoV nsp12 and EAV nsp9) purified from E. coli subsequently revealed that Zn2+ directly inhibited the in vitro activity of both nidovirus polymerases. More specifically, Zn2+ was found to block the initiation step of EAV RNA synthesis, whereas in the case of the SARS-CoV RdRp elongation was inhibited and template binding reduced. By chelating Zn2+ with MgEDTA, the inhibitory effect of the divalent cation could be reversed, which provides a novel experimental tool for in vitro studies of the molecular details of nidovirus replication and transcription.

A Complex Zinc Finger Controls the Enzymatic Activities of Nidovirus Helicases

Nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) encode a nonstructural protein, called nsp10 in arteriviruses and nsp13 in coronaviruses, that is comprised of a C-terminal superfamily 1 helicase domain and an N-terminal, putative zinc-binding domain (ZBD). Previously, mutations in the equine arteritis virus (EAV) nsp10 ZBD were shown to block arterivirus reproduction by disrupting RNA synthesis and possibly virion biogenesis. Here, we characterized the ATPase and helicase activities of bacterially expressed mutant forms of nsp10 and its human coronavirus 229E ortholog, nsp13, and correlated these in vitro activities with specific virus phenotypes. Replacement of conserved Cys or His residues with Ala proved to be more deleterious than Cys-for-His or His-for-Cys replacements. Furthermore, denaturation-renaturation experiments revealed that, during protein refolding, Zn2+ is essential for the rescue of the enzymatic activities of nidovirus helicases. Taken together, the data strongly support the zinc-binding function of the N-terminal domain of nidovirus helicases. nsp10 ATPase/helicase deficiency resulting from single-residue substitutions in the ZBD or deletion of the entire domain could not be complemented in trans by wild-type ZBD, suggesting a critical function of the ZBD in cis. Consistently, no viral RNA synthesis was detected after transfection of EAV full-length RNAs encoding ATPase/helicase-deficient nsp10 into susceptible cells. In contrast, diverse phenotypes were observed for mutants with enzymatically active nsp10, which in a number of cases correlated with the activities measured in vitro. Collectively, our data suggest that the ZBD is critically involved in nidovirus replication and transcription by modulating the enzymatic activities of the helicase domain and other, yet unknown, mechanisms.

Investigations of Remedies

<h3>Abstract</h3> The genus <i>Torovirus</i> (subfamily <i>Torovirinae</i>, family <i>Coronaviridae</i>, order <i>Nidovirales</i>) encompasses a range of species that infect domestic ungulates including cattle, sheep, goats, pigs and horses, causing an acute self-limiting gastroenteritis. Using the prototype species equine torovirus (EToV) we performed parallel RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) to analyse the relative expression levels of the known torovirus proteins and transcripts, chimaeric sequences produced via discontinuous RNA synthesis (a characteristic of the nidovirus replication cycle) and changes in host transcription and translation as a result of EToV infection. RNA sequencing confirmed that EToV utilises a unique combination of discontinuous and non-discontinuous RNA synthesis to produce its subgenomic RNAs; indeed, we identified transcripts arising from both mechanisms that would result in sgRNAs encoding the nucleocapsid. Our ribosome profiling analysis revealed that ribosomes efficiently translate two novel CUG-initiated ORFs, located within the so-called 5’ UTR. We have termed the resulting proteins U1 and U2. Comparative genomic analysis confirmed that these ORFs are conserved across all available torovirus sequences and the inferred amino acid sequences are subject to purifying selection, indicating that U1 and U2 are functionally relevant. This study provides the first high-resolution analysis of transcription and translation in this neglected group of livestock pathogens. <h3>Importance</h3> Toroviruses infect cattle, goats, pigs and horses worldwide and can cause gastrointestinal disease. There is no treatment or vaccine and their ability to spill over into humans has not been assessed. These viruses are related to important human pathogens including severe acute respiratory syndrome (SARS) coronavirus and they share some common features, however the mechanism that they use to produce subgenomic RNA molecules differs. Here we performed deep sequencing to determine how equine torovirus produces subgenomic RNAs. In doing so, we also identified two previously unknown open reading frames “hidden” within the genome. Together these results highlight the similarities and differences between this domestic animal virus and related pathogens of humans and livestock.