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Eric J. Snijder - One of the best experts on this subject based on the ideXlab platform.
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a swine Arterivirus deubiquitinase stabilizes two major envelope proteins and promotes production of viral progeny
PLOS Pathogens, 2021Co-Authors: Rui Guo, Eric J. Snijder, Ying Fang, Xingyu Yan, Jin Cui, Saurav Misra, Andrew E FirthAbstract:Arteriviruses are enveloped positive-strand RNA viruses that assemble and egress using the host cell's exocytic pathway. In previous studies, we demonstrated that most Arteriviruses use a unique -2 ribosomal frameshifting mechanism to produce a C-terminally modified variant of their nonstructural protein 2 (nsp2). Like full-length nsp2, the N-terminal domain of this frameshift product, nsp2TF, contains a papain-like protease (PLP2) that has deubiquitinating (DUB) activity, in addition to its role in proteolytic processing of replicase polyproteins. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nsp2TF localizes to compartments of the exocytic pathway, specifically endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and Golgi complex. Here, we show that nsp2TF interacts with the two major viral envelope proteins, the GP5 glycoprotein and membrane (M) protein, which drive the key process of Arterivirus assembly and budding. The PRRSV GP5 and M proteins were found to be poly-ubiquitinated, both in an expression system and in cells infected with an nsp2TF-deficient mutant virus. In contrast, ubiquitinated GP5 and M proteins did not accumulate in cells infected with the wild-type, nsp2TF-expressing virus. Further analysis implicated the DUB activity of the nsp2TF PLP2 domain in deconjugation of ubiquitin from GP5/M proteins, thus antagonizing proteasomal degradation of these key viral structural proteins. Our findings suggest that nsp2TF is targeted to the exocytic pathway to reduce proteasome-driven turnover of GP5/M proteins, thus promoting the formation of GP5-M dimers that are critical for Arterivirus assembly.
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adaptive mutations in replicase transmembrane subunits can counteract inhibition of equine arteritis virus rna synthesis by cyclophilin inhibitors
Journal of Virology, 2019Co-Authors: A.h. De ,wilde, Eric J. Snijder, Montserrat Bárcena, A.w.m. De ,jong, Linda Boomaarsvan Der A Zanden, Clara C PosthumaAbstract:Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse RNA viruses, including Arteriviruses and coronaviruses, which both belong to the order Nidovirales In this study, we aimed to identify Arterivirus proteins involved in the mode of action of cyclophilin inhibitors and to investigate how these compounds inhibit Arterivirus RNA synthesis in the infected cell. Repeated passaging of the Arterivirus prototype equine arteritis virus (EAV) in the presence of CsA revealed that reduced drug sensitivity is associated with the emergence of adaptive mutations in nonstructural protein 5 (nsp5), one of the transmembrane subunits of the Arterivirus replicase polyprotein. Introduction of singular nsp5 mutations (nsp5 Q21R, Y113H, or A134V) led to an ∼2-fold decrease in sensitivity to CsA treatment, whereas combinations of mutations further increased EAV's CsA resistance. The detailed experimental characterization of engineered EAV mutants harboring CsA resistance mutations implicated nsp5 in Arterivirus RNA synthesis. Particularly, in an in vitro assay, EAV RNA synthesis was far less sensitive to CsA treatment when nsp5 contained the adaptive mutations mentioned above. Interestingly, for increased sensitivity to the closely related drug ALV, CsA-resistant nsp5 mutants required the incorporation of an additional adaptive mutation, which resided in nsp2 (H114R), another transmembrane subunit of the Arterivirus replicase. Our study provides the first evidence for the involvement of nsp2 and nsp5 in the mechanism underlying the inhibition of Arterivirus replication by cyclophilin inhibitors.IMPORTANCE Currently, no approved treatments are available to combat infections with nidoviruses, a group of positive-stranded RNA viruses, including important zoonotic and veterinary pathogens. Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse nidoviruses (both Arteriviruses and coronaviruses), and they may thus represent a class of pan-nidovirus inhibitors. In this study, using the Arterivirus prototype equine arteritis virus, we have established that resistance to CsA and ALV treatment is associated with adaptive mutations in two transmembrane subunits of the viral replication machinery, nonstructural proteins 2 and 5. This is the first evidence for the involvement of specific replicase subunits of Arteriviruses in the mechanism underlying the inhibition of their replication by cyclophilin inhibitors. Understanding this mechanism of action is of major importance to guide future drug design, both for nidoviruses and for other RNA viruses inhibited by these compounds.
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Antiviral Innate Immune Response Interferes with the Formation of Replication-Associated Membrane Structures Induced by a Positive-Strand RNA Virus
American Society for Microbiology, 2016Co-Authors: Diede Oudshoorn, Corrine Beugeling, Eric J. Snijder, Barbara Van Der Hoeven, Ronald W A L Limpens, Montserrat Bárcena, Marjolein KikkertAbstract:Infection with nidoviruses like corona- and Arteriviruses induces a reticulovesicular network of interconnected endoplasmic reticulum (ER)-derived double-membrane vesicles (DMVs) and other membrane structures. This network is thought to accommodate the viral replication machinery and protect it from innate immune detection. We hypothesized that the innate immune response has tools to counteract the formation of these virus-induced replication organelles in order to inhibit virus replication. Here we have investigated the effect of type I interferon (IFN) treatment on the formation of Arterivirus-induced membrane structures. Our approach involved ectopic expression of Arterivirus nonstructural proteins nsp2 and nsp3, which induce DMV formation in the absence of other viral triggers of the interferon response, such as replicating viral RNA. Thus, this setup can be used to identify immune effectors that specifically target the (formation of) virus-induced membrane structures. Using large-scale electron microscopy mosaic maps, we found that IFN-β treatment significantly reduced the formation of the membrane structures. Strikingly, we also observed abundant stretches of double-membrane sheets (a proposed intermediate of DMV formation) in IFN-β-treated samples, suggesting the disruption of DMV biogenesis. Three interferon-stimulated gene products, two of which have been reported to target the hepatitis C virus replication structures, were tested for their possible involvement, but none of them affected membrane structure formation. Our study reveals the existence of a previously unknown innate immune mechanism that antagonizes the viral hijacking of host membranes. It also provides a solid basis for further research into the poorly understood interactions between the innate immune system and virus-induced replication structures
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Arterivirus nsp12 versus the coronavirus nsp16 2 o methyltransferase comparison of the c terminal cleavage products of two nidovirus pp1ab polyproteins
Journal of General Virology, 2015Co-Authors: Kathleen C Lehmann, Jessika C Zevenhovendobbe, Alexander E Gorbalenya, Eric J. Snijder, Anastasia A Gulyaeva, Dmitry V Samborskiy, Lisa Hooghiemstra, Clara C PosthumaAbstract: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 β-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.
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genome replication and
2015Co-Authors: Richard Molenkamp, Willy J. M. Spaan, Yvonne Van Der Meer, Hans Van Tol, Babette C. D. Rozier, Eric J. SnijderAbstract:The Arterivirus replicase is the only viral protein required fo
Alexander E Gorbalenya - One of the best experts on this subject based on the ideXlab platform.
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domain organization and evolution of the highly divergent 5 coding region of genomes of Arteriviruses including the novel possum nidovirus
Journal of Virology, 2017Co-Authors: Anastasia A Gulyaeva, Alexander E Gorbalenya, M Dunowska, Erik Hoogendoorn, Julia Giles, Dmitry V SamborskiyAbstract:In five experimentally characterized Arterivirus species, the 5′-end genome coding region encodes the most divergent nonstructural proteins (nsp's), nsp1 and nsp2, which include papain-like proteases (PLPs) and other poorly characterized domains. These are involved in regulation of transcription, polyprotein processing, and virus-host interaction. Here we present results of a bioinformatics analysis of this region of 14 Arterivirus species, including that of the most distantly related virus, wobbly possum disease virus (WPDV), determined by a modified 5′ rapid amplification of cDNA ends (RACE) protocol. By combining profile-profile comparisons and phylogeny reconstruction, we identified an association of the four distinct domain layouts of nsp1-nsp2 with major phylogenetic lineages, implicating domain gain, including duplication, and loss in the early nsp1 evolution. Specifically, WPDV encodes highly divergent homologs of PLP1a, PLP1b, PLP1c, and PLP2, with PLP1a lacking the catalytic Cys residue, but does not encode nsp1 Zn finger (ZnF) and “nuclease” domains, which are conserved in other Arteriviruses. Unexpectedly, our analysis revealed that the only catalytically active nsp1 PLP of equine arteritis virus (EAV), known as PLP1b, is most similar to PLP1c and thus is likely to be a PLP1b paralog. In all non-WPDV Arteriviruses, PLP1b/c and PLP1a show contrasting patterns of conservation, with the N- and C-terminal subdomains, respectively, being enriched with conserved residues, which is indicative of different functional specializations. The least conserved domain of nsp2, the hypervariable region (HVR), has its size varied 5-fold and includes up to four copies of a novel PxPxPR motif that is potentially recognized by SH3 domain-containing proteins. Apparently, only EAV lacks the signal that directs −2 ribosomal frameshifting in the nsp2 coding region. IMPORTANCE Arteriviruses comprise a family of mammalian enveloped positive-strand RNA viruses that include some of the most economically important pathogens of swine. Most of our knowledge about this family has been obtained through characterization of viruses from five species: Equine arteritis virus, Simian hemorrhagic fever virus, Lactate dehydrogenase-elevating virus, Porcine respiratory and reproductive syndrome virus 1, and Porcine respiratory and reproductive syndrome virus 2. Here we present the results of comparative genomics analyses of viruses from all known 14 Arterivirus species, including the most distantly related virus, WPDV, whose genome sequence was completed in this study. Our analysis focused on the multifunctional 5′-end genome coding region that encodes multidomain nonstructural proteins 1 and 2. Using diverse bioinformatics techniques, we identified many patterns of evolutionary conservation that are specific to members of distinct Arterivirus species, both characterized and novel, or their groups. They are likely associated with structural and functional determinants important for virus replication and virus-host interaction.
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Arterivirus nsp12 versus the coronavirus nsp16 2 o methyltransferase comparison of the c terminal cleavage products of two nidovirus pp1ab polyproteins
Journal of General Virology, 2015Co-Authors: Kathleen C Lehmann, Jessika C Zevenhovendobbe, Alexander E Gorbalenya, Eric J. Snijder, Anastasia A Gulyaeva, Dmitry V Samborskiy, Lisa Hooghiemstra, Clara C PosthumaAbstract: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 β-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.
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Domain organization of EAV nsp1.
2013Co-Authors: Danny D. Nedialkova, Alexander E Gorbalenya, Eric J. SnijderAbstract:The partial sequence alignment shows key regions in the three subdomains previously identified in the Arterivirus nsp1 region. GenBank accession numbers for the full-length Arterivirus genomes used for the alignment are as follows: EAV, NC_002532; simian hemorrhagic fever virus (SHFV), NC_003092; lactate dehydrogenase-elevating virus (LDV-P and LDV-C), NC_001639 and NC_002534; PRRSV-LV, M96262.2; PRRSV-VR, AY150564. Zinc-coordinating residues are indicated in bold font; the active-site Cys and His of PCPα and PCPβ are indicated with triangles (note the loss of the active-site Cys in EAV PCPα). The positions of amino acid clusters mutated in this study are indicated with arrows. All substitutions were with Ala, with the exception of the ZCH construct, in which Cys-25 and His-27 were swapped. The positions of mutations found in pseudorevertants are indicated with open circles.
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Nidovirus genome and region size differences.
2013Co-Authors: Chris Lauber, Eric J. Snijder, Jelle J. Goeman, Maria Del Carmen Parquet, Phan Thi Nga, Kouichi Morita, Alexander E GorbalenyaAbstract:Shown are size distributions of genomes (left part) and the three genome coding parts ORF1b, ORF1a and 3′ORFs (right part) for five small-sized Arterivirus species (small), 22 large-sized nidovirus species (large) and one intermediate-sized mesonivirus species (interm.). The distributions are represented by box-and-whisker graphs, where the box spans from the first to the third quartile and includes the median (bold line). The whiskers extend (dashed lines) to the extreme values.
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proteolytic maturation of replicase polyprotein pp1a by the nsp4 main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp7
Journal of General Virology, 2006Co-Authors: Danny Van Aken, Jessika C Zevenhovendobbe, Alexander E Gorbalenya, Eric J. SnijderAbstract:The positive-stranded RNA genome of the Arterivirus Equine arteritis virus (order Nidovirales) encodes the partially overlapping replicase polyproteins pp1a (1727 aa) and pp1ab (3175 aa). Previously, three viral proteinases were reported to cleave these large polyproteins into 12 non-structural proteins (nsps). The chymotrypsin-like viral main proteinase residing in nsp4 is responsible for eight of these cleavages. Processing of the C-terminal half of pp1a (the nsp3-8 region) was postulated to occur following either of two alternative proteolytic pathways (the 'major' and 'minor' pathways). Here, the importance of these two pathways was investigated by using a reverse-genetics system and inactivating each of the cleavage sites by site-directed mutagenesis. For all of these pp1a cleavage sites, mutations that prevented cleavage by the nsp4 proteinase were found to block or severely inhibit EAV RNA synthesis. Furthermore, our studies identified a novel nsp4 cleavage site (Glu-1575/Ala-1576) that is located within nsp7 and is conserved in Arteriviruses. The N-terminal nsp7 fragment (nsp7alpha) derived from this cleavage was detected in lysates of both EAV-infected cells and cells transiently expressing pp1a. Mutagenesis of the novel cleavage site in the context of an EAV full-length cDNA clone proved to be lethal, underlining the fact that the highly regulated, nsp4-mediated processing of the C-terminal half of pp1a is a crucial event in the Arterivirus life cycle.
Clara C Posthuma - One of the best experts on this subject based on the ideXlab platform.
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adaptive mutations in replicase transmembrane subunits can counteract inhibition of equine arteritis virus rna synthesis by cyclophilin inhibitors
Journal of Virology, 2019Co-Authors: A.h. De ,wilde, Eric J. Snijder, Montserrat Bárcena, A.w.m. De ,jong, Linda Boomaarsvan Der A Zanden, Clara C PosthumaAbstract:Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse RNA viruses, including Arteriviruses and coronaviruses, which both belong to the order Nidovirales In this study, we aimed to identify Arterivirus proteins involved in the mode of action of cyclophilin inhibitors and to investigate how these compounds inhibit Arterivirus RNA synthesis in the infected cell. Repeated passaging of the Arterivirus prototype equine arteritis virus (EAV) in the presence of CsA revealed that reduced drug sensitivity is associated with the emergence of adaptive mutations in nonstructural protein 5 (nsp5), one of the transmembrane subunits of the Arterivirus replicase polyprotein. Introduction of singular nsp5 mutations (nsp5 Q21R, Y113H, or A134V) led to an ∼2-fold decrease in sensitivity to CsA treatment, whereas combinations of mutations further increased EAV's CsA resistance. The detailed experimental characterization of engineered EAV mutants harboring CsA resistance mutations implicated nsp5 in Arterivirus RNA synthesis. Particularly, in an in vitro assay, EAV RNA synthesis was far less sensitive to CsA treatment when nsp5 contained the adaptive mutations mentioned above. Interestingly, for increased sensitivity to the closely related drug ALV, CsA-resistant nsp5 mutants required the incorporation of an additional adaptive mutation, which resided in nsp2 (H114R), another transmembrane subunit of the Arterivirus replicase. Our study provides the first evidence for the involvement of nsp2 and nsp5 in the mechanism underlying the inhibition of Arterivirus replication by cyclophilin inhibitors.IMPORTANCE Currently, no approved treatments are available to combat infections with nidoviruses, a group of positive-stranded RNA viruses, including important zoonotic and veterinary pathogens. Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse nidoviruses (both Arteriviruses and coronaviruses), and they may thus represent a class of pan-nidovirus inhibitors. In this study, using the Arterivirus prototype equine arteritis virus, we have established that resistance to CsA and ALV treatment is associated with adaptive mutations in two transmembrane subunits of the viral replication machinery, nonstructural proteins 2 and 5. This is the first evidence for the involvement of specific replicase subunits of Arteriviruses in the mechanism underlying the inhibition of their replication by cyclophilin inhibitors. Understanding this mechanism of action is of major importance to guide future drug design, both for nidoviruses and for other RNA viruses inhibited by these compounds.
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Arterivirus nsp12 versus the coronavirus nsp16 2 o methyltransferase comparison of the c terminal cleavage products of two nidovirus pp1ab polyproteins
Journal of General Virology, 2015Co-Authors: Kathleen C Lehmann, Jessika C Zevenhovendobbe, Alexander E Gorbalenya, Eric J. Snijder, Anastasia A Gulyaeva, Dmitry V Samborskiy, Lisa Hooghiemstra, Clara C PosthumaAbstract: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 β-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.
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discovery of a small Arterivirus gene that overlaps the gp5 coding sequence and is important for virus production
Journal of General Virology, 2011Co-Authors: Andrew E Firth, Jessika C Zevenhovendobbe, Eric J. Snijder, Norma M Wills, Udeni B R Balasuriya, John F Atkins, Clara C PosthumaAbstract: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.
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formation of the Arterivirus replication transcription complex a key role for nonstructural protein 3 in the remodeling of intracellular membranes
Journal of Virology, 2008Co-Authors: Clara C Posthuma, Zhengchun Lu, Ruth G Joosten, Ketil W. Pedersen, Norbert Roos, Jessika C ZevenhovendobbeAbstract: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.
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an infectious recombinant equine arteritis virus expressing green fluorescent protein from its replicase gene
Journal of General Virology, 2007Co-Authors: Erwin Van Den Born, Clara C Posthuma, Kevin Knoops, Eric J. SnijderAbstract:Thus far, systems developed for heterologous gene expression from the genomes of nidoviruses (Arteriviruses and coronaviruses) have relied mainly on the translation of foreign genes from subgenomic mRNAs, whose synthesis is a key feature of the nidovirus life cycle. In general, such expression vectors often suffered from relatively low and unpredictable expression levels, as well as genome instability. In an attempt to circumvent these disadvantages, the possibility to express a foreign gene [encoding enhanced green fluorescent protein (eGFP)] from within the nidovirus replicase gene, which encodes two large polyproteins that are processed proteolytically into the non-structural proteins (nsps) required for viral RNA synthesis, has now been explored. A viable recombinant of the Arterivirus Equine arteritis virus, EAV-GFP2, was obtained, which contained the eGFP insert at the site specifying the junction between the two most N-proximal replicase-cleavage products, nsp1 and nsp2. EAV-GFP2 replication could be launched by transfection of cells with either in vitro-generated RNA transcripts or a DNA launch plasmid. EAV-GFP2 displayed growth characteristics similar to those of the wild-type virus and was found to maintain the insert stably for at least eight passages. It is proposed that EAV-GFP2 has potential for Arterivirus vector development and as a tool in inhibitor screening. It can also be used for fundamental studies into EAV replication, which was illustrated by the fact that the eGFP signal of EAV-GFP2, which largely originated from an eGFP-nsp2 fusion protein, could be used to monitor the formation of the membrane-bound EAV replication complex in real time.
Colin A Chapman - One of the best experts on this subject based on the ideXlab platform.
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exceptional simian hemorrhagic fever virus diversity in a wild african primate community
Journal of Virology, 2013Co-Authors: Michael Lauck, Jens H. Kuhn, William M. Switzer, Samuel D Sibley, David Hyeroba, Alex Tumukunde, Geoffrey Weny, Colin A Chapman, Nelson Ting, Thomas C FriedrichAbstract:Simian hemorrhagic fever virus (SHFV) is an Arterivirus that causes severe disease in captive macaques. We describe two new SHFV variants subclinically infecting wild African red-tailed guenons (Cercopithecus ascanius). Both variants are highly divergent from the prototype virus and variants infecting sympatric red colobus (Procolobus rufomitratus). All known SHFV variants are monophyletic and share three open reading frames not present in other Arteriviruses. Our data suggest a need to modify the current Arterivirus classification.
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Genome organization of novel simian hemorrhagic fever viruses from a Ugandan red colobus monkey.
2013Co-Authors: Michael Lauck, David Hyeroba, Alex Tumukunde, Geoffrey Weny, Colin A Chapman, Thomas C Friedrich, Simon M Lank, David H. O'connor, Tony L. GoldbergAbstract:The novel variants SHFV-krc1 and SHFV-krc2 are shown in comparison to the SHFV type strain LVR 42-0/M6941 and the prototype Arterivirus, equine arteritis virus (EAV-Bucyrus strain). Boxes represent open reading frames and are drawn to scale. Shaded boxes indicate ORFs unique to SHFV, with dashed lines indicating the location of putative insertion relative to the EAV genome.
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novel divergent simian hemorrhagic fever viruses in a wild ugandan red colobus monkey discovered using direct pyrosequencing
PLOS ONE, 2011Co-Authors: Michael Lauck, David Hyeroba, Alex Tumukunde, Geoffrey Weny, Colin A Chapman, Simon M Lank, David H OconnorAbstract:Background Simian hemorrhagic fever virus (SHFV) has caused lethal outbreaks of hemorrhagic disease in captive primates, but its distribution in wild primates has remained obscure. Here, we describe the discovery and genetic characterization by direct pyrosequencing of two novel, divergent SHFV variants co-infecting a single male red colobus monkey from Kibale National Park, Uganda. Methodology/Principal Findings The viruses were detected directly from blood plasma using pyrosequencing, without prior virus isolation and with minimal PCR amplification. The two new SHFV variants, SHFV-krc1 and SHFV-krc2 are highly divergent from each other (51.9% nucleotide sequence identity) and from the SHFV type strain LVR 42-0/M6941 (52.0% and 51.8% nucleotide sequence identity, respectively) and demonstrate greater phylogenetic diversity within SHFV than has been documented within any other Arterivirus. Both new variants nevertheless have the same 3′ genomic architecture as the type strain, containing three open reading frames not present in the other Arteriviruses. Conclusions/Significance These results represent the first documentation of SHFV in a wild primate and confirm the unusual 3′ genetic architecture of SHFV relative to the other Arteriviruses. They also demonstrate a degree of evolutionary divergence within SHFV that is roughly equivalent to the degree of divergence between other Arterivirus species. The presence of two such highly divergent SHFV variants co-infecting a single individual represents a degree of within-host viral diversity that exceeds what has previously been reported for any Arterivirus. These results expand our knowledge of the natural history and diversity of the Arteriviruses and underscore the importance of wild primates as reservoirs for novel pathogens.
Erwin Van Den Born - One of the best experts on this subject based on the ideXlab platform.
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an infectious recombinant equine arteritis virus expressing green fluorescent protein from its replicase gene
Journal of General Virology, 2007Co-Authors: Erwin Van Den Born, Clara C Posthuma, Kevin Knoops, Eric J. SnijderAbstract:Thus far, systems developed for heterologous gene expression from the genomes of nidoviruses (Arteriviruses and coronaviruses) have relied mainly on the translation of foreign genes from subgenomic mRNAs, whose synthesis is a key feature of the nidovirus life cycle. In general, such expression vectors often suffered from relatively low and unpredictable expression levels, as well as genome instability. In an attempt to circumvent these disadvantages, the possibility to express a foreign gene [encoding enhanced green fluorescent protein (eGFP)] from within the nidovirus replicase gene, which encodes two large polyproteins that are processed proteolytically into the non-structural proteins (nsps) required for viral RNA synthesis, has now been explored. A viable recombinant of the Arterivirus Equine arteritis virus, EAV-GFP2, was obtained, which contained the eGFP insert at the site specifying the junction between the two most N-proximal replicase-cleavage products, nsp1 and nsp2. EAV-GFP2 replication could be launched by transfection of cells with either in vitro-generated RNA transcripts or a DNA launch plasmid. EAV-GFP2 displayed growth characteristics similar to those of the wild-type virus and was found to maintain the insert stably for at least eight passages. It is proposed that EAV-GFP2 has potential for Arterivirus vector development and as a tool in inhibitor screening. It can also be used for fundamental studies into EAV replication, which was illustrated by the fact that the eGFP signal of EAV-GFP2, which largely originated from an eGFP-nsp2 fusion protein, could be used to monitor the formation of the membrane-bound EAV replication complex in real time.
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secondary structure and function of the 5 proximal region of the equine arteritis virus rna genome
RNA, 2004Co-Authors: Erwin Van Den Born, Alexander P Gultyaev, Eric J. SnijderAbstract:Nidoviruses produce an extensive 3'-coterminal nested set of subgenomic mRNAs, which are used to express their structural proteins. In addition, Arterivirus and coronavirus mRNAs contain a common 5' leader sequence, derived from the genomic 5' end. The joining of this leader sequence to different segments (mRNA bodies) from the genomic 3'-proximal region presumably involves a unique mechanism of discontinuous minus-strand RNA synthesis. Key elements in this process are the so-called transcription-regulating sequences (TRSs), which determine a base-pairing interaction between sense and antisense viral RNA that is essential for leader-to-body joining. To identify RNA structures in the 5'-proximal region of the equine arteritis virus genome that may be involved in subgenomic mRNA synthesis, a detailed secondary RNA structure model was established using bioinformatics, phylogenetic analysis, and RNA structure probing. According to this structure model, the leader TRS is located in the loop of a prominent hairpin (leader TRS hairpin; LTH). The importance of the LTH was supported by the results of a mutagenesis study using an EAV molecular clone. Besides evidence for a direct role of the LTH in subgenomic RNA synthesis, indications for a role of the LTH region in genome replication and/or translation were obtained. Similar LTH structures could be predicted for the 5'-proximal region of all Arterivirus genomes and, interestingly, also for most coronaviruses. Thus, we postulate that the LTH is a key structural element in the discontinuous subgenomic RNA synthesis and is likely critical for leader TRS function.
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The stability of the duplex between sense and antisense transcription-regulating sequences is a crucial factor in Arterivirus subgenomic mRNA synthesis.
Journal of virology, 2003Co-Authors: Alexander O. Pasternak, Erwin Van Den Born, Willy J. M. SpaanAbstract:Subgenomic mRNAs of nidoviruses (Arteriviruses and coronaviruses) are composed of a common leader sequence and a "body" part of variable size, which are derived from the 5'- and 3'-proximal part of the genome, respectively. Leader-to-body joining has been proposed to occur during minus-strand RNA synthesis and to involve transfer of the nascent RNA strand from one site in the template to another. This discontinuous step in subgenomic RNA synthesis is guided by short transcription-regulating sequences (TRSs) that are present at both these template sites (leader TRS and body TRS). Sense-antisense base pairing between the leader TRS in the plus strand and the body TRS complement in the minus strand is crucial for strand transfer. Here we show that extending the leader TRS-body TRS duplex beyond its wild-type length dramatically enhanced the subgenomic mRNA synthesis of the Arterivirus Equine arteritis virus (EAV). Generally, the relative amount of a subgenomic mRNA correlated with the calculated stability of the corresponding leader TRS-body TRS duplex. In addition, various leader TRS mutations induced the generation of minor subgenomic RNA species that were not detected upon infection with wild-type EAV. The synthesis of these RNA species involved leader-body junction events at sites that bear only limited resemblance to the canonical TRS. However, with the mutant leader TRS, but not with the wild-type leader TRS, these sequences could form a duplex that was stable enough to direct subgenomic RNA synthesis, again demonstrating that the stability of the leader TRS-body TRS duplex is a crucial factor in Arterivirus subgenomic mRNA synthesis.