Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Can Prevent Self-Priming of Minus-Strand Strong Stop DNA by Promoting the Annealing of Short Oligonucleotides to Hairpin Sequences

Abstract

Understanding how viral components collaborate to convert the human immunodeficiency virus type 1 genome from single-stranded RNA into double-stranded DNA is critical to the understanding of viral replication. Not only must the correct reactions be carried out, but unwanted side reactions must be avoided. After minus-strand strong stop DNA (؊sssDNA) synthesis, degradation of the RNA template by the RNase H domain of reverse transcriptase (RT) produces single-stranded DNA that has the potential to self-prime at the imperfectly base-paired TAR hairpin, making continued DNA synthesis impossible. Although nucleocapsid protein (NC) interferes with ؊sssDNA self-priming in reverse transcription reactions in vitro, NC alone did not prevent self-priming of a synthetic ؊sssDNA oligomer. NC did not influence DNA bending and therefore cannot inhibit self-priming at hairpins by directly blocking hairpin formation. Using DNA oligomers as a model for genomic RNA fragments, we found that a 17-base DNA fragment annealed to the 3 end of the ؊sssDNA prevented self-priming in the presence of NC. This implies that to avoid self-priming, an RNA-DNA hybrid that is more thermodynamically stable than the hairpin must remain within the hairpin region. This suggests that NC prevents self-priming by generating or stabilizing a thermodynamically favored RNA-DNA heteroduplex instead of the kinetically favored TAR hairpin. In support of this idea, sequence changes that increased base pairing in the DNA TAR hairpin resulted in an increase in self-priming in vitro. We present a model describing the role of NC-dependent inhibition of self-priming in first-strand transfer.Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) synthesizes minus-strand strong stop DNA (ϪsssDNA) by extending tRNA 3 Lys bound at the primer binding site (PBS) in the RNA genome (8,15,37). To synthesize ϪsssDNA, RT copies the U5 and R regions; the R region contains two large hairpins known as the poly(A) hairpin and the TAR hairpin (9). When this RNA is copied into DNA, hairpins that correspond to the TAR and poly(A) hairpins of the RNA can form in the nascent ϪsssDNA; the formation of these hairpins depends on the digestion of the template RNA (17). The RNase H activity of RT cleaves the RNA portion of the RNA-DNA heteroduplex during polymerization, and there is additional RNase H cleavage after ϪsssDNA synthesis is complete (8,12,18,27,33). Although it is possible for either the nascent TAR and poly(A) DNA to form hairpins that could self-prime, such self-priming events are not detected when the HIV-1 genome is copied into DNA in infected cells. Because the TAR DNA hairpin forms at the end of R, the likelihood of self-priming is higher for TAR than for the poly(A) hairpin. Instead of self-priming, the 3Ј end of the nascent DNA is efficiently transferred to the R sequence on the 3Ј end of the template RNA, where synthesis continues. This event is known as the first-strand transfer. Nucleocapsid protein (NC) has been shown to prevent synthesis of selfpri...

Mechanism of Minus Strand Strong Stop Transfer in HIV-1 Reverse Transcription

“…Interestingly, the 2Ј-O-methyl modification greatly reduced the self-priming product in both the donor extension and strand transfer reactions (data not shown). This is not surprising, since the uncleaved 16-nt RNA fragment would remain annealed to the 3Ј-end of cDNA and thereby inhibit it from folding back and self-priming, as shown previously by the Hughes group (50,58,77).…”

“…A ladder of pause sites was observed from position 47 through 53. Analysis of the predicted secondary structure of this region suggested that the pausing ladder was due to displacement synthesis through an alternate hairpin structure formed from refolding of the template, as shown previously (50).…”

“…Formation of self-priming products is an indication of donor 5Ј-end cleavage (50,58). The Hughes group proposed that the uncleaved 14-nt 5Ј-end fragment serves to prevent the ϪsssDNA from folding back on itself and self-priming and that NC serves to keep the terminal 14-nt fragment annealed to the donor template (50,58). In contrast, with the shorter 19-nt homology system, the bulk of transfers occurred after substantial cleavage near the RNA 5Ј-end (Fig.…”

“…The strand annealing and chaperone activities of NC plus its effects on RT-RNase H have been implicated in facilitating the transfer reaction (34,35,39,(43)(44)(45)(46)(47)(48). Analyses in vitro have been used to show that NC inhibits the formation of dead end, self-primed products from the ϪsssDNA, allowing it to be used for transfers (38,49,50). In addition, NC has been shown to stimulate RT-RNase H activity, especially on a bluntended substrate (33,43).…”

Single-molecule stretching studies of RNA chaperones

“…Studies have shown that all these three steps need the participation of NC proteins, especially the minus stand transfer step. 66,[78][79][80] The effect of NC's chaperone activity on these processes is detailed in the review by Levin and Musier-Forsyth in this special issue.…”