Post translational modification of proteins is a critical requirement that regulates function. Among the diverse kinds of protein post translational modifications, phosphorylation plays essential roles in protein folding, protein:protein interactions, signal transduction, intracellular localization, transcription regulation, cell cycle progression, survival and apoptosis. Protein phosphorylation is also essential for many intracellular pathogens to establish a productive infection cycle. Preservation of protein phosphorylation moieties in pathogens in a manner that mirrors the host components underscores the co-evolutionary trajectory of pathogens and hosts, and sheds light on how successful pathogens have usurped, either in part or as a whole, the host enzymatic machinery. Phosphorylation of viral proteins for many acute RNA viruses including Flaviviruses and Alphaviruses has been demonstrated to be critical for protein functionality. This review focuses on phosphorylation modifications that have been documented to occur on viral proteins with emphasis on acutely infectious, single stranded RNA viruses. The review additionally explores the possibility of repurposing Food and Drug Administration (FDA) approved inhibitors as antivirals for the treatment of acute RNA viral infections.

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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.

Many viruses have been implicated in utilizing or modulating the Ubiquitin Proteasome System (UPS) to enhance viral multiplication and/or to sustain a persistent infection. The mosquito-borne Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is an important biodefense pathogen and select agent. There are currently no approved vaccines or therapies for VEEV infections; therefore, it is imperative to identify novel targets for therapeutic development. We hypothesized that a functional UPS is required for efficient VEEV multiplication. We have shown that at non-toxic concentrations Bortezomib, a FDA-approved inhibitor of the proteasome, proved to be a potent inhibitor of VEEV multiplication in the human astrocytoma cell line U87MG. Bortezomib inhibited the virulent Trinidad donkey (TrD) strain and the attenuated TC-83 strain of VEEV. Additional studies with virulent strains of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) demonstrated that Bortezomib is a broad spectrum inhibitor of the New World alphaviruses. Time-of-addition assays showed that Bortezomib was an effective inhibitor of viral multiplication even when the drug was introduced many hours post exposure to the virus. Mass spectrometry analyses indicated that the VEEV capsid protein is ubiquitinated in infected cells, which was validated by confocal microscopy and immunoprecipitation assays. Subsequent studies revealed that capsid is ubiquitinated on K48 during early stages of infection which was affected by Bortezomib treatment. This study will aid future investigations in identifying host proteins as potential broad spectrum therapeutic targets for treating alphavirus infections.