Venezuelan equine encephalitis virus (VEEV) belongs to the genus Alphavirus, family Togaviridae. VEEV infection is characterized by extensive inflammation and studies from other laboratories implicated an involvement of the NF-κB cascade in the in vivo pathology. Initial studies indicated that at early time points of VEEV infection, the NF-κB complex was activated in cells infected with the TC-83 strain of VEEV. One upstream kinase that contributes to the phosphorylation of p65 is the IKKβ component of the IKK complex. Our previous studies with Rift valley fever virus, which exhibited early activation of the NF-κB cascade in infected cells, had indicated that the IKKβ component underwent macromolecular reorganization to form a novel low molecular weight form unique to infected cells. This prompted us to investigate if the IKK complex undergoes a comparable macromolecular reorganization in VEEV infection. Size-fractionated VEEV infected cell extracts indicated a macromolecular reorganization of IKKβ in VEEV infected cells that resulted in formation of lower molecular weight complexes. Well-documented inhibitors of IKKβ function, BAY-11-7082, BAY-11-7085 and IKK2 compound IV, were employed to determine whether IKKβ function was required for the production of infectious progeny virus. A decrease in infectious viral particles and viral RNA copies was observed with inhibitor treatment in the attenuated and virulent strains of VEEV infection. In order to further validate the requirement of IKKβ for VEEV replication, we over-expressed IKKβ in cells and observed an increase in viral titers. In contrast, studies carried out using IKKβ−/− cells demonstrated a decrease in VEEV replication. In vivo studies demonstrated that inhibitor treatment of TC-83 infected mice increased their survival. Finally, proteomics studies have revealed that IKKβ may interact with the viral protein nsP3. In conclusion, our studies have revealed that the host IKKβ protein may be critically involved in VEEV replication.
Alphaviruses, including Venezuelan Equine Encephalitis Virus (VEEV), cause disease in both equine and humans that exhibit overt encephalitis in a significant percentage of cases. Features of the host immune response and tissue-specific responses may contribute to fatal outcomes as well as the development of encephalitis. It has previously been shown that VEEV infection of mice induces transcription of pro-inflammatory cytokines genes (e.g., IFN-γ, IL-6, IL-12, iNOS and TNF-α) within 6 h. GSK-3β is a host protein that is known to modulate pro-inflammatory gene expression and has been a therapeutic target in neurodegenerative disorders such as Alzheimer's. Hence inhibition of GSK-3β in the context of encephalitic viral infections has been useful in a neuroprotective capacity. Small molecule GSK-3β inhibitors and GSK-3β siRNA experiments indicated that GSK-3β was important for VEEV replication. Thirty-eight second generation BIO derivatives were tested and BIOder was found to be the most potent inhibitor, with an IC 50 of ∼0.5 µM and a CC 50 of >100 µM. BIOder was a more potent inhibitor of GSK-3β than BIO, as demonstrated through in vitro kinase assays from uninfected and infected cells. Size exclusion chromatography experiments demonstrated that GSK-3β is found in three distinct complexes in VEEV infected cells, whereas GSK-3β is only present in one complex in uninfected cells. Cells treated with BIOder demonstrated an increase in the anti-apoptotic gene, survivin, and a decrease in the pro-apoptotic gene, BID, suggesting that modulation of pro- and anti-apoptotic genes contributes to the protective effect of BIOder treatment. Finally, BIOder partially protected mice from VEEV induced mortality. Our studies demonstrate the utility of GSK-3β inhibitors for modulating VEEV infection.
Background: Rift Valley fever virus is a single-stranded RNA virus that causes disease in humans and livestock. Results: Rift Valley fever virus infection activates the host NFB signaling cascade. Conclusion: NFB inhibitors, particularly curcumin, down-regulate virus in both in vitro and in vivo models. Significance: Novel versions of host components resulting from an infection make them ideal therapeutic targets.
The aerosol form of the bacterium Yersinia pestis causes the pneumonic plague, a rapidly fatal disease. At present, no plague vaccines are available for use in the United States. One candidate for the development of a subunit vaccine is the Y. pestis virulence (V) antigen, a protein that mediates the function of the Yersinia outer protein virulence factors and suppresses inflammatory responses in the host. On the basis of the knowledge that adenovirus (Ad) gene-transfer vectors act as adjuvants in eliciting host immunity against the transgene they carry, we tested the hypothesis that a single administration of a replication-defective Ad gene-transfer vector encoding the Y. pestis V antigen (AdsecV) could stimulate strong protective immune responses without a requirement for repeat administration. AdsecV elicited specific T cell responses and high IgG titers in serum within 2 weeks after a single intramuscular immunization. Importantly, the mice were protected from a lethal intranasal challenge of Y. pestis CO92 from 4 weeks up to 6 months after immunization with a single intramuscular dose of AdsecV. These observations suggest that an Ad gene-transfer vector expressing V antigen is a candidate for development of an effective anti-plague vaccine.
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