Mitochondria are sentinel organelles that are impacted by various forms of cellular stress, including viral infections. While signaling events associated with mitochondria, including those activated by pathogen associated molecular patterns (PAMPs), are widely studied, alterations in mitochondrial distribution and changes in mitochondrial dynamics are also beginning to be associated with cellular insult. Cells of neuronal origin have been demonstrated to display remarkable alterations in several instances, including neurodegenerative disorders. Venezuelan Equine Encephalitis Virus (VEEV) is a New World alphavirus that infects neuronal cells and contributes to an encephalitic phenotype. We demonstrate that upon infection by the vaccine strain of VEEV (TC-83), astrocytoma cells experience a robust drop in mitochondrial activity, which corresponds with an increased accumulation of reactive oxygen species (ROS) in an infection-dependent manner. Infection status also corresponds with a prominent perinuclear accumulation of mitochondria. Cellular enzymatic machinery, including PINK1 and Parkin, appears to be enriched in mitochondrial fractions as compared with uninfected cells, which is indicative of mitochondrial damage. Dynamin related protein 1 (Drp1), a protein that is associated with mitochondrial fission, demonstrated a modest enrichment in mitochondrial fractions of infected cells. Treatment with an inhibitor of mitochondrial fission, Mdivi-1, led to a decrease in caspase cleavage, suggesting that mitochondrial fission was likely to contribute to apoptosis of infected cells. Finally, our data demonstrate that mitophagy ensues in infected cells. In combination, our data suggest that VEEV infection results in significant changes in the mitochondrial landscape that may influence pathological outcomes in the infected cell.

Alphaviruses are members of the Togaviridae family and are grouped into two categories: arthritogenic and encephalitic. Arthritogenic alphavirus infections, as the name implies, are associated with arthritic outcomes while encephalitic alphavirus infections can lead to encephalitic outcomes in the infected host. Of the non-structural proteins (nsPs) that the viruses code for, nsP3 is the least understood in terms of function. Alphavirus nsP3s are characterized by regions with significantly conserved domain structure along with regions of high variability. Interactions of nsP3 with several host proteins have been documented including, stress granule-related proteins, dead box proteins, heat shock proteins, and kinases. In some cases, in addition to the interaction, requirement of the interaction to support infection has been demonstrated. An understanding of the proteomic network of nsP3 and the mechanisms by which these interactions support the establishment of a productive infection would make alphavirus nsP3 an interesting target for design of effective medical countermeasures.

The mosquito-borne New World alphavirus, Venezuelan equine encephalitis virus (VEEV) is a Category B select agent with no approved vaccines or therapies to treat infected humans. Therefore it is imperative to identify novel targets that can be targeted for effective therapeutic intervention. We aimed to identify and validate interactions of VEEV nonstructural protein 3 (nsP3) with host proteins and determine the consequences of these interactions to viral multiplication. We used a HA tagged nsP3 infectious clone (rTC-83-nsP3-HA) to identify and validate two RNA helicases: DDX1 and DDX3 that interacted with VEEV-nsP3. In addition, DDX1 and DDX3 knockdown resulted in a decrease in infectious viral titers. Furthermore, we propose a functional model where the nsP3:DDX3 complex interacts with the host translational machinery and is essential in the viral life cycle. This study will lead to future investigations in understanding the importance of VEEV-nsP3 to viral multiplication and apply the information for the discovery of novel host targets as therapeutic options.

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