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. Our previous results showed that most HIV-1 particles have two copies of viral RNA genome, indicating that the packaging is tightly regulated. We have performed studies and showed that HIV-1 packaging is not regulated by RNA mass but by recognizing a dimeric RNA. We have now defined the sequences necessary and sufficient for packaging RNA into HIV-1 virions. Additionally, we have determined the role of several highly structured RNA elements in HIV-1 replication. We are also examining the translation of the full-length RNA, and delineating the host factors that affect HIV-1 RNA nuclear export and the dynamics of HIV-1 RNA export from the nucleus to the cytoplasm. These experiments seek to gain insights into how HIV-1 RNA serves its functions. ___BACKGROUND: The full-length HIV-1 RNA (hereafter referred to as HIV-1 RNA) serves as a template for Gag/Gag-Pol translation and as the virion genome. HIV-1 RNA needs to negotiate through the complex cellular regulation of the host to be exported from the nucleus to the cytoplasm. Once exported, HIV-1 RNA can be translated and/or packaged and needs to strike a balance between these two functions. In this project, we seek to gain a better understanding of how HIV-1 RNA serves its roles. ___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. ___It was recently shown that HIV-1 uses heterogenous transcriptional start sites and the usage of the sites can affect RNA function. We have established a collaboration with Dr. Karin Musier-Forsyth to examine the impact of HIV-1 transcription start site usage on RNA structures and functions. Additionally, we are collaborating with Dr. Brandon Keele to study the conservation of the usage of transcription start sites to control RNA functions. ___ACCOMPLISHMENTS: To gain a better understanding of HIV-1 gene expression, we tracked translation of individual HIV-1 RNAs in living cells. We found that, on average, half of the cytoplasmic HIV-1 RNAs are being actively translated at a given time. Furthermore, translating and nontranslating RNAs are well mixed in the cytoplasm; thus, Gag biogenesis occurs throughout the cytoplasm without being constrained to particular subcellular locations. Because Gag is an RNA-binding protein and packages HIV-1 RNA, a long-standing question regarding HIV-1 gene expression is whether Gag modulates its own translation. We observed that despite its RNA-binding ability, Gag expression does not alter the proportion of translating HIV-1 RNA. Although both translating and nontranslating RNAs can travel to the plasma membrane, the major HIV-1 assembly site, Gag selectively packages nontranslating RNA into the assembly complex. These studies illustrate that although HIV-1 RNA serves two functions, as a translation template and as a viral genome, individual RNA molecules carry out only one function at a time. ____ 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.