Elucidating the Regulation of HIV RNA Functions: Translation Genome Packaging

Division Of Basic Sciences - Nci

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Abstract

Retroviral full-length RNA serves two important roles in viral replication: the template for Gag/Gag-Pol translation and the genome in the virion. Recent studies from our group and others demonstrated that HIV-1 transcription initiates from multiple neighboring sites, generating RNA species that only differ by a few nucleotides at the 5' end, including those with one (1G) or three (3G) 5' guanosines. Strikingly, 1G RNA is preferentially packaged into virions over 3G RNA. We used biochemical and virological assays to investigate how HIV-1 distinguishes between these two nearly identical HIV-1 RNAs. We found that 1G RNA, but not 3G RNA, mainly folds into structures that expose elements important for RNA:RNA and RNA:Gag interactions. Additionally, we have identified mutants in which 1G and 3G RNAs fold into similar structures, resulting in efficient packaging of 3G RNA. Thus, HIV-1 selects its viral genome based on its capacity to adopt structures that facilitate RNA dimerization and Gag binding. Unspliced HIV-1 RNA serves two important roles during viral replication: as the virion genome and as the template for translation of Gag/Gag-Pol. Previous studies of two HIV-1 molecular clones have concluded that the TSS usage affects unspliced HIV-1 RNA structures and functions. To investigate the evolutionary origin of this replication strategy, we determined transcription start sites (TSS) of HIV-1 RNA in infected cells and virions for 15 primate lentiviruses. All the HIV-1 isolates examined, including several transmitted founder viruses, utilized multiple TSS and selected a particular RNA species for packaging. Furthermore, these features were observed in SIVs related to the progenitors of HIV-1, suggesting that these characteristics originated from the ancestral viruses. HIV-2, SIVs related to HIV-2, and other SIVs also exhibited multiple TSS and preferential packaging of specific unspliced RNA species. These findings indicate that multiple TSS usage and selective packaging of a particular unspliced RNA species predate the emergence of HIV-1. We have determined that transcription initiation is regulated by the distance between the CATA/TATA box and the three guanosines, as well as the sequence context of the initiation sites. We have constructed two HIV-1 mutants, each with two-nucleotide substitution, that predominantly express 3G or 1G RNA. We found that both mutants can replicate; however, both exhibited defects during stages of replication cycle; furthermore neither virus can replicate as well as the wildtype virus, indicating defects in replication fitness. These results indicate that HIV-1 optimizes its replication fitness by using multiple transcription start sites to generate RNAs that serve different functions. We are currently studying how the 5' context of the HIV-1 RNA affects RNA translation and protein production as well as RNA splicing. As an unspliced RNA, HIV-1 RNA needs to bypass the cellular gatekeepers to be exported from the nucleus and reach the cytoplasm. HIV-1 RNA contains an RNA structure, the Rev responsive element (RRE). The viral protein Rev binds to the RRE and interacts with the host protein CRM1 to allow for the export of HIV-1 RNA. Recent studies revealed that the regulation of RNA export may be more complex than previously envisioned and may involve multiple host factors other than CRM1 and RanGTP. We are studying how export pathways affect cytoplasmic HIV-1 RNA transport and proteins associated with the RNA. In summary, we seek to gain a better understanding of how HIV-1 RNA serves its critical roles in generating infectious viruses and how latent viruses can be activated. This knowledge can potentially help us design novel antiviral or cure strategies.