| Due to a limited genome size, Alphavirus replication and protein expression are dependent upon a number of cellular factors. While a handful of viral RNA-interacting host factors have been documented, the functions of many of these proteins during the viral replication cycle remain unclear. Here we report the identification more than two hundred Alphavirus RNA-binding partners via the crosslink-assisted mRNP purification (CLAMP) coupled with tandem mass spectrometry (MS/MS). The proteins identified participate in a variety of cellular processes including DNA/RNA regulation, protein modification, and cellular metabolism. In order to identify viral RNA binding sites for these host factors, we have employed photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). This method involves the immunoprecipitation and subsequent sequencing of short fragments of viral RNA cross-linked to target proteins. We have successfully utilized PAR-CLIP to identify multiple sites of interaction within the viral RNA for the Alphavirus capsid protein, and have identified binding sites for a group of CLAMP-identified viral RNA-binding proteins, the heterogeneous ribonucleoproteins (hnRNPs). Identification of binding sites for such proteins has enabled mutational studies that have begun to inform the role of the CLAMP-identified viral RNA-binding proteins in viral replication and gene expression. |
