Abstract
Retroviral reverse transcription is accomplished by sequential strand-transfers of partial cDNA intermediates copied from viral genomic RNA. Here, we revealed an unprecedented role of 5′-end guanosine (G) of HIV-1 genomic RNA for reverse transcription. Based on current consensus for HIV-1 transcription initiation site, HIV-1 transcripts possess a single G at 5′-ends (G1-form). However, we found that HIV-1 transcripts with additional Gs at 5′-ends (G2- and G3-forms) were abundantly expressed in infected cells by using alternative transcription initiation sites. The G2- and G3-forms were also detected in the virus particle, although the G1-form predominated. To address biological impact of the 5′-G number, we generated HIV clone DNA to express the G1-form exclusively by deleting the alternative initiation sites. Virus produced from the clone showed significantly higher strand-transfer of minus strong-stop cDNA (-sscDNA). The in vitro assay using synthetic HIV-1 RNAs revealed that the abortive forms of -sscDNA were abundantly generated from the G3-form RNA, but dramatically reduced from the G1-form. Moreover, the strand-transfer of -sscDNA from the G1-form was prominently stimulated by HIV-1 nucleocapsid. Taken together, our results demonstrated that the 5′-G number that corresponds to HIV-1 transcription initiation site was critical for successful strand-transfer of -sscDNA during reverse transcription.
Highlights
Resultant + sscDNA forms a duplex with primer binding site (PBS) region of the tRNA primer
Through quantitative and qualitative analysis of cDNA intermediates, which were generated within infected cells and in vitro reaction, we revealed that the 5′ -G that corresponded to initiation site of human immunodeficiency virus type 1 (HIV-1) genomic RNA might have a critical role for successful 1st strand-transfer during reverse transcription
The 5′ -end nucleotide of HIV-1 RNA is defined by transcription initiation site, which is located at U3/R junction of integrated HIV-1 DNA
Summary
Analysis of 5′-end nucleotides of HIV-1 transcripts. The 5′ -end nucleotide of HIV-1 RNA is defined by transcription initiation site, which is located at U3/R junction of integrated HIV-1 DNA. We reproduced significant stimulatory effect of NC on 1st strand-transfer under reaction with 10 μ M dNTPs corresponding to the concentration within activated T cells (Fig. 7C,D) and with 10- or 100-fold dilution of the G1-form of HIV-sRNA, pbs-sRNA and rRT concentrations (Fig. 7E) These results suggested that impact of sNC on the G1-form of RNA in vitro might have a physiologic relevance. In vitro reconstitution assay revealed that number of G at 5′ -end of HIV-1 RNA contributed to successful 1st strand-transfer by reducing abortive cDNA generation of -sscDNA. We detected other forms of viral RNAs with additional 5′ -Gs in cells persistently infected with HIV-1 or transfected with HIV-1 clone DNA These results suggested that HIV-1 transcripts were efficiently transcribed at 1 or 2 nt upstream of the currently proposed initiation site. The in vitro reconstitution assay using the G1-form of HIV-sRNA would serve as a useful assay to evaluate additional cis- and trans-acting factors to reproduce entire reverse transcription
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