Alternate structures regulate transcription and translation of RNA viral genomes

Abstract

With the emergence of new RNA-structure related techniques, it is becoming increasingly clear that viral RNA genomes are structurally and functionally plastic, which drive multiple phases of the viral lifecycle. My doctoral work investigates how alternate RNA structures regulate transcription and translation of human immunodeficiency virus – 1 (HIV-1) and translation of both severe acute respiratory syndrome-related coronavirus (SARS-CoV-1) and severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2). During transcription of the HIV-1 genome, the viral Tat protein must compete with the cellular 7SK:HEXIM interaction to hijack the positive transcriptional elongation factor pTEFb and recruit it to the TAR RNA motif on stalled viral transcripts. My work on this system finds that 7SK is peppered with arginine sandwich motifs (ASMs) previously thought to be exclusive to retroviruses and is conformationally plastic, shedding light on its role as a molecular scaffold capable of interacting with a diverse number of factors. The HEXIM protein causes specific local destabilization of 7SK upon binding – a feature that is then exploited by Tat to both bind and remodel the RNA more efficiently and displace HEXIM. Furthermore, Tat also remodels the TAR bulge into an ASM that is identical to how it remodels 7SK. Overall, my work on HIV-1 transcription has uncovered that HEXIM primes for its own displacement by locally destabilizing and increasing the conformational sampling of 7SK. Additionally, I find that HIV-1 has evolved a dual structural mimicry wherein structural motifs present in HEXIM and 7SK are co-opted for productive transcription of its genome. During translation of their genomes, HIV-1, SARS-CoV-1, and SARS-CoV-2 precisely regulate the ratio of enzymatic and structural proteins needed for viral assembly using -1 programmed ribosomal frameshifting (-1 PRF) wherein the ribosome moves one nucleotide backward during a fraction of translation events to reveal a new open reading frame. My work on -1 PRF in HIV-1 provides evidence that a minor conformation of the HIV frameshifting signal folds into a pseudoknotted asymmetric heterodimer to regulate frameshifting. Additionally, my work on -1 PRF in SARS-CoV-1 and CoV-2 shows that a single protonation event can partition the frameshifting signal of SARS-Coronaviruses into two distinct active and inactive configurations. Taken together, my studies show that the HIV-1 and SARS frameshifting signals sample multiple conformations to regulate the fraction of ribosomes that undergo -1 PRF.