Investigations into viral RNA replication

RNA viruses are incredibly important to study because they make up 44% of all emerging infectious diseases and novel RNA viruses are also being discovered at a rate of 2 to 3 each year. Understanding the basis of viral RNA replication has remained elusive due to the difficulty in studying this dynamic process. The viral RNA genome is highly plastic and must change conformations to accommodate the different steps of the viral life cycle. This body of work investigates viral replication in two RNA viruses, HIV-1 and dengue. HIV-1 has been studied for decades, however we still don't understand the details of the initiation phase of replication. Replication of the HIV-1 genome involves two primary steps, the initiation step, copying the first 200 nucleotides and the later elongation phase, copying the rest of the 10kb genome. Initiation is 200X slower than elongation and is slow and non-processive. Efforts to understand initiation in structural studies using X-ray crystallography and cryogenic electron microscopy (cryo-EM) of the reverse transcriptase initiation complex (RTIC), comprised of reverse transcriptase (RT) in complex with a vRNA-tRNALys3 duplex, have remained limited at 4 Å resolution. At this resolution we are unable to understand the molecular mechanisms that underlie initiation. Drug inhibition of the elongation phase has been thoroughly studied, however it is unknown whether drugs target initiation, a phase that might be especially vulnerable to drug inhibition. In this work we report cryo-EM structures of the core RTIC, RTIC-nevirapine, and RTIC-efavirenz at 2.8, 3.1 and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA-tRNALys3 initiation complex and reveal specific stabilization of the nucleic acid via several hydrophobic contacts in the RNaseH region. Our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription. Dengue is a part of the flaviviral family and there are currently no antivirals available to treat this class of mosquito-borne viruses. This is due in part to our limited understanding of its life cycle and pathogenesis. Flaviviruses are a group of single-stranded, positive sense RNA viruses with a conserved 11kb genome containing a single open reading frame (ORF) flanked by two highly structure 5' and 3' ends. The 5' UTR, 5' capsid coding region and 3' UTR contain inverted complementary sequences that have the potential to mediate long range non-covalent RNA-RNA interactions forming a circularized genome. Genetic studies testing disruption of base pairing in the 5'-3' UAR or 5'-3' CS complementary regions have severely impaired replication efficiency, and the hypothesis is that cyclization of the genome is impaired. Studies investigating dengue cyclization have been limited as it's difficult to detect long-range interactions in the genome. NS5 is the polymerase responsible for replicating the dengue viral genome and has been implicated in altering the conformation of the viral RNA. The promoter site is located at the 5' end, however replication initiates at the 3' end. It has been hypothesized that cyclization of the genome brings the two ends together so that NS5 can get to the 3' end to initiate replication. In this work we investigate the cyclization dynamics of the dengue viral RNA using time-resolved single-molecule FRET (smFRET). We reveal that the vRNA can exist in at least three conformations a linear state, a mid-cyclized state and a high-FRET state. Cyclization mutants in the 5'-3' UAR or 5'-3' CS regions shift the conformational equilibrium toward the mid-cyclized and linear states. In this complete body of work, we found that HIV-1 reverse transcription initiation is a highly plastic process and that vRNAs are very flexible making them difficult to study. We must employ a variety of methods to uncover conformational states relevant for replication. Finally, we found evidence that viral proteins play a role in vRNA structure

Advisor: Puglisi Joseph D.
Brünger Axel T.
Bryant Zev David
Puglisi Elisabetta