Crystal Structure of Porcine Reproductive and Respiratory Syndrome Virus Leader Protease Nsp1α

Sun, Yuna; X, Fei; Guo, Yu; Ma, Ming; Hao, Nanjing; Zhang, Xuejun C.; Lou, Zhiyong; Li, Xuemei; Rao, Zihe

doi:10.1128/jvi.02579-08

Cited by 62 publications

(96 citation statements)

References 37 publications

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“…Lines of evidence have shown that nsp1 is processed in vitro into nsp1␣ and nsp1␤ by cis-active PCP1␣ and PCP1␤, respectively, and that this processing occurs cotranslationally and rapidly (8,30,34). The nsp2 protein of PRRSV strain VR-2332, situated immediately downstream of nsp1, is a large multidomain protein of about 1,197 amino acids (aa) containing an N-terminal PL2 cysteine protease, a 500-to 700-aa middle hypervariable region, a putative transmembrane (TM) domain, and a C-terminal tail (13,15).…”

mentioning

confidence: 99%

Proteolytic Products of the Porcine Reproductive and Respiratory Syndrome Virus nsp2 Replicase Protein

Han

Rutherford²,

Faaberg

2010

J Virol

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The nsp2 replicase protein of porcine reproductive and respiratory syndrome virus (PRRSV) was recently demonstrated to be processed from its precursor by the PL2 protease at or near the G 1196 ԽG 1197 dipeptide in transfected CHO cells. Here the proteolytic cleavage of PRRSV nsp2 was further investigated in virally infected MARC-145 cells by using two recombinant PRRSVs expressing epitope-tagged nsp2. The data revealed that PRRSV nsp2 exists as different isoforms, termed nsp2a, nsp2b, nsp2c, nsp2d, nsp2e, and nsp2f, during PRRSV infection. Moreover, on the basis of deletion mutagenesis and antibody probing, these nsp2 species appeared to share the same N terminus but to differ in their C termini. The largest protein, nsp2a, corresponded to the nsp2 product identified in transfected CHO cells. nsp2b and nsp2c were processed within or near the transmembrane (TM) region, presumably at or near the conserved sites G 981 ԽG 982 and G 828 ԽG 829 ԽG 830 , respectively. The C termini for nsp2d, -e, and -f were mapped within the nsp2 middle hypervariable region, but no conserved cleavage sites could be definitively predicted. The larger nsp2 species emerged almost simultaneously in the early stage of PRRSV infection. Pulse-chase analysis revealed that all six nsp2 species were relatively stable and had low turnover rates. Deletion mutagenesis revealed that the smaller nsp2 species (e.g., nsp2d, nsp2e, and nsp2f) were not essential for viral replication in cell culture. Lastly, we identified a cellular chaperone, named heat shock 70-kDa protein 5 (HSPA5), that was strongly associated with nsp2, which may have important implications for PRRSV replication. Overall, these findings indicate that PRRSV nsp2 is increasingly emerging as a multifunctional protein and may have a profound impact on PRRSV replication and viral pathogenesis.

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mentioning

confidence: 99%

Proteolytic Products of the Porcine Reproductive and Respiratory Syndrome Virus nsp2 Replicase Protein

Han

Rutherford²,

Faaberg

2010

J Virol

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“…cleaved internally into nsp1␣ and nsp1␤ in the case of PRRSV (and also LDV;denBoon et al, 1995). In type 2 PRRSV, previous studies revealed that the nsp1␣/1␤ cleavage site is located at 180 M/A 181 (P1/P1 ; Chen et al, 2010;Sun et al, 2009). In this study, using the type 1 virus SD01-08, we identified 180 H/S 181 as the corresponding cleavage site in the other PRRSV genotype.…”

Section: Discussionmentioning

confidence: 76%

“…For genotype 2 PRRSV, the nsp1␣/1␤ cleavage site was previously identified to be 180 M/A 181 Sun et al, 2009), but these P1 and P1 residues are not conserved in genotype 1 viruses ( Fig. 2A).…”

Section: Proteolytic Processing Between Prrsv Nsp1˛ and Nsp1ǐmentioning

confidence: 93%

“…Important differences in ORF1a expression have already been discovered, including the number of ORF1a-encoded proteases involved, three in EAV in contrast to four in PRRSV. In addition to the papain-like protease (PLP1␤) cleaving the nsp1␤/nsp2 junction , the nsp1 region of PRRSV (and LDV) was found to contain an additional papain-like proteinase (PLP1␣), which has apparently lost its activity in EAV and mediates internal cleavage of the PRRSV/LDV nsp1 sequence into nsp1␣ and nsp1␤ (den Boon et al, 1995;Sun et al, 2009;Chen et al, 2010;Fig. 1).…”

Section: Introductionmentioning

confidence: 96%

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Proteolytic processing of the porcine reproductive and respiratory syndrome virus replicase

et al. 2015

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The porcine reproductive and respiratory syndrome virus (PRRSV) replicase polyproteins pp1a and pp1ab are proteolytically processed by four proteases encoded in ORF1a. In this study, a large set of PRRSV replicase cleavage products were identified and pp1a cleavage sites were verified by using a combination of bioinformatics, proteomics, immunoprecipitation, and site-directed mutagenesis. For genotype 1 PRRSV (isolate SD01-08), proteomic analysis identified H180/S181, G385/A386, and G1446/A1447 as the cleavage sites separating nsp1α/1β, nsp1β/nsp2, and nsp2/nsp3, respectively. Transient expression of nsp2-8, nsp3-8, nsp4-8, nsp5-8 (using the recombinant vaccinia virus/T7 RNA polymerase system) and immunoprecipitation identified the cleavage end products nsp2, nsp3, nsp4, nsp7α and nsp7β, and various processing intermediates. Our studies also revealed the existence of alternative proteolytic processing pathways for the processing of the nsp3-8 region, depending on the presence or absence of nsp2 as a co-factor. The identity of most cleavage products was further corroborated by site-directed mutagenesis of individual cleavage sites in constructs expressing nsp3-8 or nsp4-8. This study constitutes the first in-depth experimental analysis of PRRSV replicase processing and the data are discussed against the background of the processing scheme previously derived for the arterivirus prototype, the distantly related equine arteritis virus (EAV). Despite several differences between the two viruses, of which the functional significance remains to be studied, our study demonstrates the general conservation of the replicase pp1a processing scheme between EAV and PRRSV, and likely also the other members of the arterivirus family.

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“…RT-PCR was performed to amplify the full-length cDNA using sets of specific primers for hpPRRSV, as described previously [26]. The full-length genome was divided into 18 overlapping cDNA segments.…”

Section: Rna Isolation Rt-pcr and Sequence Analysismentioning

confidence: 99%

An infectious clone of the highly pathogenic porcine reproductive and respiratory syndrome virus: Topology of glycoprotein 3 (GP3) addressing the intrachain disulfide bonds

et al. 2011

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The highly pathogenic porcine reproductive and respiratory virus (hpPRRSV) with discontinuous 30 amino acid (aa) deletion as a gene marker has caused great economic loss in pig industry and since 2007 has become the dominant strain prevalent in China and Vietnam since 2007. Reverse genetics method is a powerful tool urgently needed to be used on the hpPRRSV to study the intriguing molecular mechanism of transcription, replication and the virulence determinant factors. In our study, we successfully constructed a full-length infectious clone, prBJSY07, based on hpPRRSV isolate, BJSY07. The rescued virus, vrBJSY07, showed similar growth characters to those of the parental virus, BJSY07. We also found that a rescued virus vBJSY07 generated from pBJSY07 was viable but displayed decreased and delayed reproductive capacity, which might be caused by two amino acids mutations, S83C and S117C in glycoprotein 3 (GP3), acquired during the preparation of the infectious clone. The topology of the wild type GP3 and the mutant were further analyzed by bioinformatics and revealed that the mutated GP3 possessed slightly altered structure, most likely by forming a new disulfide bond between the two new cysteine residues. As GP3 is a cysteine-rich glycoprotein, common for viral glycoproteins, our results show that GP3 can accommodate even more cysteine mutations. Based on this, a topological model of GP3 is proposed by addressing the intrachain disulfides.

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Crystal Structure of Porcine Reproductive and Respiratory Syndrome Virus Leader Protease Nsp1α

Cited by 62 publications

References 37 publications

Proteolytic Products of the Porcine Reproductive and Respiratory Syndrome Virus nsp2 Replicase Protein

Proteolytic Products of the Porcine Reproductive and Respiratory Syndrome Virus nsp2 Replicase Protein

Proteolytic processing of the porcine reproductive and respiratory syndrome virus replicase

An infectious clone of the highly pathogenic porcine reproductive and respiratory syndrome virus: Topology of glycoprotein 3 (GP3) addressing the intrachain disulfide bonds

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