Virion-associated, host-derived DHX9/RNA helicase A enhances the processivity of HIV-1 reverse transcriptase on genomic RNA.

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 tRNALys3-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.

Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity.

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3'-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

Transcriptome analysis of malate-induced Schizochytrium sp. FJU-512 reveals a novel pathway for biosynthesis of docosahexaenoic acid with enhanced expression of genes responsible for acetyl-CoA and NADPH accumulation.

Schizochytrium is one of the few oleaginous microalgae that produce docosahexaenoic acid (DHA)-rich lipids. In this study, global changes in gene expression levels of Schizochytrium sp. FJU-512 cultured with malate in a 15 l-bioreactor was analyzed using comparative transcriptomics. The changes were found mainly in the genes involved in oxidative phosphorylation, ß-oxidation, and pentose phosphate pathways. Consequently, the global changes in genes associated with the pathways could lead to an increase in the influx throughputs of pyruvate, branched-chain amino acids, fatty acids, and vitamin B6. Our transcriptome analysis indicated pyruvate dehydrogenase E2 component and acetolactate synthase I/II/III large subunit as major contributors to acetyl-CoA biosynthesis, whereas glucose-6-phosphate dehydrogenase was indicated as the major contributor to the biosynthesis of NADPH. An increase in DHA titer of up to 22% was achieved with the addition of malate to the fed-batch culture of Schizochytrium sp. FJU-512. This study provides an alternate method to enhance DHA production in Schizochytrium sp. FJU-512 through malate induced upregulation of genes responsible for acetyl-CoA and NADPH biosynthesis.