HIV-1 viral RNA structure regulates both initiation of reverse transcription and protein synthesis

HIV-1, a single-stranded RNA retrovirus, has devastated millions of people worldwide. Understanding how this virus infects cells and replicates is critical for both understanding retroviruses and making next generation antiretrovirals that can improve quality of life for people living with HIV. While decades of research have been dedicated to the understanding of this virus, key mechanistic information remains to be elucidated. RNA structure plays a key role in regulating different stages of the HIV replication cycle. HIV-1 genomic RNA contains a highly structured and conserved 5' untranslated region that is the hub for multiple functions during viral replication. Reverse transcription, the process by which HIV converts its single-stranded RNA genome into double-stranded DNA, represents an early stage in the virus' replication cycle where the viral genome is simultaneously copied and prepared for integration into the host cell's genome. Translation of the genomic RNA transcripts allows production of the proteins that comprise new HIV virions. Both processes initiate in the 5' UTR region of the genomic RNA. There are many open questions surrounding mechanism, efficiency, and kinetics of these processes due to the structural and functional density of the 5'UTR. Furthermore, many conserved structures and RNA interactions that have been demonstrated to be important in cell cultures have yet to be placed within the context of viral replication processes. Here I present my thesis work to understand how RNA structure in this region of the viral genome regulates reverse transcription and translation initiation. Reverse transcription begins upon entry of a viral capsid into a host cell cytoplasm. The process can be divided into initiation and elongation phases. The initiation phase is very slow and non-processive while the elongation phase is quite rapid and processive. To understand why the initiation phase is so slow, a minimal construct was crosslinked to reverse transcriptase (RT) and purified for cryogenic electron microscopy (cryoEM). The complexes captured a reverse transcription initiation complex (RTIC) after three and six rounds of incorporation. The +3 RTIC represents a significant stalling point for the process, with an approximately 10- fold decrease in rate compared to the other steps in the initiation phase. The +6 RTIC represents the transition from the slow, non-processive initiation phase to elongation phase. The resulting data showed conformational heterogeneity after three rounds of incorporation. This heterogeneity showed intermediate states that represent off-pathway intermediates that are the result of a stable hairpin in the templating viral RNA. After six rounds of incorporation, the complex shows novel RNA density corresponding to a previously demonstrated regulatory RNA interaction. Additionally, the primer terminus is poised for catalysis, relative to previous RTIC structures. These structural changes are likely responsible for the steep rise in processivity, and speed observed after 6 rounds of dNTP incorporation. In the latter phase of viral replication, viral proteins and genomic RNA must be assembled into virions and bud from the cell. To achieve this, viral mRNAs are transcribed from HIV genes and host cell ribosomes are used to make viral factors. However, the mechanisms used to initiate translation of HIV proteins required for viral assembly and budding remain poorly defined. Here we focused on the regulatory role of the 5'UTR structure and sequence in HIV mRNA translation initiation. To understand how HIV translation initiation differs from standard translation initiation, the 5'UTR was cloned in front of a nano luciferase (nLuc) luminescence reporter gene. The bulk translation activity of the resulting mRNA was assayed in HeLa cell extracts. RNAs containing the HIV 5'UTR had a significant drop in translation efficiency. While they translate poorly relative to housekeeping genes, there is no significant difference between the translation efficiency, mean synthesis rate, and mean synthesis time for capped and uncapped HIV mRNAs. Using single-molecule fluorescence to track translation components during initiation, the kinetics of translation initiation for the housekeeping gene control and the HIV mRNA constructs were similar, however, the small ribosomal subunit struggles to load onto the structured 5'-UTR. This results in most small ribosomal subunit binding events not leading to successful translation initiation for HIV mRNAs. Together, these findings provide evidence for RNA structure as playing a regulatory role in translation initiation. This inefficiency might be advantageous for the virus to maintain a slow yet steady pace for virion assembly such that it minimally triggers immune system sensors

Keywords: Medicine.
Structural Biology.
Advisor: Puglisi Joseph D.
Brünger Axel T.
Fire Andrew Zachary