Self-priming of retroviral minus-strand strong-stop DNAs

Abstract

After minus-strand strong-stop DNA (-sssDNA) is synthesized, the RNA template is degraded by the RNase H activity of the reverse transcriptase (RT), generating a single-stranded DNA. The 3′ end of -sssDNA from HIV-1 can form a hairpin; this hairpin will self-prime in vitro. We previously used a model substrate, -R ssDNA, which corresponds to the 3′ end of the -sssDNA of HIV-1, to show that the self-priming of this model substrate could be prevented by annealing a 17-nt-long DNA oligonucleotide to the 3′ end of -R ssDNA in the presence of HIV-1 nucleocapsid (NC) protein. Similar model substrates were prepared for HIV-2 and HTLV-1; the R regions of these two viruses are longer and form more complex structures than the R region of the HIV-1 genome. However, the size of the R region and the complexity of the secondary structures they can form do not affect self-priming or its prevention. The efficiency of the self-priming is related to the relative stabilities of the conformations of -R ssDNA that can and cannot induce self-priming. For the three viruses (HIV-1, HIV-2, and HTLV-1), the size of the DNA oligonucleotide needed to block self-priming in the presence of NC is similar to the expected size of the piece of RNA left after degradation of the RNA template during reverse transcription. We also found that when the 3′ end of -R ssDNA is annealed to a complementary DNA oligonucleotide, it is a good substrate for efficient nonspecific strand transfer to other single-stranded DNA molecules.