Impact of 5′-end nucleotide modifications of HIV-1 genomic RNA on reverse transcription

Abstract

Reverse transcription of retroviral RNA is accomplished through a minus-strand strong stop cDNA (-sscDNA) synthesis and subsequent strand-transfer reactions. We have previously reported a critical role of guanosine (G) number at 5′-terminal of HIV-1 RNA for successful strand-transfer of -sscDNA. In this study, role(s) of the cap consisting of 7-methyl guanosine (7mG), a hallmark of transcripts generated by RNA polymerase II, at the 5′-end G nucleotide (5′-G) of HIV-1 RNA were examined. In parallel, contribution of highly conserved GGG tract located at the U3/R boundary in 3′ terminal region of viral RNA (3′-GGG tract) was also addressed. The in vitro reverse transcription analysis using synthetic HIV-1 RNAs possessing the 5′-G with cap or triphosphate form demonstrated that the 5′-cap significantly increased strand-transfer efficiency of -sscDNA. Meanwhile, effect of the 5′-cap on the strand-transfer was retained in the reaction using mutant HIV-1 RNAs in which two Gs were deleted from the 3′-GGG tract. Lack of apparent contribution of the 3′-GGG tract during strand-transfer events in vitro was reproduced in the context of HIV-1 replication within cells. Instead, we noticed that the 3′-GGG tract might be required for efficient gene expression from proviral DNA. These results indicated that 7mG of the cap on HIV-1 RNA might not be reverse-transcribed and a possible role of the 3′-GGG tract to accept the non-template nucleotide addition during -sscDNA synthesis might be less likely. The 5′-G modifications of HIV-1 RNAs by the cap- or phosphate-removal enzyme revealed that the cap or monophosphate form of the 5′-G was preferred for the 1st strand-transfer compared to the triphosphate or non-phosphate form. Taken together, a status of the 5′-G determined strand-transfer efficiency of -sscDNA without affecting the non-template nucleotide addition, probably by affecting association of the 5′-G with 3′-end region of viral RNA.