Interaction of cellular factors with intragenic cis-acting repressive sequences within the HIV genome

Expression of the human immunodeficiency virus (HIV) structural gene products is suppressed in the absence of the Rev protein. The block to expression reflects, in part, nuclear retention of those mRNAs which encode the structural proteins. The presence of intragenic cis-acting repressive sequences (CRS) and inefficient splicing of the primary viral transcript are thought to contribute to nuclear entrapment of viral RNA. To elucidate the mechanism for repression of HIV gene expression, the ability of a 270-bp segment of the pol gene shown previously to repress gene expression to interact with cellular factors was investigated. Incubation of RNA corresponding to the 270-bp CRS element with nuclear extract prepared from human T-cells revealed a strong and specific interaction with several cellular factors. Covalent cross-linking of the RNA-protein complex demonstrated the presence of at least three proteins, the predominant one having a molecular weight of approximately 42 kDa. A monoclonal antibody raised against hnRNP C, a component of the splicing machinery, recognized the CRS-protein complex, suggesting that hnRNP C or a closely related gene product interacts with CRS-containing RNA. Consistent with this conclusion, addition of RNA corresponding to a beta-globin intron sequence in the binding reaction completely blocked formation of the CRS-protein complex. These findings raise the possibility that the CRS elements elicit nuclear entrapment of viral RNA through formation of RNA-protein complexes that are not accessible to nuclear export pathways.