Significance: A nucleotide-specific ² H-edited NMR approach was used to determine the nature of the intermolecular interface in the intact, dimeric HIV type-1 (HIV-1) 5′-leader RNA (230 kD). The studies distinguish between previously proposed extended duplex and kissing hairpin models and identify additional intermolecular interaction sites. A one-dimensional ² H-edited NMR method that allows temporal monitoring of intermolecular base-pair formation revealed that the observed “extended dimer interface” forms rapidly, even in the absence of RNA chaperones. In addition to addressing long-standing questions about retroviral genome dimerization, these studies illustrate the utility of ² H-edited NMR for determining the structures and folding kinetics of relatively large RNAs.

Significance: The proliferation of viral pathogens is restricted by hosts, but resilient pathogens antagonize the restriction by hosts. Findings explain that HIV-1 blocked mono-methylated guanosine cap by hypermethylation and engaged novel cap-binding complex for virion protein translation unaffected by global translation inhibition. The hypermethylated cap activity required RNA-structure-dependent binding of RNA helicase A/DHX9. eIF4E interaction proceeded on completely spliced HIV messenger RNA templates encoding viral regulatory proteins, thus eIF4E inactivation by catalytic site mTOR inhibitor suppressed regulatory protein translation, while structural/accessory protein translation was maintained. Two mutually exclusive translation pathways antagonize hosts and facilitate HIV-1 proliferation in primary CD4 ⁺ T cells to the detriment of hosts. eIF4E inactivation imposed an operational rheostat that suppressed regulatory proteins, while maintaining virion production in immune cells.

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. HIV^NL4-3 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.

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 single-nucleotide 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.

Coronaviruses (CoVs) are positive-stranded RNA viruses that infect humans and animals. Infection by CoVs such as HCoV-229E, -NL63, -OC43 and -HKU1 leads to the common cold, short lasting rhinitis, cough, sore throat and fever. However, CoVs such as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and the newest SARS-CoV-2 (the causative agent of COVID-19) lead to severe and deadly diseases with mortality rates ranging between ~1 to 35% depending on factors such as age and pre-existing conditions. Despite continuous global health threats to humans, there are no approved vaccines or drugs targeting human CoVs, and the recent outbreak of COVID-19 emphasizes an urgent need for therapeutic interventions. Using computational and bioinformatics tools, here we present the feasibility of reported broad-spectrum RNA polymerase inhibitors as anti- SARS-CoV-2 drugs targeting its main RNA polymerase, suggesting that investigational and approved nucleoside RNA polymerase inhibitors have potential as anti-SARS-CoV-2 drugs. However, we note that it is also possible for SARS-CoV-2 to evolve and acquire drug resistance mutations against these nucleoside inhibitors.