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Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA

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Masuda, Takao, Sato, Yoko, Huang, Yu-Lun, Koi, Satoshi, Takahata, Tatsuro, Hasegawa, Atsuhiko, Kawai, Gota, Kannagi, Mari·2015·Scientific Reports
DOI 10.1038/srep17680PMC PMC4668388
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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.

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  • HIV-1 generates multiple unspliced RNA species (e.g. 1 G versus 3 G RNAs) that differ only slightly at the 5′ end, but fold into distinct structures with differing packaging efficiencies.

    “HIV-1 generates multiple unspliced RNA species (e.g. 1 G versus 3 G RNAs) that differ only slightly at the 5′ end, but fold into distinct structures with differing packaging efficiencies.”

    From Beyond reverse transcription: molecular mechanisms and emerging paradigms in retroviral replication (2026) · cites this paper as Masuda et al. 2015

    The Results (‘Analysis of 5′-end nucleotides of HIV-1 transcripts’; page unavailable) identify G1 and G3 transcripts and show preferential packaging of G1 RNA. The Discussion reports only a preliminary prediction that the 5′-G number might affect RNA structure; it says the exact structures remain undetermined. The claim overstates that prediction as an established difference in folding. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • The 1 G RNA preferentially adopts conformations that leave the DIS and Gag-binding motifs such as unpaired purines fully exposed thus favoring gRNA packaging.

    “The 1 G RNA preferentially adopts conformations that leave the DIS and Gag-binding motifs such as unpaired purines fully exposed thus favoring gRNA packaging.”

    From Beyond reverse transcription: molecular mechanisms and emerging paradigms in retroviral replication (2026) · cites this paper as Masuda et al. 2015

    The Results report preferential packaging of G1-form RNA in virus particles, and the Discussion suggests that differences in 5′-leader structure might help explain this advantage. But the Discussion says the exact structure of each RNA form remains undetermined; it does not establish that G1 RNA exposes the DIS or unpaired purine Gag-binding motifs. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • The 3 G RNA adopts a different conformation in which DIS and Gag binding sites are obscured, rendering it more disposed to translation than to packaging.

    “On the other hand, the 3 G RNA adopts a different conformation in which DIS and Gag binding sites are obscured, rendering it more disposed to translation than to packaging”

    From Beyond reverse transcription: molecular mechanisms and emerging paradigms in retroviral replication (2026) · cites this paper as Masuda et al. 2015

    The Results (Analysis of 5′-end nucleotides; page unavailable) show that G3-form RNA predominates in cells while G1-form RNA is preferentially packaged. The Discussion (page unavailable) only tentatively suggests that 5′-G number may affect leader-region structure; it says the exact structures remain undetermined. It does not establish that DIS and Gag binding sites are obscured in G3 RNA or that G3 RNA is more disposed to translation. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

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Cited by

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Authors
Masuda, T., Sato, Y., Huang, Y.-L., Koi, S., Takahata, T., Hasegawa, A., Kawai, G., & Kannagi, M.
Affiliations
  • Tokyo Medical and Dental University (JP)
  • Chiba Institute of Technology (JP)
Corresponding author
Masuda, Takao
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2015-12-03
Volume
5
Issue
1
Pages
17680-17680
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/srep17680
PMC
PMC4668388
OpenAlex
W2188834155
Cited by
73
References
2
Citations by year
2026: 9 · 2025: 7 · 2024: 11 · 2023: 5 · 2022: 13 · 2021: 7 · 2020: 9 · 2019: 4 · 2018: 2 · 2017: 2 · 2016: 4
FWCI
1.35
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Japan Agency for Medical Research and Development
  • Japan Society for the Promotion of Science (15K15139)
Topics
  • Life Sciences › Immunology and Microbiology › Virology › HIV Research and Treatment
  • Health Sciences › Medicine › Infectious Diseases › HIV/AIDS drug development and treatment
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and Retrovirus Studies
Keywords
HIV-1, reverse transcription, transcription start sites, strand transfer, nucleocapsid protein, viral RNA genome
MeSH terms
Humans, RNA, Viral, HIV-1, DNA, Complementary, Nucleocapsid Proteins, Transcription Initiation Site, Reverse Transcription, HEK293 Cells

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