Study of HIV‐2 primer–template initiation complex using antisense oligonucleotides

Abstract

HIV‐2 reverse transcription is initiated by the retroviral DNA polymerase (reverse transcriptase) from a cellular tRNALys3 partially annealed to the primer binding site in the 5′‐region of viral RNA. The HIV‐2 genome has two A‐rich regions upstream of the primer binding site. In contrast to HIV‐1 RNA, no direct evidence of interactions with the U‐rich anticodon loop of tRNALys3 has been described to date. Here we address the question of the potential role of the interactions between these highly structured regions in the initiation of viral DNA synthesis. To evaluate this we used an antisense approach, first validated in our in vitro HIV‐1 reverse transcription system. Annealing of the antisense oligonucleotides to the pre‐primer binding site (the upstream region contiguous to the HIV‐2 primer binding site) was determined in the presence of native tRNALys3 or synthetic primers. Using natural and chemically modified antisense oligonucleotides we found that interactions between the anticodon of tRNALys3 and an A‐rich loop of viral RNA led to an important destabilization of the pre‐primer binding site; this region became accessible to anti‐pre‐primer binding site oligonucleotides in a cooperative manner. These studies allowed to identify an A‐rich region in HIV‐2ROD RNA capable of interacting with tRNALys3. Better knowledge of these interactions is very important for understanding the primer/template positioning in the early steps of HIV‐2 reverse transcription.

Cellular Uptake of ODNs in HIV-1 Human-Infected Cells: A Role for Viral Particles in DNA Delivery?

“…Similar results were described previously with antisense oligonucleotides in our laboratory (Boulme et al, 2000;Freund et al, 2001). One of the first questions we tried to answer was whether the ODN alone was able to enter the cell.…”

Direct and Indirect Contributions of RNA Secondary Structure Elements to the Initiation of HIV-1 Reverse Transcription

“…In addition to this "general" tRNA-PBS interaction, "virusspecific" interactions between the primer tRNA and the genomic RNA have been demonstrated in avian retroviruses (7)(8)(9)(10), HIV types 1 (11)(12)(13)(14)(15)(16)(17)(18) and 2 (19,20), feline immunodeficiency virus (21), and the yeast retrotransposons Ty1 (22)(23)(24) and Ty3 (25). These additional interactions are required for efficient replication (9, 24, 26 -28) and in vivo initiation of reverse transcription (29 -31).…”

Frequent Dual Initiation in Human Immunodeficiency Virus-Based Vectors Containing Two Primer-Binding Sites: a Quantitative In Vivo Assay for Function of Initiation Complexes

“…The structures of the U5-IR and U5-⌿ stems as well the existence of an unpaired A-loop have been confirmed by chemical probing of viral genomic RNA (14). The base pairing of the A-loop of viral RNA with the AC loop of tRNA Lys3 was proposed based on studies with antisense oligonucleotides targeted to these regions (16). Interestingly, in vitro studies of HIV-2 reverse transcription showed significant amounts of elongated products in the absence of AC-loop interactions (17).…”

“…Mutations H5 and H6 disrupted the proposed interactions with the T⌿C and the AC loops, respectively (14,16). The results suggest that these U5 sequences are important for efficiency of initiation; however, it will be necessary to restore these interactions by using mutant tRNAs to verify that the U5 sequences indeed interact with elements of the primer tRNA.…”

“…Mutation H5 targeted a portion of the U5 region that was previously proposed to interact with the T⌿C arm of tRNA Lys3 (30). Mutation H6 was designed to block the proposed interaction between the A-rich loop of viral RNA with the AC loop of the primer tRNA (16,30). Finally, mutation H7 disrupted annealing of the 3Ј-terminal CCA of the tRNA with the 5Ј region of the HIV-2 PBS.…”