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

Yan Chen,Mini Balakrishnan,Bernard P Roques,Philip J Fay,Robert A Bambara

doi:10.1074/jbc.m210959200

Yan Chen, Mini Balakrishnan + Show 3 more

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https://doi.org/10.1074/jbc.m210959200

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Abstract

Retrovirus minus strand strong stop transfer (minus strand transfer) requires reverse transcriptase-associated RNase H, R sequence homology, and viral nucleocapsid protein. The minus strand transfer mechanism in human immunodeficiency virus-1 was examined in vitro with purified protein and substrates. Blocking donor RNA 5'-end cleavage inhibited transfers when template homology was 19 nucleotides (nt) or less. Cleavage of the donor 5'-end occurred prior to formation of transfer products. This suggests that when template homology is short, transfer occurs through a primer terminus switch-initiated mechanism, which requires cleavage of the donor 5' terminus. On templates with 26-nt and longer homology, transfer occurred before cleavage of the donor 5' terminus. Transfer was unaffected when donor 5'-end cleavages were blocked but was reduced when internal cleavages within the donor were restricted. Based on the overall data, we conclude that in human immunodeficiency virus-1, which contains a 97-nt R sequence, minus strand transfer occurs through an acceptor invasion-initiated mechanism. Transfer is initiated at internal regions of the homologous R sequence without requiring cleavage at the donor 5'-end. The acceptor invades at gaps created by reverse transcriptase-RNase H in the donor-cDNA hybrid. The fragmented donor is eventually strand-displaced by the acceptor, completing the transfer.

Highlights

Reverse transcription, during which the single-stranded viral genomic RNA is converted into the double-stranded DNA, is an essential step in viral replication
Synthesis proceeds to the 5Ј-end of the genomic RNA, creating the minus strand strong stop DNA (ϪsssDNA), which comprises all of the U5 and R sequences from the 5Ј-untranslated region
Minus strand synthesis, reverse transcriptase (RT) simultaneously cleaves the copied RNA using its RNase H activity. This frees the ϪsssDNA, enabling its transfer to the homologous R sequence in the 3Ј-untranslated region and continuation of minus strand synthesis. This step is called minus strand strong stop transfer, and it is obligatory for reverse transcription and viral replication

Summary

Introduction

Reverse transcription, during which the single-stranded viral genomic RNA is converted into the double-stranded DNA, is an essential step in viral replication (see Ref. 1 and reference therein). The overall data support that minus strand transfer in retroviruses with long R sequences, such as HIV-1, occurs through an acceptor invasion-initiated mechanism that does not require terminal cleavages at the 5Ј-end of R.

Results

Conclusion