| Porcine reproductive and respiratory syndrome virus (PRRSV) causes late-term abortion in sows and pneumonia in growing piglets. PRRSV evades the host immune response by several mechanisms, including the modulation of cytokine secretions in infected pigs, which is the subject of this dissertation. Particularly, PRRSV reduces the secretion of the pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha) but increases the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10). The latter effect, however, is PRRSV strain-specific. In this dissertation, we have examined mechanisms by which PRRSV regulates TNF-alpha and IL-10 expressions. The pathogenic strain FL12, derived from a PRRSV infectious clone, consistently suppressed TNF-alpha but failed to induce IL-10 in infected swine cells. In addition, no significant IL-10 production takes place in pig tissues following infection with vFL12. Using a TNF-alpha promoterreporter gene construct, we demonstrated that the viral non-structural proteins, Nsp1alpha and Nsp1beta reduce the TNF-alpha promoter activity in transiently transfected cells, mainly by inhibiting the cellular transcription factors Nuclear Factor-kappaB (NF-kappaB) and Specificity Protein 1 (Sp1) respectively. Furthermore, screening of Nsp1alpha mutant constructs revealed that five amino acid residues (Gly90, Asn91, Arg97, Arg100, and Arg124) are important for inhibiting the TNF-alpha promoter activity. Nsp1alpha also reduced TNF-alpha protein levels in an in vitro translation assay, and Gly90 was important for this activity. Screening of Nsp1beta mutant constructs showed that multiple amino acid stretches in all domains are important for inhibiting the TNF-alpha promoter activity. We obtained two mutant vFL12 strains with substitutions at Nsp1alphaGly90 and Nsp1beta 70SMVRE74 positions. Both mutant viruses increased TNF-alpha mRNA levels at early times after infection when compared to parental vFL12 strain in infected macrophages. However, only Nsp1alpha mutant virus induced higher TNF-alpha protein levels when compared to vFL12 in infected macrophage cultures. Moreover, Nsp1beta mutant virus was attenuated in infected pigs. In summary, we have identified proteins that regulate the expression of TNF-alpha. These results may be useful for designing novel prophylactics and therapeutics for PRRSV. |
