1141. The N1 Vaccinia Virus Virulence Factor Inhibits Host Immune Responses
Session: Poster Abstract Session: Innate and Adaptive Immunity to Infections
Saturday, October 22, 2011
Room: Poster Hall B1
  • Marshall-1141.pdf (292.5 kB)
  • Background:  Poxviruses encode proteins that increase viral virulence by modulating host immune responses, including a nine-member family of proteins thought to modulate signaling by the innate immune system. N1 was the first member of this family to be identified and N1 enhances vaccinia virus (VACV) virulence by 10,000-fold in animal models of infection. The crystal structure of the N1 protein reveals a bcl-2-like fold. Similarly to cellular bcl-2, N1 inhibits apoptosis. N1 also inhibits signaling to NF-kB and IRF3.

    Methods: AP-1 and NF-kB luciferase assays were performed in HEK293 cells transfected with N1 and N1 mutants. A low-density array was utilized to quantify expression of mRNAs for cytokine and co-stimulatory molecules in samples from human macrophages infected with wild type and N1-deficient VACV. Immunoblots of VACV infected cells were probed with anti-bcl-xl and anti-N1 antibody. HeLa cells transfected with an N1 and N1 mutants were treated +/- 1 nM stuarosporine  and assessed for apoptosis by Annexin V staining.

    Results: N1 inhibited in vitro signaling to AP-1, a transcription factor key to cell survival, antiviral cytokine production, and cellular and humoral immunity. Furthermore, AP-1 and NF-kB dependent transcripts such as IL-1 alpha, CD80, CD54 and bcl-xl were suppressed in human macrophages infected with wild type, but not N1-deficient, VACV. In cells infected with wild type VACV, bcl-xl protein was suppressed coincident with an increase in N1 protein.  Finally, N1 was also found to be phosphorylated by a host-dependent kinase at an evolutionarily conserved threonine residue located near the binding cleft where N1 binds to, and neutralizes, proapoptotic proteins. Mutation of N1 modulated only the antiapoptotic properties of N1. Inhibition of NF-kB signaling by N1 was independent of its phosphorylation.

    Conclusion: The robust virulence properties of N1 are consistent with extensive N1-mediated suppression of cellular signaling to anti-viral genes critical to innate and adaptive immune responses. These findings support the hypothesis that inhibition of apoptosis by the N1 phosphoprotein compensates for the anti-survival effects of N1-mediated suppression of NF-kB and AP-1 signaling during VACV infection.

    Subject Category: E. Innate and adaptive immunity to infections, including vaccine immunology

    William Marshall, MD, University of Massachusetts Medical School, Worcester, MA


    W. Marshall, None

    Findings in the abstracts are embargoed until 12:01 a.m. EST Thursday, Oct. 20 with the exception of research findings presented at IDSA press conferences.