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Kay S Faaberg - One of the best experts on this subject based on the ideXlab platform.
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porcine reproductive and respiratory syndrome virus Nonstructural Protein 2 nsp2 topology and selective isoform integration in artificial membranes
Virology, 2015Co-Authors: Matthew A Kappes, Cathy L Miller, Kay S FaabergAbstract:The membrane insertion and topology of Nonstructural Protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) strain VR-2332 was assessed using a cell free translation system in the presence or absence of artificial membranes. Expression of PRRSV nsp2 in the absence of all other viral factors resulted in the genesis of both full-length nsp2 as well as a select number of C-terminal nsp2 isoforms. Addition of membranes to the translation stabilized the translation reaction, resulting in predominantly full-length nsp2 as assessed by immunoprecipitation. Analysis further showed full-length nsp2 strongly associates with membranes, along with two additional large nsp2 isoforms. Membrane integration of full-length nsp2 was confirmed through high-speed density fractionation, protection from protease digestion, and immunoprecipitation. The results demonstrated that nsp2 integrated into the membranes with an unexpected topology, where the amino (N)-terminal (cytoplasmic) and C-terminal (luminal) domains were orientated on opposite sides of the membrane surface.
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the votu domain of highly pathogenic porcine reproductive and respiratory syndrome virus displays a differential substrate preference
Virology, 2014Co-Authors: Michelle K Deaton, Allyn Spear, Kay S Faaberg, Scott D PeganAbstract:Arterivirus genus member Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically devastating disease, recently exacerbated by the emergence of highly pathogenic strains (HP-PRRSV). Within the Nonstructural Protein 2 of PRRSV is a deubiquitinating enzyme domain belonging to the viral ovarian tumor (vOTU) protease superfamily. vOTUs, which can greatly vary in their preference for their host ubiquitin (Ub) and Ub-like substrates such as interferon stimulated gene 15 (ISG15), have been implicated as a potential virulence factor. Since various strains of PRRSV have large variations in virulence, the specificity of vOTUs from two PRRSV strains of varying virulence were determined. While both vOTUs showed de-ubiquitinating activity and markedly low deISGylating activity, HP-PRRSV demonstrated a strong preference for lysine 63-linked poly-Ubiquitin, tied to innate immune response regulation. This represents the first report of biochemical activity unique to HP-PRRSV that has implications for a potential increase in immunosuppression and virulence.
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highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of Nonstructural Protein 2 into virions
Journal of Virology, 2013Co-Authors: Matthew A Kappes, Cathy L Miller, Kay S FaabergAbstract:Viral structural Proteins form the critical intermediary between viral infection cycles within and between hosts, function to initiate entry, participate in immediate early viral replication steps, and are major targets for the host adaptive immune response. We report the identification of Nonstructural Protein 2 (nsp2) as a novel structural component of the porcine reproductive and respiratory syndrome virus (PRRSV) particle. A set of custom α-nsp2 antibodies targeting conserved epitopes within four distinct regions of nsp2 (the PLP2 protease domain [OTU], the hypervariable domain [HV], the putative transmembrane domain [TM], and the C-terminal region [C]) were obtained commercially and validated in PRRSV-infected cells. Highly purified cell-free virions of several PRRSV strains were isolated through multiple rounds of differential density gradient centrifugation and analyzed by immunoelectron microscopy (IEM) and Western blot assays using the α-nsp2 antibodies. Purified viral preparations were found to contain pleomorphic, predominantly spherical virions of uniform size (57.9 nm ± 8.1 nm diameter; n = 50), consistent with the expected size of PRRSV particles. Analysis by IEM indicated the presence of nsp2 associated with the viral particle of diverse strains of PRRSV. Western blot analysis confirmed the presence of nsp2 in purified viral samples and revealed that multiple nsp2 isoforms were associated with the virion. Finally, a recombinant PRRSV genome containing a myc-tagged nsp2 was used to generate purified virus, and these particles were also shown to harbor myc-tagged nsp2 isoforms. Together, these data identify nsp2 as a virion-associated structural PRRSV Protein and reveal that nsp2 exists in or on viral particles as multiple isoforms.
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genomic sequence and virulence comparison of four type 2 porcine reproductive and respiratory syndrome virus strains
Virus Research, 2012Co-Authors: Susan L Brockmeier, Crystal L Loving, A C Vorwald, Marcus E Kehrli, Rodney B Baker, Tracy L Nicholson, Kelly M Lager, Laura C Miller, Kay S FaabergAbstract:Porcine reproductive and respiratory syndrome virus (PRRSV) is a ubiquitous and costly virus that exhibits substantial sequence and virulence disparity among diverse isolates. In this study, we compared the whole genomic sequence and virulence of 4 Type 2 PRRSV isolates. Among the 4 isolates, SDSU73, MN184, and NADC30 were all clearly more virulent than NADC31, and among the 3 more virulent isolates, there were subtle differences based on viral replication, lung lesions, lymphadenopathy, febrile response, decreased weight gains, and cytokine responses in the lung. Lesions consistent with bacterial bronchopneumonia were present to varying degrees in pigs infected with PRRSV, and bacteria typically associated with the porcine respiratory disease complex were isolated from the lung of these pigs. Genomic sequence evaluation indicates that SDSU73 is most similar to the nucleotide sequence of JA142, the parental strain of Ingelvac(®) PRRS ATP, while the nucleotide sequences of NADC30 and NADC31 are more similar to strain MN184. Both the NADC30 and NADC31 isolates of PRRSV, isolated in 2008, maintain the Nonstructural Protein 2 (nsp2) deletion seen in MN184 that was isolated in 2001, but NADC31 has two additional 15 and 36 nucleotide deletions, and these strains are 8-14% different on a nucleotide basis from the MN184 strain. These results indicate that newer U.S. Type 2 strains still exhibit variability in sequence and pathogenicity and although PRRSV strains appear to be reducing the size of the nsp2 over time, this does not necessarily mean that the strain is more virulent.
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the porcine reproductive and respiratory syndrome virus nsp2 cysteine protease domain possesses both trans and cis cleavage activities
Journal of Virology, 2009Co-Authors: Jun Han, Mark S Rutherford, Kay S FaabergAbstract:The N terminus of the replicase Nonstructural Protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a putative cysteine protease domain (PL2). Previously, we demonstrated that deletion of either the PL2 core domain (amino acids [aa] 47 to 180) or the immediate downstream region (aa 181 to 323) is lethal to the virus. In this study, the PL2 domain was found to encode an active enzyme that mediates efficient processing of nsp2-3 in CHO cells. The PL2 protease possessed both trans- and cis-cleavage activities, which were distinguished by individual point mutations in the protease domain. The minimal size required to maintain these two enzymatic activities included nsp2 aa 47 to 240 (Tyr(47) to Cys(240)) and aa 47 to 323 (Tyr(47) to Leu(323)), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys(55)- His(124) catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys(111), Cys(142), and Cys(147)). The conserved aspartic acids (e.g., Asp(89)) were essential for the PL2 protease trans-cleavage activity. Reverse genetics revealed that the PL2 trans-cleavage activity played an important role in the PRRSV replication cycle in that mutations that impaired the PL2 protease trans function, but not the cis activity, were detrimental to viral viability. Lastly, the potential nsp2/3 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G(1196)|G(1197) dipeptide.
Ying Fang - 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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porcine reproductive and respiratory syndrome virus Nonstructural Protein 2 contributes to nf κb activation
Virology Journal, 2012Co-Authors: Ying Fang, Liurong Fang, Dang Wang, Huanchun Chen, Yang Wang, Yingying Lei, Rui Luo, Shaobo XiaoAbstract:Nuclear factor-kappaB (NF-κB) is an inducible transcription factor that plays a key role in inflammation and immune responses, as well as in the regulation of cell proliferation and survival. Previous studies by our group and others have demonstrated that porcine reproductive and respiratory syndrome virus (PRRSV) infection could activate NF-κB in MARC-145 cells and alveolar macrophages. The nucleocapsid (N) Protein was identified as an NF-κB activator among the structural Proteins encoded by PRRSV; however, it remains unclear whether the Nonstructural Proteins (Nsps) contribute to NF-κB activation. In this study, we identified which Nsps can activate NF-κB and investigated the potential mechanism(s) by which they act. By screening the individual Nsps of PRRSV strain WUH3, Nsp2 exhibited great potential to activate NF-κB in MARC-145 and HeLa cells. Overexpression of Nsp2 induced IκBα degradation and nuclear translocation of NF-κB. Furthermore, Nsp2 also induced NF-κB-dependent inflammatory factors, including interleukin (IL)-6, IL-8, COX-2, and RANTES. Compared with the Nsp2 of the classical PRRSV strain, the Nsp2 of highly pathogenic PRRSV (HP-PRRSV) strains that possess a 30 amino acid (aa) deletion in Nsp2 displayed greater NF-κB activation. However, the 30-aa deletion was demonstrated to not be associated with NF-κB activation. Further functional domain analyses revealed that the hypervariable region (HV) of Nsp2 was essential for NF-κB activation. Taken together, these data indicate that PRRSV Nsp2 is a multifunctional Protein participating in the modulation of host inflammatory response, which suggests an important role of Nsp2 in pathogenesis and disease outcomes.
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Nonstructural Protein 2 of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon stimulated gene 15
Journal of Virology, 2012Co-Authors: Zhi Sun, Russell Ransburgh, Eric J Snijder, Ying FangAbstract:Type I interferon (alpha/beta interferon [IFN-α/β]) stimulates the expression of interferon-stimulated gene 15 (ISG15), which encodes a ubiquitin-like Protein, ISG15. Free ISG15 and ISG15 conjugates function in diverse cellular pathways, particularly regulation of antiviral innate immune responses. In this study, we demonstrate that ISG15 overexpression inhibits porcine reproductive and respiratory syndrome virus (PRRSV) replication in cell culture and that the antiviral activity of interferon is reduced by inhibition of ISG15 conjugation. PRRSV Nonstructural Protein 2 (nsp2) was previously identified as a potential antagonist of ISG15 production and conjugation. The Protein contains a papain-like protease domain (PLP2) that plays a crucial role in the proteolytic cleavage of the PRRSV replicase polyProteins. PLP2 was also proposed to belong to the ovarian tumor domain-containing superfamily of deubiquitinating enzymes (DUBs), which is capable of inhibiting ISG15 production and counteracting ISG15 conjugation to cellular Proteins. To determine whether this immune antagonist function could be selectively inactivated, we engineered a panel of mutants with deletions and/or mutations at the N-terminal border of the nsp2 PLP2-DUB domain. A 23-amino-acid deletion (amino acids 402 to 424 of the ORF1a-encoded Protein) largely abolished the inhibitory effect of nsp2 on ISG15 production and conjugation, but no viable recombinant virus was recovered. A 19-amino-acid deletion (amino acids 402 to 420), in combination with a downstream point mutation (S465A), partially relieved the ISG15 antagonist function and yielded a viable recombinant virus. Taken together, our data demonstrate that ISG15 and ISGylation play an important role in the response to PRRSV infection and that nsp2 is a key factor in counteracting the antiviral function of ISG15.
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porcine reproductive and respiratory syndrome virus Nonstructural Protein 2 contributes to nf κb
2012Co-Authors: Ying Fang, Liurong Fang, Dang Wang, Huanchun Chen, Yang Wang, Yingying Lei, Rui Luo, Shaobo XiaoAbstract:Background: Nuclear factor-kappaB (NF-κB) is an inducible transcription factor that plays a key role in inflammation and immune responses, as well as in the regulation of cell proliferation and survival. Previous studies by our group and others have demonstrated that porcine reproductive and respiratory syndrome virus (PRRSV) infection could activate NF-κB in MARC-145 cells and alveolar macrophages. The nucleocapsid (N) Protein was identified as an NF-κB activator among the structural Proteins encoded by PRRSV; however, it remains unclear whether the Nonstructural Proteins (Nsps) contribute to NF-κB activation. In this study, we identified which Nsps can activate NF-κB and investigated the potential mechanism(s) by which they act. Results: By screening the individual Nsps of PRRSV strain WUH3, Nsp2 exhibited great potential to activate NF-κ Bi n MARC-145 and HeLa cells. Overexpression of Nsp2 induced IκBα degradation and nuclear translocation of NF-κB. Furthermore, Nsp2 also induced NF-κB-dependent inflammatory factors, including interleukin (IL)-6, IL-8, COX-2, and RANTES. Compared with the Nsp2 of the classical PRRSV strain, the Nsp2 of highly pathogenic PRRSV (HP-PRRSV) strains that possess a 30 amino acid (aa) deletion in Nsp2 displayed greater NF-κB activation. However, the 30-aa deletion was demonstrated to not be associated with NF-κB activation. Further functional domain analyses revealed that the hypervariable region (HV) of Nsp2 was essential for NF-κB activation. Conclusions: Taken together, these data indicate that PRRSV Nsp2 is a multifunctional Protein participating in the modulation of host inflammatory response, which suggests an important role of Nsp2 in pathogenesis and disease outcomes.
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the cysteine protease domain of porcine reproductive and respiratory syndrome virus Nonstructural Protein 2 possesses deubiquitinating and interferon antagonism functions
Journal of Virology, 2010Co-Authors: Zhi Sun, Zhenhai Chen, Steven R Lawson, Ying FangAbstract:Porcine reproductive and respiratory syndrome (PRRS) virus Nonstructural Protein 2 (nsp2) contains a cysteine protease domain at its N terminus, which belongs to the ovarian tumor (OTU) protease family. In this study, we demonstrated that the PRRSV nsp2 OTU domain antagonizes the type I interferon induction by interfering with the NF-κB signaling pathway. Further analysis revealed that the nsp2 OTU domain possesses ubiquitin-deconjugating activity. This domain has the ability to inhibit NF-κB activation by interfering with the polyubiquitination process of IκBα, which subsequently prevents IκBα degradation. To determine whether the nsp2 Protein antagonist function can be ablated from the virus, we introduced point mutations into the OTU domain region by use of reverse genetics. The D458A, S462A, and D465A mutations targeting on a B-cell epitope in the OTU domain region generated the viable recombinant viruses, and the S462A and D465A mutants were attenuated for growth in cell culture. The OTU domain mutants were examined to determine whether mutations in the nsp2 OTU domain region altered virus ability to inhibit NF-κB activation. The result showed that certain mutations lethal to virus replication impaired the ability of nsp2 to inhibit NF-κB activation but that the viable recombinant viruses, vSD-S462A and vSD-D465A, were unable to inhibit NF-κB activation as effectively as the wild-type virus. This study represents a fundamental step in elucidating the role of nsp2 in PRRS pathogenesis and provides an important insight in future modified live-virus vaccine development.
Andres Merits - One of the best experts on this subject based on the ideXlab platform.
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structural insights into rna recognition by the chikungunya virus nsp2 helicase
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Jie Zheng, Sainan Wang, Ming Wei Chen, Patrick R Griffin, Andres MeritsAbstract:Chikungunya virus (CHIKV) is transmitted to humans through mosquitoes and causes Chikungunya fever. Nonstructural Protein 2 (nsP2) exhibits the protease and RNA helicase activities that are required for viral RNA replication and transcription. Unlike for the C-terminal protease, the structure of the N-terminal RNA helicase (nsP2h) has not been determined. Here, we report the crystal structure of the nsP2h bound to the conserved 3′-end 14 nucleotides of the CHIKV genome and the nonhydrolyzable transition-state nucleotide analog ADP-AlF4. Overall, the structural analysis revealed that nsP2h adopts a uniquely folded N-terminal domain followed by a superfamily 1 RNA helicase fold. The conserved helicase motifs establish polar contacts with the RNA backbone. There are three hydrophobic residues (Y161, F164, and F287) which form stacking interactions with RNA bases and thereby bend the RNA backbone. An F287A substitution that disrupted these stacking interactions increased the basal ATPase activity but decreased the RNA binding affinity. Furthermore, the F287A substitution reduced viral infectivity by attenuating subgenomic RNA synthesis. Replication of the mutant virus was restored by pseudoreversion (A287V) or adaptive mutations in the RecA2 helicase domain (T358S or V410I). Y161A and/or F164A substitutions, which were designed to disrupt the interactions with the RNA molecule, did not affect the ATPase activity but completely abolished the replication and transcription of viral RNA and the infectivity of CHIKV. Our study sheds light on the roles of the RNA helicase region in viral replication and provides insights that might be applicable to alphaviruses and other RNA viruses in general.
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molecular determinants of substrate specificity for semliki forest virus Nonstructural protease
Journal of Virology, 2006Co-Authors: Tero Ahola, Aleksei Lulla, Valeria Lulla, Kairit Tints, Andres MeritsAbstract:The C-terminal cysteine protease domain of Semliki Forest virus Nonstructural Protein 2 (nsP2) regulates the virus life cycle by sequentially cleaving at three specific sites within the virus-encoded replicase polyProtein P1234. The site between nsP3 and nsP4 (the 3/4 site) is cleaved most efficiently. Analysis of Semliki Forest virus-specific cleavage sites with shuffled N-terminal and C-terminal half-sites showed that the main determinants of cleavage efficiency are located in the region preceding the cleavage site. Random mutagenesis analysis revealed that amino acid residues in positions P4, P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much variation, whereas in the P5 position most residues were permitted. When mutations affecting cleavage efficiency were introduced into the 2/3 and 3/4 cleavage sites, the resulting viruses remained viable but had similar defects in P1234 processing as observed in the in vitro assay. Complete blockage of the 3/4 cleavage was found to be lethal. The amino acid in position P1′ had a significant effect on cleavage efficiency, and in this regard the protease markedly preferred a glycine residue over the tyrosine natively present in the 3/4 site. Therefore, the cleavage sites represent a compromise between protease recognition and other requirements of the virus life cycle. The protease recognizes at least residues P4 to P1′, and the P4 arginine residue plays an important role in the fast cleavage of the 3/4 site.
Robert E. Johnston - One of the best experts on this subject based on the ideXlab platform.
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nuclear import and export of venezuelan equine encephalitis virus Nonstructural Protein 2
Journal of Virology, 2007Co-Authors: Stephanie A. Montgomery, Robert E. JohnstonAbstract:Many RNA viruses, which replicate predominantly in the cytoplasm, have nuclear components that contribute to their life cycle or pathogenesis. We investigated the intracellular localization of the multifunctional Nonstructural Protein 2 (nsP2) in mammalian cells infected with Venezuelan equine encephalitis virus (VEE), an important, naturally emerging zoonotic alphavirus. VEE nsP2 localizes to both the cytoplasm and the nucleus of mammalian cells in the context of infection and also when expressed alone. Through the analysis of a series of enhanced green fluorescent Protein fusions, a segment of nsP2 that completely localizes to the nucleus of mammalian cells was identified. Within this region, mutation of the putative nuclear localization signal (NLS) PGKMV diminished, but did not obliterate, the ability of the Protein to localize to the nucleus, suggesting that this sequence contributes to the nuclear localization of VEE nsP2. Furthermore, VEE nsP2 specifically interacted with the nuclear import Protein karyopherin-α1 but not with karyopherin-α2, -3, or -4, suggesting that karyopherin-α1 transports nsP2 to the nucleus during infection. Additionally, a novel nuclear export signal (NES) was identified, which included residues L526 and L528 of VEE nsP2. Leptomycin B treatment resulted in nuclear accumulation of nsP2, demonstrating that nuclear export of nsP2 is mediated via the CRM1 nuclear export pathway. Disruption of either the NLS or the NES in nsP2 compromised essential viral functions. Taken together, these results establish the bidirectional transport of nsP2 across the nuclear membrane, suggesting that a critical function of nsP2 during infection involves its shuttling between the cytoplasm and the nucleus.
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Ribosomal Protein S6 Associates with Alphavirus Nonstructural Protein 2 and Mediates Expression from Alphavirus Messages
Journal of virology, 2006Co-Authors: Stephanie A. Montgomery, Peter Berglund, Clayton W. Beard, Robert E. JohnstonAbstract:Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular Proteins are poorly understood. Although the alphavirus Nonstructural Protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular Proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal Protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase Proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural Proteins nor the presence of the other Nonstructural Proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other Nonstructural Proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 Protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.
Yihong Xiao - One of the best experts on this subject based on the ideXlab platform.
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characterization of the interactome of the porcine reproductive and respiratory syndrome virus Nonstructural Protein 2 reveals the hyper variable region as a binding platform for association with 14 3 3 Proteins
Journal of Proteome Research, 2016Co-Authors: Yihong Xiao, Enmin Zhou, Weining Wu, Nikki Smith, Christine Burkard, Minxia Zhang, Chengbao Wang, Alan Archibald, Paul Digard, Julian A HiscoxAbstract:Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry worldwide and hence global food security, exacerbated by a newly emerged highly pathogenic (HP-PRRSV) strain from China. PRRSV Nonstructural Protein 2 (nsp2) is a multifunctional polypeptide with strain-dependent influences on pathogenicity. A number of discrete functional regions have been identified on the Protein. Quantitative label free proteomics was used to identify cellular binding partners of nsp2 expressed by HP-PRRSV. This allowed the identification of potential cellular interacting partners and the discrimination of nonspecific interactions. The interactome data were further investigated and validated using biological replicates and also compared with nsp2 from a low pathogenic (LP) strain of PRRSV. Validation included both forward and reverse pulldowns and confocal microscopy. The data indicated that nsp2 interacted with a number of cellular Proteins including 14–3–3, CD2AP, and other components...
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characterization of the interactome of the porcine reproductive and respiratory syndrome virus Nonstructural Protein 2 reveals the hyper variable region as a binding platform for association with 14 3 3 Proteins
Journal of Proteome Research, 2016Co-Authors: Yihong Xiao, Nikki Smith, Christine Burkard, Minxia Zhang, Chengbao Wang, Alan Archibald, Jiming Gao, Dong Xia, Paul DigardAbstract:Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry worldwide and hence global food security, exacerbated by a newly emerged highly pathogenic (HP-PRRSV) strain from China. PRRSV Nonstructural Protein 2 (nsp2) is a multifunctional polypeptide with strain-dependent influences on pathogenicity. A number of discrete functional regions have been identified on the Protein. Quantitative label free proteomics was used to identify cellular binding partners of nsp2 expressed by HP-PRRSV. This allowed the identification of potential cellular interacting partners and the discrimination of nonspecific interactions. The interactome data were further investigated and validated using biological replicates and also compared with nsp2 from a low pathogenic (LP) strain of PRRSV. Validation included both forward and reverse pulldowns and confocal microscopy. The data indicated that nsp2 interacted with a number of cellular Proteins including 14-3-3, CD2AP, and other components of cellular aggresomes. The hyper-variable region of nsp2 Protein was identified as a binding platform for association with 14-3-3 Proteins.
