Open Reading Frame 1a-Encoded Subunits of the Arterivirus Replicase Induce Endoplasmic Reticulum-Derived Double-Membrane Vesicles Which Carry the Viral Replication Complex

Pedersen, Ketil W.; Meer, Yvonne van der; Roos, Norbert; Snijder, Eric J.

doi:10.1128/jvi.73.3.2016-2026.1999

Cited by 265 publications

(272 citation statements)

References 62 publications

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“…The conservation of these domains within the nidoviruses further emphasizes their significance. The hydrophobic domains in the arteriviruses have been shown to dramatically alter the host-cell membrane architecture in addition to mediating the association of the replication complex with cellular membranes (Pedersen et al, 1999;van der Meer et al, 1998). A similar role can be conjectured in the toroviruses.…”

Section: Discussionmentioning

confidence: 91%

The complete sequence of the bovine torovirus genome

et al. 2006

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Viruses in the family Coronaviridae have elicited new interest, with the outbreaks caused by SARS-HCoV in 2003 and the recent discovery of a new human coronavirus, HCoV-NL63. The genus Torovirus, within the family Coronaviridae, is less well characterized, in part because toroviruses cannot yet be grown in cell culture (except for the Berne virus). In this study, we determined the sequence of the complete genome of Breda-1 (BoTV-1), a bovine torovirus. This is the first complete torovirus genome sequence to be reported. BoTV-1 RNA was amplified using long RT-PCR and the amplicons sequenced. The genome has a length of 28.475 kb and consisted mainly of the replicase gene ( approximately 20.2 kb) which contains two large overlapping ORFs, ORF1a and ORF1b, encoding polyproteins pp1a and pp1b, respectively. Sequence analysis identified conserved domains within the predicted sequences of pp1a and pp1b. Sequence alignments and protein secondary structure prediction data suggest the presence of a 3C-like serine protease domain with similarity to the arterivirus 3C-like serine protease and a single papain-like cysteine protease domain with similarity to the picornavirus leader protease. The ADRP (APPR-1'') domain - unique to the Coronaviridae - was also located in BoTV pp1a. In addition, several hydrophobic domains were identified that are typical of a nidovirus replicase. Within the pp1b sequence the polymerase and helicase domains were identified, as well as sequences predicted to be involved in ribosomal frameshifting, including the conserved slippery sequence UUUAAAC and two potential pseudoknot structures.

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Section: Discussionmentioning

confidence: 91%

The complete sequence of the bovine torovirus genome

et al. 2006

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“…5A-B; van der Meer et al, 1998;Kroese et al, 2008;Fang et al, unpublished data). They are associated with intracellular membranes, likely derived from the endoplasmic reticulum (ER), which are modified into vesicular double-membrane structures with which the viral replication and transcription complex (RTC) is thought to be associated (Pedersen et al, 1999). The formation of closely paired membranes and double membrane vesicles (DMVs; Fig.…”

Section: Hydrophobic Nsps Inducing Extensive Reorganization Of Intracmentioning

confidence: 99%

“…The key enzymes for arterivirus RNA synthesis are encoded in ORF1b, in particular the viral RNA-dependent RNA polymerase (RdRp; nsp9) and RNA helicase (Hel; nsp10). Together with putative "accessory subunits", these enzymes assemble into a membrane-associated viral replication and transcription complex (RTC; Pedersen et al, 1999), which mediates both genome replication and the synthesis of a nested set of subgenomic (sg) mRNAs. The latter, a hallmark of all nidoviruses, form a 5 -and 3 -coterminal nested set (de Vries et al, 1990) and is required to express the arterivirus structural protein genes (Snijder and Meulenberg, 1998;Pasternak et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

The PRRSV replicase: Exploring the multifunctionality of an intriguing set of nonstructural proteins

2010

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Our knowledge about the structure and function of the nonstructural proteins (nsps) encoded by the arterivirus replicase gene has advanced in recent years. The continued characterization of the nsps of the arterivirus prototype equine arteritis virus has not only corroborated several important functional predictions, but also revealed various novel features of arteriviral replication. For porcine reproductive and respiratory syndrome virus (PRRSV), based on bioinformatics predictions and experimental studies, a processing map for the pp1a and pp1ab replicase polyproteins has been developed. Crystal structures have been resolved for two of the PRRSV nonstructural proteins that possess proteinase activity (nsp1α and nsp4). The functional characterization of the key enzymes for arterivirus RNA synthesis, the nsp9 RNA polymerase and nsp10 helicase, has been initiated. In addition, progress has been made on nsp functions relating to the regulation of subgenomic mRNAs synthesis (nsp1), the induction of replication-associated membrane rearrangements (nsp2 and nsp3), and an intriguing replicative endoribonuclease (nsp11) for which the natural substrate remains to be identified. The role of nsps in viral pathogenesis and host immunity is also being explored, and specific nsps (including nsp1α/β, nsp2, nsp4, nsp7, and nsp11) have been implicated in the modulation of host immune responses to PRRSV infection. The nsp3-8 region was identified as containing major virulence factors, although mechanistic information is scarce. The biological significance of PRRSV nsps in virus-host interactions and the technical advancements in engineering the PRRSV genome by reverse genetics are also reflected in recent developments in the area of vaccines and diagnostic assays.

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“…Based on considerable research on equine arteritis virus (EAV), the nsp2 protein is processed from the ORF1 protein by the action of the papain-like protease in nsp1␤ and the self-encoded PL2 cysteine protease, and acts as a co-factor for the nsp4 serine protease (3CL) in processing downstream viral cleavages (den Boon et al, 1995;Snijder et al, 1994Snijder et al, , 1995avan Hemert and Snijder, 2008;Wassenaar et al, 1997). In addition, EAV nsp2 induces double membrane vesicles, probably through its predicted transmembrane spanning regions, and serves as an anchor for the virus replication complex (Pedersen et al, 1999;Snijder et al, 2001). Nsp2 of both genotypes of PRRSV is considerably larger than that of EAV and contains, in addition to the signature motifs described above, an extended N-terminal hypervariable region (HV1) and a middle hypervariable region (HV2).…”

Section: Introductionmentioning

confidence: 99%

In vivo growth of porcine reproductive and respiratory syndrome virus engineered nsp2 deletion mutants

et al. 2010

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Prior studies on PRRSV strain VR-2332 non-structural protein 2 (nsp2) had shown that as much as 403 amino acids could be removed from the hypervariable region without losing virus viability in vitro. We utilized selected nsp2 deletion mutants to examine in vivo growth. Young swine (4 pigs/group; 5 control swine) were inoculated intramuscularly with one of 4 nsp2 deletion mutants (rΔ727-813, rΔ543-726, rΔ324-523, rΔ324-726) or full-length recombinant virus (rVR-2332). Serum samples were collected on various days post-inoculation and analyzed by HerdChek* ELISA, PRRSV real time RT-PCR, gamma interferon (IFN-γ) ELISA, and nucleotide sequence analysis of the entire nsp2 coding region. Tracheobronchial lymph node weight compared to body weight was recorded for each animal and used as a clinical measurement of viral pathogenesis. Results showed that all deletion mutants grew less robustly than full-length recombinant virus, yet all but the large deletion virus (rΔ324-726) recovered to parental viral RNA levels by study end. Swine receiving the rΔ727-813 mutants had a significant decrease in lymph node enlargement compared to rVR-2332. While swine infection with rVR-2332 caused a rapid rise in serum IFN-γ levels, the IFN-γ protein produced by infection with 3 of the 4 deletion mutant viruses was significantly reduced, perhaps due to differences in viral growth kinetics. The rΔ543-726 nsp2 mutant virus, although growth impaired, mimicked rVR-2332 in inducing a host serum IFN-γ response but exhibited a 2-week delay. Targeted sequencing showed that all deletions were stable in the region coding for nsp2 after one swine passage. The data suggested that the selected nsp2 deletion mutants were growth attenuated in swine, altered the induction of serum IFN-γ, an innate cytokine of unknown function in PRRSV clearance, and pointed to a domain that may influence tracheobronchial lymph node size.

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Open Reading Frame 1a-Encoded Subunits of the Arterivirus Replicase Induce Endoplasmic Reticulum-Derived Double-Membrane Vesicles Which Carry the Viral Replication Complex

Cited by 265 publications

References 62 publications

The complete sequence of the bovine torovirus genome

The complete sequence of the bovine torovirus genome

The PRRSV replicase: Exploring the multifunctionality of an intriguing set of nonstructural proteins

In vivo growth of porcine reproductive and respiratory syndrome virus engineered nsp2 deletion mutants

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