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

Abstract

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.

Ushering in the era of tRNA medicines

“…( 89 ) used small-angle X-ray scattering to evaluate the impact of single nucleotide changes on tRNA folding and stability, whereas Gremminger et al . ( 90 ) used microscale thermophoresis to monitor interactions between HIV-1 viral RNA and tRNA Lys3 .…”

Clonally expanded HIV-1 proviruses with 5′-leader defects can give rise to nonsuppressible residual viremia

“…Some defective proviruses have small deletions in the 5'-Leader (5'-L) upstream of gag (23,32,33). This region contains regulatory elements orchestrating genomic RNA dimerization and packaging, reverse transcription, proviral gene expression, and RNA splicing and translation (34)(35)(36)(37)(38)(39). Proviruses with small 5'-L deletions comprise 5-10% of proviral populations, are found in most PLWH, often in expanded clones (23,32,(40)(41)(42), and may produce viral mRNA and p24 protein (26,33).…”

Retroviral PBS-segment sequence and structure: Orchestrating early and late replication events

“…1C, orange) are highly conserved in HIV-1 [3], and an analogous bulge is observed in other retroviruses, such as HIV-2 and simian immunodeficiency virus (SIV) that is situated at a comparable distance from the PBS [13,14]. The intermolecular interaction between the A-rich loop residues of vRNA and anticodon residues of tRNA Lys3 is supported by chemical and enzymatic probing studies, as well as solution NMR [5,[15][16][17]. When mutations were introduced within the 18 nt PBS of vRNA and near the A-rich loop region to be complementary to the anticodon residues of an alternative tRNA, virus replication was observed (Fig.…”

“…Other in vitro assays revealed that the long-range vRNA: tRNA complex requiring the A-rich loop motif diminished affinity for RT, but protected the vRNA from degradation by the RNase H activity of RT in the absence of active reverse transcription [17]. Since RNase H activity of RT can slowly cleave RNA/RNA in the absence of a DNA/RNA hybrid, forming a complex engendered by A-rich loop: anticodon interaction and reducing interaction with RT when reverse transcription is hindered in an environment lacking dNTPs could safeguard the integrity of the vRNA within the virion from the RNase H nuclease activity of RT [17,33]. Upon the virus entering a host and gaining access to a sufficient amount of dNTPs to support efficient reverse transcription into complementary DNA (cDNA), the RNase H activity directed by cDNA/ RNA hybrids would then digest the vRNA [17].…”