Sakthivel Subramaniam

Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1β Modulates Host Innate Immune Response by Antagonizing IRF3 Activation

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine leads to a serious disease characterized by a delayed and defective adaptive immune response. It is hypothesized that a suboptimal innate immune response is responsible for the disease pathogenesis. In the study presented here we tested this hypothesis and identified several nonstructural proteins (NSPs) with innate immune evasion properties encoded by the PRRS viral genome. Four of the total ten PRRSV NSPs tested were found to have strong to moderate inhibitory effects on beta interferon (IFN-β) promoter activation. The strongest inhibitory effect was exhibited by NSP1 followed by, NSP2, NSP11, and NSP4. We focused on NSP1α and NSP1β (self-cleavage products of NSP1 during virus infection) and NSP11, three NSPs with strong inhibitory activity. All of three proteins, when expressed stably in cell lines, strongly inhibited double-stranded RNA (dsRNA) signaling pathways. NSP1β was found to inhibit both IFN regulatory factor 3 (IRF3)- and NF-κB-dependent gene induction by dsRNA and Sendai virus. Mechanistically, the dsRNA-induced phosphorylation and nuclear translocation of IRF3 were strongly inhibited by NSP1β. Moreover, when tested in a porcine myelomonocytic cell line, NSP1β inhibited Sendai virus-mediated activation of porcine IFN-β promoter activity. We propose that this NSP1β-mediated subversion of the host innate immune response plays an important role in PRRSV pathogenesis.

RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses

Negative-strand (NS) RNA viruses comprise many pathogens that cause serious diseases in humans and animals. Despite their clinical importance, little is known about the host factors required for their infection. Using vesicular stomatitis virus (VSV), a prototypic NS RNA virus in the family Rhabdoviridae, we conducted a human genome-wide siRNA screen and identified 72 host genes required for viral infection. Many of these identified genes were also required for infection by two other NS RNA viruses, the lymphocytic choriomeningitis virus of the Arenaviridae family and human parainfluenza virus type 3 of the Paramyxoviridae family. Genes affecting different stages of VSV infection, such as entry/uncoating, gene expression, and assembly/release, were identified. Depletion of the proteins of the coatomer complex I or its upstream effectors ARF1 or GBF1 led to detection of reduced levels of VSV RNA. Coatomer complex I was also required for infection of lymphocytic choriomeningitis virus and human parainfluenza virus type 3. These results highlight the evolutionarily conserved requirements for gene expression of diverse families of NS RNA viruses and demonstrate the involvement of host cell secretory pathway in the process.

Porcine reproductive and respiratory syndrome virus non-structural protein 1 suppresses tumor necrosis factor-alpha promoter activation by inhibiting NF-κB and Sp1

The objective of this study was to identify porcine reproductive and respiratory syndrome virus (PRRSV)-encoded proteins that are responsible for the inhibition of TNF-α expression and the mechanism(s) involved in this phenomenon. Using a TNF-α promoter reporter system, the non-structural protein 1 (Nsp1) was found to strongly suppress the TNF-α promoter activity. Such inhibition takes place especially at the promoters proximal region. Both Nsp1α and Nsp1β, the two proteolytic fragments of Nsp1, were shown to be involved in TNF-α promoter suppression. Furthermore, using reporter plasmids specific for transcription factors (TFs) that bind to TNF-α promoter, Nsp1α and Nsp1β were demonstrated to inhibit the activity of the TFs that bind CRE-κB(3) and Sp1 elements respectively. Subsequent analyses showed that Nsp1α moderately inhibits NF-κB activation and that Nsp1β completely abrogates the Sp1 transactivation. These findings reveal one of the important mechanisms underlying the innate immune evasion by PRRSV during infection.

miR-27b*, an oxidative stress-responsive microRNA modulates nuclear factor-kB pathway in RAW 264.7 cells

Reactive oxygen species (ROS) produced in macrophages is critical for microbial killing, but they also take part in inflammation and antigen presentation functions. MicroRNAs (miRNAs) are endogenous regulators of gene expression, and they can control immune responses. To dissect the complex nature of ROS-mediated effects in macrophages, we sought to characterize miRNAs that are responsive to oxidative stress-induced with hydrogen peroxide (H2O2) in the mouse macrophage cell line, RAW 264.7. We have identified a set of unique miRNAs that are differentially expressed in response to H2O2. These include miR-27a*, miR-27b*, miR-29b*, miR-24-2*, and miR-21*, all of which were downregulated except for miR-21*. By using luciferase reporter vector containing nuclear factor-kB (NF-kB) response elements, we demonstrate that overexpression of miR-27b* suppresses lipopolysaccharide-induced activation of NF-kB in RAW 264.7 cells. Our data suggest that macrophage functions can be regulated by oxidative stress-responsive miRNAs by modulating the NF-kB pathway.

Computer-aided codon-pairs deoptimization of the major envelope GP5 gene attenuates porcine reproductive and respiratory syndrome virus.

Synthetic attenuated virus engineering (SAVE) is an emerging technology that enables rapid attenuation of viruses. In this study, by using SAVE we demonstrated rapid attenuation of an arterivirus, porcine reproductive and respiratory syndrome virus (PRRSV). The major envelope GP5 gene of PRRSV was codon-pair deoptimized aided by a computer algorithm. The codon-pair deoptimized virus, designated as SAVE5 with a deoptimized GP5 gene, was successfully rescued in vitro. The SAVE5 virus replicated at a lower level in vitro with a significant decrease of GP5 protein expression compared to the wild-type PRRSV VR2385 virus. Pigs experimentally infected with the SAVE5 virus had significantly lower viremia level up to 14 days post-infection as well as significantly reduced gross and histological lung lesions when compared to wild-type PRRSV VR2385 virus-infected pigs, indicating the attenuation of the SAVE5 virus. This study proved the feasibility of rapidly attenuating PRRSV by SAVE.

Identification of amino acid residues important for anti-IFN activity of porcine reproductive and respiratory syndrome virus non-structural protein 1

Abstract The non-structural protein 1 (nsp1) of porcine reproductive and respiratory syndrome virus is partly responsible for inhibition of type I interferon (IFN) response by the infected host. By performing alanine-scanning mutagenesis, we have identified amino acid residues in nsp1α and nsp1β (the proteolytic products of nsp1) that when substituted with alanine(s) exhibited significant relief of IFN-suppression. A mutant virus (16-5A, in which residues 16–20 of nsp1β were substituted with alanines) encoding mutant nsp1β recovered from infectious cDNA clone was shown to be attenuated for growth in vitro and induced significantly higher amount of type I IFN transcripts in infected macrophages. In infected pigs, the 16-5A virus exhibited reduced growth at early times after infection but quickly regained wild type growth properties as a result of substitutions within the mutated sequences. The results indicate a strong selection pressure towards maintaining the IFN-inhibitory property of the virus for successful propagation in pigs.

A virulent strain of porcine reproductive and respiratory syndrome virus does not up-regulate interleukin-10 levels in vitro or in vivo.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure and respiratory illness in infected pigs. It has been postulated that the ability of PRRSV to induce the anti-inflammatory cytokine interleukin-10 (IL-10) in macrophages of infected pigs would be important for PRRSV immunopathogenesis, although this property would be variable and might be dependent on the strain. Several strains were reported to induce IL-10 in infected macrophages while others would not. In this study, we analyzed the IL-10 expression during in vitro and in vivo infections by a well-characterized virulent strain of PRRSV, vFL12, which is derived from an infectious clone. Our results showed that the vFL12 strain did not up-regulate IL-10 at mRNA or protein levels in either infected macrophages or dendritic cells in vitro. Furthermore, immunofluorescence staining for IL-10 on tonsil sections of PRRSV-infected pigs did not produce any evidence of IL-10 induction in PRRSV-infected cells or in bystander cells of the lymphoid tissues. Hence, based on these results obtained with a well-characterized highly pathogenic PRRSV strain it may be concluded that the induction of IL-10 release is not a part of the PRRSV virulence mechanisms.

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

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.

Broadening the Heterologous Cross-Neutralizing Antibody Inducing Ability of Porcine Reproductive and Respiratory Syndrome Virus by Breeding the GP4 or M genes

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens, which causes reproductive failure in sows and respiratory disease in piglets. A major hurdle to control PRRSV is the ineffectiveness of the current vaccines to confer protection against heterologous strains. Since both GP4 and M genes of PRRSV induce neutralizing antibodies, in this study we molecularly bred PRRSV through DNA shuffling of the GP4 and M genes, separately, from six genetically different strains of PRRSV in an attempt to identify chimeras with improved heterologous cross-neutralizing capability. The shuffled GP4 and M genes libraries were each cloned into the backbone of PRRSV strain VR2385 infectious clone pIR-VR2385-CA. Three GP4-shuffled chimeras and five M-shuffled chimeras, each representing sequences from all six parental strains, were selected and further characterized in vitro and in pigs. These eight chimeric viruses showed similar levels of replication with their backbone strain VR2385 both in vitro and in vivo, indicating that the DNA shuffling of GP4 and M genes did not significantly impair the replication ability of these chimeras. Cross-neutralization test revealed that the GP4-shuffled chimera GP4TS14 induced significantly higher cross-neutralizing antibodies against heterologous strains FL-12 and NADC20, and similarly that the M-shuffled chimera MTS57 also induced significantly higher levels of cross-neutralizing antibodies against heterologous strains MN184B and NADC20, when compared with their backbone parental strain VR2385 in infected pigs. The results suggest that DNA shuffling of the GP4 or M genes from different parental viruses can broaden the cross-neutralizing antibody-inducing ability of the chimeric viruses against heterologous PRRSV strains. The study has important implications for future development of a broadly protective vaccine against PRRSV.

Distribution and characterization of IL-10-secreting cells in lymphoid tissues of PCV2-infected pigs.

Distribution and characterization of interlukin-10 (IL-10)-secreting cells in lymphoid tissues of pigs naturally infected with porcine circovirus type 2 (PCV2) were evaluated in accordance with PCV2 antigen detection. After screening a total of 56 pigs showing the symptoms of postweaning multisystemic wasting syndrome (PMWS), 15 pigs were PCV2 positive and 5 pigs, which showed stronger positive signals over multiples tissues were further investigated. This study showed that in PCV2-infected lymphoid tissues, particularly mandibular lymph node, spleen and tonsil, IL-10 expression was mainly localized in T-cell rich areas but rarely in B cell rich areas. IL-10 was highly expressed in bystander cells but rarely in PCV2-infected cells. Elevated IL-10 expression was predominantly associated with T cells, but rarely with B cells or with macrophages. The results of this study provide evidence for the role of IL-10 in chronic PCV2 infection and its relation to PCV2 antigen in affected tissues. Constantly elevated levels of IL-10 lead to immunosuppression in persistent and chronic viral infections. The increased IL-10 expression observed in PCV2 infection in this study suggests that IL-10-mediated immunosuppression may play an important role in the pathogenesis and maintenance of naturally occurring PCV2 infection.