Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication.

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5' exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3'-to-5' ExoN domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14's enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis.

Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection.

The Enzymatic Activity of the nsp14 Exoribonuclease Is Critical for Replication of MERS-CoV and SARS-CoV-2.

Coronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) domains. While the latter presumably supports mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously reported to have crippled but viable hypermutation phenotypes. Remarkably, using reverse genetics, a large set of corresponding ExoN knockout mutations has now been found to be lethal for another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). For 13 mutants, viral progeny could not be recovered, unless-as happened occasionally-reversion had first occurred. Only a single mutant was viable, likely because its E191D substitution is highly conservative. Remarkably, a SARS-CoV-2 ExoN knockout mutant was found to be unable to replicate, resembling observations previously made for alpha- and gammacoronaviruses, but starkly contrasting with the documented phenotype of ExoN knockout mutants of the closely related SARS-CoV. Subsequently, we established in vitro assays with purified recombinant MERS-CoV nsp14 to monitor its ExoN and N7-MTase activities. All ExoN knockout mutations that proved lethal in reverse genetics were found to severely decrease ExoN activity while not affecting N7-MTase activity. Our study strongly suggests that CoV nsp14 ExoN has an additional function, which apparently is critical for primary viral RNA synthesis and thus differs from the proofreading function that, based on previous MHV and SARS-CoV studies, was proposed to boost longer-term replication fidelity.IMPORTANCE The bifunctional nsp14 subunit of the coronavirus replicase contains 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase domains. For the betacoronaviruses MHV and SARS-CoV, ExoN was reported to promote the fidelity of genome replication, presumably by mediating a form of proofreading. For these viruses, ExoN knockout mutants are viable while displaying an increased mutation frequency. Strikingly, we have now established that the equivalent ExoN knockout mutants of two other betacoronaviruses, MERS-CoV and SARS-CoV-2, are nonviable, suggesting an additional and critical ExoN function in their replication. This is remarkable in light of the very limited genetic distance between SARS-CoV and SARS-CoV-2, which is highlighted, for example, by 95% amino acid sequence identity in their nsp14 sequences. For (recombinant) MERS-CoV nsp14, both its enzymatic activities were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.

SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology.

The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of non-structural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.

Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses.

RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that catalyzes the synthesis of their RNA(s). In the case of positive-stranded RNA viruses belonging to the order Nidovirales, the RdRp resides in a replicase subunit that is unusually large. Bioinformatics analysis of this non-structural protein has now revealed a nidoviral signature domain (genetic marker) that is N-terminally adjacent to the RdRp and has no apparent homologs elsewhere. Based on its conservation profile, this domain is proposed to have nucleotidylation activity. We used recombinant non-structural protein 9 of the arterivirus equine arteritis virus (EAV) and different biochemical assays, including irreversible labeling with a GTP analog followed by a proteomics analysis, to demonstrate the manganese-dependent covalent binding of guanosine and uridine phosphates to a lysine/histidine residue. Most likely this was the invariant lysine of the newly identified domain, named nidovirus RdRp-associated nucleotidyltransferase (NiRAN), whose substitution with alanine severely diminished the described binding. Furthermore, this mutation crippled EAV and prevented the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in cell culture, indicating that NiRAN is essential for nidoviruses. Potential functions supported by NiRAN may include nucleic acid ligation, mRNA capping and protein-primed RNA synthesis, possibilities that remain to be explored in future studies.

One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities.

In addition to members causing milder human infections, the Coronaviridae family includes potentially lethal zoonotic agents causing severe acute respiratory syndrome (SARS) and the recently emerged Middle East respiratory syndrome. The ∼30-kb positive-stranded RNA genome of coronaviruses encodes a replication/transcription machinery that is unusually complex and composed of 16 nonstructural proteins (nsps). SARS-CoV nsp12, the canonical RNA-dependent RNA polymerase (RdRp), exhibits poorly processive RNA synthesis in vitro, at odds with the efficient replication of a very large RNA genome in vivo. Here, we report that SARS-CoV nsp7 and nsp8 activate and confer processivity to the RNA-synthesizing activity of nsp12. Using biochemical assays and reverse genetics, the importance of conserved nsp7 and nsp8 residues was probed. Whereas several nsp7 mutations affected virus replication to a limited extent, the replacement of two nsp8 residues (P183 and R190) essential for interaction with nsp12 and a third (K58) critical for the interaction of the polymerase complex with RNA were all lethal to the virus. Without a loss of processivity, the nsp7/nsp8/nsp12 complex can associate with nsp14, a bifunctional enzyme bearing 3'-5' exoribonuclease and RNA cap N7-guanine methyltransferase activities involved in replication fidelity and 5'-RNA capping, respectively. The identification of this tripartite polymerase complex that in turn associates with the nsp14 proofreading enzyme sheds light on how coronaviruses assemble an RNA-synthesizing machinery to replicate the largest known RNA genomes. This protein complex is a fascinating example of the functional integration of RNA polymerase, capping, and proofreading activities.

Arterivirus nsp12 versus the coronavirus nsp16 2'-O-methyltransferase: comparison of the C-terminal cleavage products of two nidovirus pp1ab polyproteins.

The 3'-terminal domain of the most conserved ORF1b in three of the four families of the order Nidovirales (except for the family Arteriviridae) encodes a (putative) 2'-O-methyltransferase (2'-O-MTase), known as non structural protein (nsp) 16 in the family Coronaviridae and implicated in methylation of the 5' cap structure of nidoviral mRNAs. As with coronavirus transcripts, arterivirus mRNAs are assumed to possess a 5' cap although no candidate MTases have been identified thus far. To address this knowledge gap, we analysed the uncharacterized nsp12 of arteriviruses, which occupies the ORF1b position equivalent to that of the nidovirus 2'-O-MTase (coronavirus nsp16). In our in-depth bioinformatics analysis of nsp12, the protein was confirmed to be family specific whilst having diverged much further than other nidovirus ORF1b-encoded proteins, including those of the family Coronaviridae. Only one invariant and several partially conserved, predominantly aromatic residues were identified in nsp12, which may adopt a structure with alternating α-helices and ß-strands, an organization also found in known MTases. However, no statistically significant similarity was found between nsp12 and the twofold larger coronavirus nsp16, nor could we detect MTase activity in biochemical assays using recombinant equine arteritis virus (EAV) nsp12. Our further analysis established that this subunit is essential for replication of this prototypic arterivirus. Using reverse genetics, we assessed the impact of 25 substitutions at 14 positions, yielding virus phenotypes ranging from WT-like to non-viable. Notably, replacement of the invariant phenylalanine 109 with tyrosine was lethal. We concluded that nsp12 plays an essential role during EAV replication, possibly by acting as a co-factor for another enzyme.

Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture.

Coronaviruses can cause respiratory and enteric disease in a wide variety of human and animal hosts. The 2003 outbreak of severe acute respiratory syndrome (SARS) first demonstrated the potentially lethal consequences of zoonotic coronavirus infections in humans. In 2012, a similar previously unknown coronavirus emerged, Middle East respiratory syndrome coronavirus (MERS-CoV), thus far causing over 650 laboratory-confirmed infections, with an unexplained steep rise in the number of cases being recorded over recent months. The human MERS fatality rate of ∼ 30% is alarmingly high, even though many deaths were associated with underlying medical conditions. Registered therapeutics for the treatment of coronavirus infections are not available. Moreover, the pace of drug development and registration for human use is generally incompatible with strategies to combat emerging infectious diseases. Therefore, we have screened a library of 348 FDA-approved drugs for anti-MERS-CoV activity in cell culture. If such compounds proved sufficiently potent, their efficacy might be directly assessed in MERS patients. We identified four compounds (chloroquine, chlorpromazine, loperamide, and lopinavir) inhibiting MERS-CoV replication in the low-micromolar range (50% effective concentrations [EC(50)s], 3 to 8 µM). Moreover, these compounds also inhibit the replication of SARS coronavirus and human coronavirus 229E. Although their protective activity (alone or in combination) remains to be assessed in animal models, our findings may offer a starting point for treatment of patients infected with zoonotic coronaviruses like MERS-CoV. Although they may not necessarily reduce viral replication to very low levels, a moderate viral load reduction may create a window during which to mount a protective immune response.

Characterization of SARS-CoV-2 replication in human H1299/ACE2 cells: A versatile and practical infection model for antiviral research and beyond.

A range of cell culture infection models have been used to study SARS-CoV-2 and perform antiviral drug research. Commonly used African green monkey Vero, human lung-derived Calu-3 and ACE2+TMPRSS2-expressing A549 cells, each have their limitations. Here, we describe human ACE2-expressing H1299 lung cells as a more efficient and robust model for SARS-CoV-2 research. These cells are as easy to handle as Vero cells, support SARS-CoV-2 replication to high titers, display a functional innate immune response and are suitable for plaque assays, microscopy, the production of (genetically stable) virus stocks and antiviral assays. H1299/ACE2-based (CPE reduction) assays can be performed without adding a P-gP drug efflux pump inhibitor, which is often required in Vero-based assays. Moreover, H1299/ACE2 cells allowed us to perform CPE reduction assays with omicron variants that did not work in Vero-based assays. In summary, H1299/ACE2 cells are a versatile infection model to study SARS-CoV-2 replication in the context of antiviral drug development and virus-host interaction studies.

Arterivirus minor envelope proteins are a major determinant of viral tropism in cell culture.

Arteriviruses are enveloped positive-strand RNA viruses for which the attachment proteins and cellular receptors have remained largely controversial. Arterivirus particles contain at least eight envelope proteins, an unusually large number among RNA viruses. These appear to segregate into three groups: major structural components (major glycoprotein GP5 and membrane protein [M]), minor glycoproteins (GP2a, GP3, and GP4), and small hydrophobic proteins (E and the recently discovered ORF5a protein). Biochemical studies previously suggested that the GP5-M heterodimer of porcine reproductive and respiratory syndrome virus (PRRSV) interacts with porcine sialoadhesin (pSn) in porcine alveolar macrophages (PAM). However, another study proposed that minor protein GP4, along with GP2a, interacts with CD163, another reported cellular receptor for PRRSV. In this study, we provide genetic evidence that the minor envelope proteins are the major determinant of arterivirus entry into cultured cells. A PRRSV infectious cDNA clone was equipped with open reading frames (ORFs) encoding minor envelope and E proteins of equine arteritis virus (EAV), the only known arterivirus displaying a broad tropism in cultured cells. Although PRRSV and EAV are only distantly related and utilize diversified transcription-regulating sequences (TRSs), a viable chimeric progeny virus was rescued. Strikingly, this chimeric virus (vAPRRS-EAV2ab34) acquired the broad in vitro cell tropism of EAV, demonstrating that the minor envelope proteins play a critical role as viral attachment proteins. We believe that chimeric arteriviruses of this kind will be a powerful tool for further dissection of the arterivirus replicative cycle, including virus entry, subgenomic RNA synthesis, and virion assembly.

Development and RNA-synthesizing activity of coronavirus replication structures in the absence of protein synthesis.

The RNA replication and transcription complex of coronaviruses is associated with an elaborate reticulovesicular network (RVN) of modified endoplasmic reticulum. Using cycloheximide and puromycin, we have studied the effect of translation inhibition on the RNA synthesis of severe acute respiratory syndrome coronavirus and mouse hepatitis virus. Both inhibitors prevented the usual exponential increase in viral RNA synthesis, with immunofluorescence and electron microscopy indicating that RVN development came to a standstill. Nevertheless, limited RNA synthesis was supported, implying that continued translation is not an absolute requirement and suggesting a direct link between RVN formation and accumulation of coronavirus proteins.

Structure and genetic analysis of the arterivirus nonstructural protein 7alpha.

Arterivirus replicase polyproteins are cleaved into at least 13 mature nonstructural proteins (nsps), and in particular the nsp5-to-nsp8 region is subject to a complex processing cascade. The function of the largest subunit from this region, nsp7, which is further cleaved into nsp7α and nsp7ß, is unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we determined the solution structure of nsp7α of equine arteritis virus, revealing an interesting unique fold for this protein but thereby providing little clue to its possible functions. Nevertheless, structure-based reverse genetics studies established the importance of nsp7/nsp7α for viral RNA synthesis, thus providing a basis for future studies.

Discovery of a small arterivirus gene that overlaps the GP5 coding sequence and is important for virus production.

The arterivirus family (order Nidovirales) of single-stranded, positive-sense RNA viruses includes porcine respiratory and reproductive syndrome virus and equine arteritis virus (EAV). Their replicative enzymes are translated from their genomic RNA, while their seven structural proteins are encoded by a set of small, partially overlapping genes in the genomic 3'-proximal region. The latter are expressed via synthesis of a set of subgenomic mRNAs that, in general, are functionally monocistronic (except for a bicistronic mRNA encoding the E and GP2 proteins). ORF5, which encodes the major glycoprotein GP5, has been used extensively for phylogenetic analyses. However, an in-depth computational analysis now reveals the arterivirus-wide conservation of an additional AUG-initiated ORF, here termed ORF5a, that overlaps the 5' end of ORF5. The pattern of substitutions across sequence alignments indicated that ORF5a is subject to functional constraints at the amino acid level, while an analysis of substitutions at synonymous sites in ORF5 revealed a greatly reduced frequency of substitution in the portion of ORF5 that is overlapped by ORF5a. The 43-64 aa ORF5a protein and GP5 are probably expressed from the same subgenomic mRNA, via a translation initiation mechanism involving leaky ribosomal scanning. Inactivation of ORF5a expression by reverse genetics yielded a severely crippled EAV mutant, which displayed lower titres and a tiny plaque phenotype. These defects, which could be partially complemented in ORF5a-expressing cells, indicate that the novel protein, which may be the eighth structural protein of arteriviruses, is expressed and important for arterivirus infection.

Cyclosporin A inhibits the replication of diverse coronaviruses.

Low micromolar, non-cytotoxic concentrations of cyclosporin A (CsA) strongly affected the replication of severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus 229E and mouse hepatitis virus in cell culture, as was evident from the strong inhibition of GFP reporter gene expression and a reduction of up to 4 logs in progeny titres. Upon high-multiplicity infection, CsA treatment rendered SARS-CoV RNA and protein synthesis almost undetectable, suggesting an early block in replication. siRNA-mediated knockdown of the expression of the prominent CsA targets cyclophilin A and B did not affect SARS-CoV replication, suggesting either that these specific cyclophilin family members are dispensable or that the reduced expression levels suffice to support replication.

SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.

Positive-strand RNA viruses, a large group including human pathogens such as SARS-coronavirus (SARS-CoV), replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs) were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200-300 nm), and "vesicle packets" apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this "replication network" will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions.

Coronavirus discovery by metagenomic sequencing: a tool for pandemic preparedness.

INTRODUCTION: The SARS-CoV-2 pandemic of 2020 is a prime example of the omnipresent threat of emerging viruses that can infect humans. A protocol for the identification of novel coronaviruses by viral metagenomic sequencing in diagnostic laboratories may contribute to pandemic preparedness. AIM: The aim of this study is to validate a metagenomic virus discovery protocol as a tool for coronavirus pandemic preparedness. METHODS: The performance of a viral metagenomic protocol in a clinical setting for the identification of novel coronaviruses was tested using clinical samples containing SARS-CoV-2, SARS-CoV, and MERS-CoV, in combination with databases generated to contain only viruses of before the discovery dates of these coronaviruses, to mimic virus discovery. RESULTS: Classification of NGS reads using Centrifuge and Genome Detective resulted in assignment of the reads to the closest relatives of the emerging coronaviruses. Low nucleotide and amino acid identity (81% and 84%, respectively, for SARS-CoV-2) in combination with up to 98% genome coverage were indicative for a related, novel coronavirus. Capture probes targeting vertebrate viruses, designed in 2015, enhanced both sequencing depth and coverage of the SARS-CoV-2 genome, the latter increasing from 71% to 98%. CONCLUSION: The model used for simulation of virus discovery enabled validation of the metagenomic sequencing protocol. The metagenomic protocol with virus probes designed before the pandemic, can assist the detection and identification of novel coronaviruses directly in clinical samples.

A molecular pore spans the double membrane of the coronavirus replication organelle.

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo-electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target.

Myristoylation of the arterivirus E protein: the fatty acid modification is not essential for membrane association but contributes significantly to virus infectivity.

The envelope of equine arteritis virus (EAV) contains two glycoprotein complexes (GP2b/GP3/GP4 and GP5/M) and the small, non-glycosylated E protein. As E is essential for the production of infectious progeny but dispensable for assembly and release of virus-like particles, it probably mediates virus entry into cells, putatively in concert with the GP2b/GP3/GP4 complex. The E protein contains a central hydrophobic domain and a conserved potential site for N-terminal myristoylation, a hydrophobic modification usually pivotal for membrane targeting of the modified protein. Here, it was shown by radiolabelling that E is myristoylated at glycine-2, both in transfected cells as a fusion protein with yellow fluorescent protein (YFP) and in virus particles. Biochemical fractionation revealed that E-YFP with an inactivated acylation site was still completely membrane-bound, indicating that the putative transmembrane domain of E mediates membrane targeting. Confocal microscopy showed that both myristoylated and non-myristoylated E-YFP were localized to the endoplasmic reticulum and Golgi complex, the membranes from which EAV buds. The presence of a myristoylation inhibitor during replication of EAV, whilst completely blocking E acylation, reduced virus titres by 1.5 log(10). Similarly, a mutant EAV with non-myristoylatable E grew to a titre five- to sevenfold lower than that of the wild-type virus and exhibited a reduced plaque size. Western blotting of cell-culture supernatants showed that N and M, the major structural proteins of EAV, are released in similar amounts by cells transfected with wild-type and mutant genomes. Thus, E myristoylation is not required for budding of particles and probably has a function during virus entry.

Formation of the arterivirus replication/transcription complex: a key role for nonstructural protein 3 in the remodeling of intracellular membranes.

The replication/transcription complex of the arterivirus equine arteritis virus (EAV) is associated with paired membranes and/or double-membrane vesicles (DMVs) that are thought to originate from the endoplasmic reticulum. Previously, coexpression of two putative transmembrane nonstructural proteins (nsp2 and nsp3) was found to suffice to induce these remarkable membrane structures, which are typical of arterivirus infection. Here, site-directed mutagenesis was used to investigate the role of nsp3 in more detail. Liberation of the hydrophobic N terminus of nsp3, which is normally achieved by cleavage of the nsp2/3 junction by the nsp2 protease, was nonessential for the formation of DMVs. However, the substitution of each of a cluster of four conserved cysteine residues, residing in a predicted luminal loop of nsp3, completely blocked DMV formation. Some of these mutant nsp3 proteins were also found to be highly cytotoxic, in particular, exerting a dramatic effect on the endoplasmic reticulum. The functionality of an engineered N glycosylation site in the cysteine-containing loop confirmed both its presence in the lumen and the transmembrane nature of nsp3. This mutant displayed an interesting intermediate phenotype in terms of DMV formation, with paired and curved membranes being formed, but DMV formation apparently being impaired. The effect of nsp3 mutations on replicase polyprotein processing was investigated, and several mutations were found to influence processing of the region downstream of nsp3 by the nsp4 main protease. When tested in an EAV reverse genetics system, none of the nsp3 mutations was tolerated, again underlining the crucial role of the protein in the arterivirus life cycle.

Production of monospecific rabbit antisera recognizing nidovirus proteins.

The importance of monospecific antisera for the experimental analysis of viral proteins is undisputed. They make it possible to identify and analyze the target protein against a background of a large number of other proteins, either in whole fixed cells or in cell lysates. This chapter describes our experience with the production of such rabbit antisera directed against proteins of coronaviruses and other nidoviruses. The use as antigens of either synthetic peptides (coupled to a carrier protein) or proteins expressed in Escherichia coli is described, and detailed protocols for immunization and preparation of test bleeds are provided. For screening of the immune response following immunization, detailed protocols for three commonly used techniques are described, all of which are based on the use of infected cells or cells expressing the protein of interest, side by side with appropriate controls. The in situ immunodetection of the target in fixed cells by immunofluorescence microscopy is described, as are protocols for techniques that can be applied to cell lysates containing the target protein (Western blotting and immunoprecipitation). The latter techniques are performed in combination with polyacrylamide gel electrophoresis, thus allowing confirmation of the molecular weight of the target that is recognized by the antiserum.