Cellular Immune Evasion Mechanisms of New World Encephalitic Alphaviruses
Open AccessOne requisite characteristic of viruses that cause disease is that they possess mechanisms to circumvent mammalian pathogen sensing and cellular innate immune pathways in order to establish a productive infection cycle. As evasion of early innate immune recognition is critical for viruses to replicate in a host organism, elucidating these viral countermeasures identifies potential targets for therapeutic interventions designed to limit the virus’s ability to evade cellular immunity. Encephalitic alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV) and Eastern Equine Encephalitis Virus (EEEV) are a significant cause of morbidity and mortality in equids. They also cause periodic outbreaks in humans resulting in neuro-invasive disease, potentially chronic neurological sequela, and in some cases death. Due to the debilitating nature of the diseases they cause, certain alphaviruses have been developed as biological weapons during the Cold War by both the United States and Soviet Union. To date there are no licensed vaccines or therapeutic interventions to treat infection with these dangerous pathogens and any research utilizing live agent is strictly controlled. In order to study how alphaviruses circumvent pathogen sensing mechanisms and cellular immune pathways, we stimulated infected cells or cells expressing specific viral proteins with various cytokines or PAMP (pathogen associated molecular pattern) molecules and assayed for induction of cellular immune activity. We observed for the first time that VEEV blocks RNA-mediated induction of interferon-stimulated genes (ISGs). Presence of the VEEV E2 glycoprotein alone is sufficient to block polyinosinic:polycytidylic acid (poly(I:C))-induced transcription of ISGs, a phenotype independent of prior roles for VEEV viral capsid-mediated host transcription inhibition. Analysis of host pathway protein expression suggests the point of impingement to be at the level of targeted loss of cytosolic innate signaling molecules including TANK-binding kinase 1 (TBK-1) and interferon regulatory factor 3 (IRF3). Overexpression of the VEEV E2 alone resulted in decreased expression of both phosphorylated and total TBK-1 as well as IRF3. This mechanism of targeted loss of innate signaling molecules appears to be conserved for other New World alphaviruses such as EEEV and Western equine encephalitis viruses (WEEV) but was not observed in studies with Old World alphaviruses [i.e. Chikungunya (CHIKV)]. Detailed mapping of the VEEV E2 domains defined that the C-terminal 33 amino acids of VEEV E2, which briefly reside in the cytoplasm during Golgi transport, is critical for mediating the loss of TBK-1/IRF3 expression. Furthermore, we observed that switching the C-terminal E2 domain between VEEV and CHIKV resulted in both a loss-of- and gain-of-function of targeting cytosolic innate signaling molecules, respectively. Finally, to develop a suite of tools to further explore the immune evasion capabilities of multiple viral pathogens, we generated reporter cell lines designed to co-express green fluorescent protein (GFP) and firefly luciferase in response to appropriate immune stimuli. These were validated by comparing the immune modulatory effects of previously characterized pathogens; in this case VEEV Trinidad Donkey and its attenuated derivative strain, TC83. Additionally, gain-of-function mutants of TC83 were created to better tease out the point mutations responsible for the phenotypic differences between the two viral strains. It is the author’s hope that tools such as these can be used to help identify viral mechanisms of immune evasion in an effort to develop novel treatments.
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