The integrated HIV-1 proviral genome is transcribed by the host transcription machinery into a single 9.2 kb primary transcript. To express the nine different gene products required for viral replication, HIV has developed a number of strategies to regulate splicing and to circumvent cellular mechanisms restricting unspliced RNA in the cytoplasm. The regulatory protein Rev promotes transport of the incompletely spliced viral RNAs to the cytoplasm, binding to the Rev Responsive Element (RRE) on the viral RNA. Mechanisms regulating the appearance of unspliced and spliced viral RNAs, and their transport to the cytoplasm, involve interactions between viral RNA sequences and host cell factors. The characterization of these viral/host interactions could allow the design of novel therapeutic compounds. hnRNP H proteins have been shown to be necessary for HIV-1 RNA splicing in a series of in vitro experiments. They may also regulate RNA export and trafficking in infected cells. The role of single members of the hnRNP H protein family in HIV-1 RNA processing in vivo will be investigated by modulating hnRNP H protein expression in cells transfected with HIV-1-derived plasmids, bearing mutations in regulatory sequences. Several viral sequences that regulate viral RNA splicing in vitro have been identified. These will be tested to determine whether they are necessary for the regulation of viral RNA splicing in vivo. Preliminary data indicates that viral sequences interact with the RRE-Rev complex and, possibly, have a role in the Rev-dependent viral RNA transport pathway. These interactions will be characterized by a combination of in vivo and in vitro assays. Transcription and splicing have been shown to be closely coupled in other systems. Preliminary data indicates that the viral promoter influences splicing of viral substrates in vitro. Possible coupling of HIV-1 transcription and splicing will be investigated, using both a coupled transcription-splicing assay and transient transfection experiments.
