A Proteomics Analysis of In Vitro Host-Protein Binding Partners of Western Equine Encephalitis Virus (WEEV) Capsid Protein

Western equine encephalitis virus (WEEV) is a New World (NW) encephalitic alphavirus. WEEV has been documented in North and South America with most human cases occurring in the Western U.S. and epizootic episodes occurring in S. America. WEEV is transmitted to humans via mosquito and clinical manifestations may include mild flu-like symptoms or more extreme manifestations including encephalitis, seizures or neurological complications that contribute to 3-5% fatality rates. NW alphaviruses including WEEV have 11 kB, positive-sense single-stranded RNA genomes that encode non-structural proteins and produce structural proteins from a subgenomic promoter. A critical structural protein, capsid, is the first protein transcribed from the subgenomic promoter and is composed of 240 repeating capsid monomers that contribute to the structure of the viral nucleocapsid. Other NW alphavirus capsid proteins such as VEEV capsid, have been previously elucidated as critical modulators in inhibition of host-cell mRNA transcription. In addition, VEEV capsid interacts with host-cell importin-alpha and -beta along with exportin-1 (CRM1) to traverse and block the nuclear pore, which contributes to its transcriptional inhibition activity. Specific information regarding pathogenesis and the role of WEEV capsid has not been identified. The current study investigates the specific role of WEEV capsid in pathogenesis through proteomic-interaction studies. A WEEV capsid expression construct was designed, transfected and expressed in Vero and BSR-T7 cells. Following validation of protein expression in both cell types, WEEV capsid was immunoprecipitated with V5 antibody and utilized for mass spectrometry (MS) analysis of protein interactions. Several proteins were identified as potential interactors by MS including those involved in cellular roles involving nuclear import and export, composition of the nuclear pore complex, mRNA splicing, and proteins involved in cellular RNA metabolism. Exportin-1 (CRM1) and importin-alpha, host nuclear export and import proteins were identified and validated by IP/WB as interacting with WEEV capsid. Future studies will include validation of other MS-identified protein interactors and observation of knockdown of CRM1 and importin-alpha and subsequent effect on capsid cellular localization. Studies involving CRM1 and importin-alpha could lead to further insight regarding the pathogenesis of WEEV and provide foundation for host-based therapeutics that target capsid-host protein interactions.

Author

• Callahan, Victoria

• Western equine encephalitis
• Alphavirus
• Exportin-1
• Togaviridae
• Capsid
• Encephalitis

Identifier

hdl:1920/11652

OAI identifier oai:identifier

oai:MARS:1920/11652