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.
Highlights
In retrovirus reverse transcription, the single-stranded genomic RNA is converted into a double-stranded DNA by reverse transcriptase (RT)3 [1, 2]
We considered whether the region of the human immunodeficiency virus 1 (HIV-1) genome 3Ј of the primer binding site (PBS) could influence the efficiency of minus strand transfer
It was similar to D199 in that there was no change in homology between the donor and acceptor, and the primer still annealed to the PBS
Summary
Materials—DNA oligonucleotides were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). Generation of RNA Templates—Genomic sequences from the HIV-1 pNL4 –3 strain were amplified by PCR using the Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) to create double-stranded DNA templates for generation of RNA transcripts. The donor RNA template D199 and acceptor RNA templates A97h, A82h, and A70h were generated by run-off transcription in vitro as described previously [21]. Transfer Assay—The PBS DNA primer or RNA templates were radiolabeled at the 5Ј-end as described previously for either primer extension assay or donor and acceptor RNA templates degradation assays, respectively [21, 24]. Distribution of Transfer Products—To determine where the primer terminus switched between the donor and acceptor templates, transfer products were isolated and amplified as described previously [47, 48]. Products were separated on an 8% polyacrylamide-urea gel, and visualized as described above
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
