Murine Norovirus Manipulation of the Immune Response

Abstract

Human norovirus is a significant human pathogen that causes morbidity and mortality worldwide. Noroviruses are a member of the Caliciviridae family of positive sense, single-stranded RNA viruses. The inability to study human norovirus in cell culture has meant that advancement in prophylactic and therapeutic treatment has been slow. The discovery of the closely related murine norovirus has allowed investigation into the pathogenesis of norovirus infection as well as the host response to infection to be studied in detail. While research has focused on the essential innate immune response and to a lesser extent the adaptive immune response, little is known about the chemokine response initiated during norovirus infection. This PhD thesis has investigated the chemokine response to murine norovirus during infection of macrophages and has identified virus-specific up-regulation of selected chemokines. This virus-induced chemokine response biases the immune system towards a Th1-type response to viral infection. This response may explain the limited efficacy of antibody responses generated during norovirus infection, resulting in poor protection against infection during an outbreak. Moreover, research identified that mRNA and protein levels of selected chemokines do not show matched up-regulation during MNV infection, suggesting viral interference with host protein production, stability or secretion. Other caliciviruses, including human norovirus, are known to interfere with host protein secretion during infection. Therefore, protein secretion was investigated during MNV infection using a secretion reporter cell line (RAW-Blue cells) in addition to examination of intracellular build up of proteins. It was determined that MNV does not affect cellular secretion during viral infection. Observations from experiments using RAW-Blue cells discovered that MNV infection was unable to induce production of the innate immune response induced reporter construct. This construct is inducible by NFκB and AP-1 therefore, MNV interactions with intracellular signaling pathways, specifically NFκB activation, were examined. The NFκB regulator protein IκBα was studied and experiments demonstrated a lack of IκBα phosphorylation and degradation during MNV infection. This revealed that MNV infection leads to impaired activation of the NFκB pathway. Moreover, investigation into the mechanism of impairment discovered that the viral protein VF1 is in part responsible, as infection of RAW-Blue cells with a mutant virus lacking VF1 restored signaling through the NFκB pathway. NFκB is a global transcription factor involved in the regulation of over 100 immune genes, thus impaired NFκB activation will likely result in delayed induction of the antiviral response. The lack of NFκB activation may partly be responsible for chemokine profile observed during MNV infection, as chemokines that are predominately inducible by interferon showed much greater up-regulation than chemokines primarily induced by NFκB. Overall, this thesis describes the interaction of murine norovirus with the host cellular response and characterizes previously unknown mechanisms of norovirus immune evasion.