Stimulation of HIV-1 minus strand strong stop DNA transfer by genomic sequences 3′ of the primer binding site

Abstract

The mechanism of human immunodeficiency virus 1 (HIV-1) minus strand transfer was examined using a genomic RNA sequence-based donor-acceptor template system. The donor RNA, D199, was a 199-nucleotide sequence from the 5′-end of the genome to the primer binding site (PBS) and shared 97 nucleotides of homology with the acceptor RNA. To investigate the influence of RNA structure on transfer, a second donor RNA, D520, was generated by extending the 3′-end of D199 to include an additional 321 nucleotides of the genome. The position of priming, length of homology with the acceptor, and length of cDNA synthesized were identical with the two donors. Interestingly, at 200% NC coating, donor D520 yielded a transfer efficiency of about 75% compared with about 35% with D199. A large proportion of the D520 promoted transfers occurred after the donor RNA was copied to the end. Analysis of donor RNA cleavage, the acceptor invasion site and R homology requirements indicated that transfers with D520 involved a similar but more efficient acceptor invasion mechanism compared with D199. RNA structure probing by RNase T1 and the RT pause profile during synthesis indicated conformational differences between D199 and D520 in the starting structure, and in dynamic structures formed during synthesis within the R region. Overall observations suggest that regions 3′ of the primer binding site influence the conformation of the R region of D520 to facilitate steps that promote strand transfer. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.