Abstract
Human immunodeficiency virus type 1 minus strand transfer was measured using a genomic donor-acceptor template system in vitro. Donor RNA D199, having the minimum region required for minus strong stop DNA synthesis, was previously shown to transfer with 35% efficiency to an acceptor RNA representing the 3' repeat region. Donor D520, having an additional 321-nucleotide segment extending into gag, transferred at 75% efficiency. In this study each transfer step was analyzed to account for the difference. Measurement of terminal transfer indicated that the 3' terminus of the cDNA generated using D520 is more accessible for transfer than that of D199. Nevertheless, acceptor competition experiments demonstrated that D520 has a greater preference for invasion-driven versus terminal transfer than D199. Competition mapping showed that the base of the transactivation response element is the primary invasion site for D520, important for efficient acceptor invasion. Acceptors complementary to the invasion and terminal transfer sites, but not the region between, allowed assessment of the significance of hybrid propagation by branch migration. These bipartite acceptors showed that with D520, invasion raises the local concentration of the acceptor for efficient terminal transfer by a proximity effect. However, with D199, invasion is relatively inefficient, and the cDNA 3' terminus is not very accessible. For most transfers that occurred, the acceptor accessed the cDNA 3' end by branch migration. Results suggest that both proximity and branch migration mechanisms contribute to transfers, with the proportion determined by donor-cDNA structure. D520 transfers better because it has greater accessibility for both invasion and terminus transfer.
Highlights
Retroviruses, including human immunodeficiency virus type 1 (HIV-1),3 package two copies of single-stranded genomic RNA [1, 2]
We previously described an HIV-1 minus strand transfer system in vitro in which the donor template included a 199-nt genomic RNA extending from the primer binding site (PBS) to the natural 5Ј end (D199) [14, 16]
Even though transfers in vivo occur with high efficiency [55,56,57], the maximum transfer efficiency achieved with D199 was only about 35% [14, 16]
Summary
Materials—HIV-1 reverse transcriptase (p66/p51 heterodimer) was purified as described previously [51, 52]. The sequence of dA80h transfer efficiencies of the cDNA 3Ј-terminal regions of the two was 5Ј-AAA AAA AAA AAG GGT CTC TCT GGT TAG ACC donors, RNA acceptor template A19h was designed. The transfer reaction of the truncated DNA acceptors, which was either dA19h, occurs at a slower rate than primer extension on the donor dA26h, dA35h, dA45h, dA59h, dA70h, or dA80h (Fig. 4), the template. A series of truncated DNA acceptor templates, dA19h, dA26h, dA35h, dA45h, dA59h, dA70h, and dA80h, which shared a homology of 19, 26, 35, 45, 59, 70, and 80 nt, respectively, with D520 and D199, were constructed (Fig. 4A) These were used in competition reactions to finely map the sequence at the invasion site. Values were averaged from a minimum of three independent experiments
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