The paradigm protein synthesis rate is regulated by structural complexity of the 5′untranslated region (UTR) derives from bacterial and other riboswitches. In-solution, HIV-1 5′UTR forms two interchangeable long-range nucleotide (nt) -pairings, one sequesters the gag start codon promoting dimerization while the other sequesters the dimer initiation signal preventing dimerization. While the effect of these nt-pairings on dimerization and packaging has been documented their effect on authentic HIV translation in cellulo has remained elusive until now. HIVNL4-3 5′UTR substitutions were designed to individually stabilize the dimer-prone or monomer-prone conformations, validated in-solution, and introduced to molecular clones. The effect of 5′UTR conformation on ribosome loading to HIV unspliced RNA and rate of Gag polypeptide synthesis was quantified in cellulo. Monomer- and dimer-prone 5′UTRs displayed equivalent, basal rate of translation. Gain-of-function substitution U103, in conjunction with previously defined nt-pairings that reorient AUG to flexible nt-pairing, significantly activated the translation rate, indicating the basal translation rate is under positive selection. The observed translation up-mutation focuses attention to nt-pairings at the junction of R and U5, a poorly characterized structure upstream of the characterized HIV riboswitch and demonstrates the basal translation rate of authentic HIV RNA is regulated independently of monomer:dimer equilibrium of the 5′UTR.

The 3′-end of the genomic RNA of the hepatitis C virus (HCV) embeds conserved elements that regulate viral RNA synthesis and protein translation by mechanisms that have yet to be elucidated. Previous studies with oligo-RNA fragments have led to multiple, mutually exclusive secondary structure predictions, indicating that HCV RNA structure may be context-dependent. Here we employed a nuclear magnetic resonance (NMR) approach that involves long-range adenosine interaction detection, coupled with site-specific 2H labeling, to probe the structure of the intact 3′-end of the HCV genome (385 nucleotides). Our data reveal that the 3′-end exists as an equilibrium mixture of two conformations: an open conformation in which the 98 nucleotides of the 3′-tail (3′X) form a two-stem–loop structure with the kissing-loop residues sequestered and a closed conformation in which the 3′X rearranges its structure and forms a long-range kissing-loop interaction with an upstream cis-acting element 5BSL3.2. The long-range kissing species is favored under high-Mg2+ conditions, and the intervening sequences do not affect the equilibrium as their secondary structures remain unchanged. The open and closed conformations are consistent with the reported function regulation of viral RNA synthesis and protein translation, respectively. Our NMR detection of these RNA conformations and the structural equilibrium in the 3′-end of the HCV genome support its roles in coordinating various steps of HCV replication.

Edited by Karin Musier-Forsyth DHX9/RNA helicase A (RHA) is a host RNA helicase that participates in many critical steps of the HIV-1 life cycle. It co-assembles with the viral RNA genome into the capsid core. Virions deficient in RHA are less infectious as a result of reduced reverse transcription efficiency, demonstrating that the virion-associated RHA promotes reverse transcription before the virion gains access to the new host's RHA. Here, we quantified reverse-transcription intermediates in HIV-1-infected T cells to clarify the mechanism by which RHA enhances HIV-1 reverse transcription efficiency. Consistently, purified recombinant human RHA promoted reverse transcription efficiency under in vitro conditions that mimic the early reverse transcription steps prior to capsid core uncoating. We did not observe RHA-mediated structural remodeling of the tRNA Lys3-viral RNA-annealed complex. RHA did not enhance the DNA synthesis rate until incorporation of the first few nucleotides, suggesting that RHA participates primarily in the elongation phase of reverse transcription. Pre-steady-state and steady-state kinetic studies revealed that RHA has little impact on the kinetics of singlenucleotide incorporation. Primer extension assays performed in the presence of trap dsDNA disclosed that RHA enhances the processivity of HIV-1 reverse transcriptase (RT). The biochemical assays used here effectively reflected and explained the low RT activity in HIV-1 virions produced from RHA-depleted cells. Moreover, RT activity in our assays indicated that RHA in HIV-1 virions is required for the efficient catalysis of (؊)cDNA synthesis during viral infection before capsid uncoating. Our study identifies RHA as a processivity factor of HIV-1 RT.

Vancomycin-resistant enterococci (VRE) are among the most common causes of nosocomial infections, which can be challenging to treat. VRE have acquired a suite of resistance genes that function together to confer resistance to vancomycin. Expression of the resistance phenotype is controlled by the VanRS two-component system. This system senses the presence of the antibiotic, and responds by initiating transcription of resistance genes. VanS is a transmembrane sensor histidine kinase, and plays a fundamental role in antibiotic resistance by detecting vancomycin and then transducing this signal to VanR. Despite the critical role played by VanS, fundamental questions remain about its function, and in particular about how it senses vancomycin. Here, we focus on purified VanRS systems from the two most clinically prevalent forms of VRE, types A and B. We show that in a native-like membrane environment, the enzymatic activities of type-A VanS are insensitive to vancomycin, suggesting that the protein functions by an indirect mechanism that detects a downstream consequence of antibiotic activity. In contrast, the autokinase activity of type-B VanS is strongly stimulated by vancomycin. We additionally demonstrate that this effect is mediated by a direct physical interaction between the antibiotic and the type-B VanS protein, and localize the interacting region to the protein's periplasmic domain. This represents the first time that a direct sensing mechanism has been confirmed for any VanS protein.

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Understanding viral RNA structure and how it functions is crucial in elucidating new drug targets. There are many kinds of viruses that utilize RNA as a critical component of their life cycle, such as retroviruses, single-stranded plus or minus sense RNA viruses, and double-stranded RNA viruses. Two viruses that are studied in this thesis are human immunodeficiency virus (HIV), which is a retrovirus, and hepatitis C virus (HCV), which is a single-stranded plus sense RNA virus. It has been previously reported that a human host factor, RNA helicase A (RHA), is packaged into HIV virions by binding to the primer binding site (PBS) segment of the 5'untranslated region in the HIV genomic RNA. We determined RHA is required for efficient reverse transcription prior to capsid uncoating by utilizing cell based and in vitro techniques. It has also been suggested that RHA plays other roles during HIV infection besides reverse transcription. Utilizing NMR, we demonstrated that RHA binds to the monomeric 5'UTR at the bottom of the TAR hairpin, which is different from how it binds during viral packaging. Next, we employed NMR techniques to probe the 3'end of the HCV genome called 3'X. We determined that the 3'X is in structural equilibrium between two states: an open conformation and a closed conformation. These two conformations have been suggested to play a role in minus sense synthesis and viral protein translation, respectively. Taken together, my thesis work has elucidated how many viruses manipulate and utilize their RNA structure to modulate their outcome.