Characterization of the vaccinia virus ankyrin repeat protein K1 on NF-κB inhibition and its contributions to viral pathogenesis
All viruses strategically alter the anti-viral immune response to their benefit. The goal of this thesis was to examine how the vaccinia virus (VACV) K1 protein, which is encoded by the K1L gene, modulates the immune response in vitro and in vivo. In vitro, the K1 protein has multiple immunomodulatory effects in tissue culture models of infection. We have previously published that the K1 protein inhibits double-stranded RNA-dependent protein kinase (PKR) activation. A consequence of this function is that K1 inhibits PKR-induced NF-κB activation during VACV infection. However, transient expression of K1 also inhibits Toll-like receptor (TLR)-induced NF-κB activation. This suggested that K1 had a second NF-κB inhibitory mechanism that is PKR-independent. This possibility was explored by expressing K1 independently of infection and stimulating NF-κB under conditions that minimized or excluded PKR activation. Using these approaches, I found a second NF-κB inhibitory function for K1: K1 inhibited nuclear acetylation of the RelA (p65) subunit of NF-κB. Moreover, p65-CBP interactions were blocked in the presence of K1. However, K1 did not preclude NF-κB binding to oligonucleotides containing B binding sites. This implies that NF-κB-promoter interactions still occurred in the presence of K1, but NF-κB cannot properly trigger transcriptional activation because K1 antagonizes acetylation of RelA. These data show that K1 has multiple mechanisms to inhibit NF-κB activation and, in comparison to all known VACV NF-κB inhibitory proteins, K1 acts at one of the most downstream events of NF-κB activation. A second goal of my thesis was to understand the contribution of K1 in vivo. I determined that a K1L-less vaccinia virus (vΔK1L) was less pathogenic than wild-type VACV in intranasal and intradermal models of infection. Decreased pathogenicity correlated with diminished virus replication in intranasally-infected mice. However, in intradermally-inoculated ears, vΔK1L replicated to nearly identical levels as VACV, implying that the decreased immune response to vΔK1L infection, not virus replication, dictated lesion size. Several lines of evidence support this theory. First, vΔK1L induced slightly less edema than vK1L as revealed by histopathology and non-invasive quantitative ultrasound technology (QUS). Second, infiltrating immune cell populations were decreased in vΔK1L-infected ears. Third, cytokine and chemokine gene expression was decreased in vΔK1L-infected ears. While these results identified the biological basis for smaller lesions they remained puzzling: because K1 antagonizes NF-κB in vitro, anti-viral gene expression was expected to be higher during vΔK1L infection. Despite these diminished innate immune responses, vΔK1L vaccination induced a protective VACV-specific CD8+ T cell response and protected against a lethal VACV challenge. Thus, vΔK1L is a unique construct that limits pathogenesis, yet still elicits protective immunity. We propose that continued studies of vΔK1L may uncover novel relationships between pathogenesis and immunity, information that has practical applications for vaccine design.
Vaccinia, Poxvirus, K1, Inflammation, NF-κB
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign