An unbiased proteomics approach to identify human cytomegalovirus RNA-associated proteins

•We used an unbiased proteomics approach to identify RNA-associated proteins during HCMV infection.•We confirmed changes in abundance and activity for two host RNA-binding proteins, DHX9 and DDX3.•We identified and confirmed three HCMV-RNA binding proteins during infection, pUS22, pTRS1 and pp71.•pp71 associated with host and viral mRNAs and associated with polysomes in infected cells, and increased protein synthesis.

Post-transcriptional events regulate herpesvirus gene expression, yet few herpesvirus RNA-binding proteins have been identified. We used an unbiased approach coupling oligo(dT) affinity capture with proteomics to identify viral RNA-associated proteins during infection. Using this approach, we identified and confirmed changes in the abundance or activity of two host RNA-associated proteins, DHX9 and DDX3, in cells infected with human cytomegalovirus (HCMV). We also identified and confirmed previously unreported activities for the HCMV US22 and pp71 proteins as RNA-associated viral proteins and confirmed that a known viral RNA-binding protein, pTRS1, associates with RNA in infected cells. Further, we found that HCMV pp71 co-sedimented with polysomes, associated with host and viral RNAs, and stimulated the overall rate of protein synthesis. These results demonstrate that oligo(dT) affinity capture coupled with proteomics provides a rapid and straightforward means to identify RNA-associated viral proteins during infection that may participate in the post-transcriptional control of gene expression.