Elucidating the Regulation of HIV RNA Functions:Translation and Genome Packaging

Division Of Basic Sciences - Nci

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Abstract

...and RNA packaging, and we are dissecting the role of the Gag-RNA interactions in packaging two copies of RNAs. __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, indicating that these nearly identical RNAs are distinct in their functions. 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. Thus, HIV-1 selects its viral genome based on its capacity to adopt structures that facilitate RNA dimerization and Gag binding (Nikolaitchik et al PNAS 2021). 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. This study is published in PNAS in 2023. We have investigated the ability of different HIV-1 RNA species to be translated using polysome profiling method and found that both 1G and 3G RNA can be translated. Although similar proportions of 1G and 3G RNAs are in the polysomes; they have distributions in the polysome fractions. Compared to 3G RNA, the 1G RNA is enriched in the light polysomes and depleted in the medium and heavy polysomes. More ribosomes are associated with RNAs in medium and heavy polysomes compared to RNA in the light polysomes. These results indicate that 3G RNA is translated more efficiently than 1G RNA. Furthermore, using mutants in which the 5' UTR of 1G and 3G RNA fold into similar conformations, we showed that 1G and 3G RNA of these mutants are translated at similar efficiencies. Thus, the 5' UTR RNA structures of HIV-1 RNA regulates both genome packaging and translation efficiencies. This study is published in Journal of Virology in 2024. We have also examined samples isolated from people living with HIV and found that, while replicating in human, HIV-1 uses multiple transcription start sites to express multiple unspliced RNA species including 1G and 3G RNA. Furthermore, 1G RNA is selected over 3G RNA to be packaged as the viral genome. These studies extend our observation from the cell culture system to the behavior of HIV-1 in humans. This study was published in the mBio Journal in 2025. 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.