Control of HIV-1 RNA processing is central to the replication of the virus. Previously, we demonstrated that the cellular protein Sam68 enhances HIV-1 structural protein expression and RNA 3' end processing. In this report, we show that Sam68 interacts with unspliced HIV-1 RNA and that other members of the STAR/GSG protein family also promote viral RNA 3' end processing. We define a portion of the GSG domain (Sam 97-255) as sufficient for enhancement of Rev-dependent expression. In contrast to Sam68, Sam 97-255 increases unspliced RNA processing only in the presence of Rev in 293T cells. In a different cell line, Sam 97-255 enhances HIV-1 gene expression without enhancing RNA 3' end processing, suggesting that stimulation of 3' end processing is not required for enhancement of HIV-1 gene expression. Overall, these results indicate that Sam68 and the mutants described affect the composition of the viral RNP to enhance viral protein synthesis.