Amino acid residues in the non-structural protein 1 of porcine reproductive and respiratory syndrome virus involved in down-regulation of TNF-α expression in vitro and attenuation in vivo

School of Veterinary Medicine & Biomedical Sciences and Nebraska Center for Virology, University of Nebraska-Lincoln, NE 68583, USA.
Virology (Impact Factor: 3.32). 06/2012; 432(2):241-9. DOI: 10.1016/j.virol.2012.05.014
Source: PubMed


Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses tumor necrosis factor-alpha (TNF-α) production at both transcriptional and post-transcriptional levels by its non-structural proteins 1α and 1β (Nsp1α and Nsp1β). To identify the amino acid residues responsible for this activity, we generated several alanine substitution mutants of Nsp1α and Nsp1β. Examination of the mutant proteins revealed that Nsp1α residues Gly90, Asn91, Arg97, Arg100 and Arg124 were necessary for TNF-α promoter suppression, whereas several amino acids spanning the entire Nsp1β were found to be required for this activity. Two mutant viruses, with mutations at Nsp1α Gly90 or Nsp1β residues 70-74, generated from infectious cDNA clones, exhibited attenuated viral replication in vitro and TNF-α was found to be up regulated in infected macrophages. In infected pigs, the Nsp1β mutant virus was attenuated in growth. These studies provide insights into how PRRSV evades the effector mechanisms of innate immunity during infection.

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    • "Thus, both Nsp1 and Nsp11 of PRRSV are suggested to be the factors contributing to the pathogenesis of PRRSV. A previous study had shown that mutant viruses with mutations at Nsp1 Gly90 or Nsp1 residues 70–74, generated from infectious cDNA clones, exhibited attenuated viral replication in vitro and TNF-was found to be up regulated in infected macrophages (Subramaniam et al., 2012). However, no differences were found by aligning the amino acids of these sites between JXwn06 and HB-1/3.9. "
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    ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to be one of the most important pathogens severely affecting global swine industry. An increasingly number of studies has paid much attention to the diverse roles of its nonstructural proteins (Nsps) in regulating the innate immune response of host upon PRRSV infection. In the present study, we first discovered that highly pathogenic PRRSV (HP-PRRSV) and low pathogenic PRRSV (LP-PRRSV) infection exhibited a differential TNF-α expression in pulmonary alveolar macrophages (PAMs), showing that HP-PRRSV infection induces lower TNF-α production at protein level in PAMs, compared with LP-PRRSV. Next, HP-PRRSV was confirmed to strongly suppress TNF-α production by inhibiting ERK signaling pathway. Finally, both Nsp1β and Nsp11 were demonstrated to be responsible for the inhibitory effect on TNF-α production induced by HP-PRRSV and the differential TNF-α production in PAMs. These findings contribute to the understanding of the pathogenesis of the Chinese HP-PRRSV. Copyright © 2015. Published by Elsevier B.V.
    Virus Research 02/2015; 201. DOI:10.1016/j.virusres.2015.02.014 · 2.32 Impact Factor
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    • "It has been recently shown that nsp1 of PRRSV modulates the expression of type I interferon (Beura et al, 2010; Beura et al, 2012; Chen et al, 2010; Wang et al, 2013; Kim et al, 2010) and TNF-α (Subramaniam et al, 2012), which leads to persistent infection (Lamontagne et al, 2003). In addition, PRRSV infects and destroys porcine alveolar macrophage (PAM), which is an active producer of cytokines and leukotrienes and has important roles in inducing pro-and anti-inflammatory responses in the alveolus (Gordon and Read, 2002) and innate immune response against respiratory infections (Pribul et al, 2008). "
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    ABSTRACT: Although it has been generally accepted that porcine reproductive and respiratory syndrome virus (PRRSV) induces weak and delayed protective immunity after infection, it is unclear that the same immunological features can be applicable to all PRRS viruses because huge genetic variation exists even among the same genotypes of PRRSV (Type 1 and 2). In the current study, two genetically distinct type 2 PRRSV strains (VR-2332 and JA142) which showed approximately 90% nucleotide homology based on ORF5 sequences were characterized by both in vitro and in vivo assessments to determine the immunological features of the viruses. For in vitro assessment, porcine alveolar macrophages (PAM) were infected with the viruses at multiplicity of infection (MOI) and then supernatants and cells were collected separately at 36 hrs post infection to determine the relative expression levels of IL-, IL-12, TNF- and INF- by quantitative RT-PCR. In addition, five PRRSV-free pigs were inoculated with either of JA142 or VR2332 for in vivo assessment. Serum samples were collected every week until 6 weeks post challenge. The serum samples were analyzed for the levels of viremia, PRRSV nucleocapsid-specific antibody and virus neutralizing antibody. Based on those assessments, the two viruses showed different patterns of cytokine expression in PAM and immune responses in pigs after infection. These results indicate that genetically distinct PRRSV strains have different immunological features, which might be criteria for virus classification and selection of candidate virus strains for vaccine development in the future.
    03/2014; 37(1). DOI:10.7853/kjvs.2014.37.1.1
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    • "Promoter and post-transcriptional downregulation of TNF-a has been associated with PRRSV non-structural proteins (NSPs) 1a, 1b and 2 (Chen et al., 2010; Subramaniam et al., 2010, 2012; Darwich et al., 2011). Specific amino acid residues of NSP1a and NSP1b have been identified that affect TNF-a promoter activity; substitution of specific NSP1b amino acids is potential tool for the generation of attenuated PRRSV vaccines (Subramaniam et al., 2012). The limited expression of TNF-a in experimental infections with some PRRSV strains may be a mechanism by which these strains impair the host immune response and prevent viral clearance (Table 1; Fig. 1). "
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    ABSTRACT: Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) impairs local pulmonary immune responses by damaging the mucociliary transport system, impairing the function of porcine alveolar macrophages andinducing apoptosis of immune cells. An imbalance between pro- and anti-inflammatory cytokines, including tumour necrosis factor-α and interleukin-10, in PRRS may impair the immune response of the lung. Pulmonary macrophage subpopulations have a range of susceptibilities to different PRRSV strains and different capacities to express cytokines. Infection with PRRSV decreases the bactericidal activity of macrophages, which increases susceptibility to secondary bacterial infections. PRRSV infection is associated with an increase in concentrations of haptoglobin, which may interact with the virus receptor (CD163) and induce the synthesis of anti-inflammatory mediators. The balance between pro- and anti-inflammatory cytokines modulates the expression of CD163, which may affect the pathogenicity and replication of the virus in different tissues. With the emergence of highly pathogenic PRRSV, there is a need for more information on the immunopathogenesis of different strains of PRRS, particularly to develop more effective vaccines.
    The Veterinary Journal 12/2012; 195(2). DOI:10.1016/j.tvjl.2012.11.012 · 1.76 Impact Factor
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