HIV-1 single transcription start site mutants display complementary replication functions that are restored by
1Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Biorxiv : the Preprint Server for Biology
|December 16, 2024
Summary
The human immunodeficiency virus type 1 (HIV-1) uses two transcription start sites, producing distinct RNA 5' ends. While both can function as mRNA and genomic RNA, the cap3G form shows replication defects, highlighting the importance of dual transcription start site use for efficient HIV-1 replication.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- HIV-1 transcription initiates at two distinct positions, yielding RNAs with either cap1G or cap3G 5' ends.
- These RNA isoforms have different replication fates: cap1G RNAs are preferentially packaged into viral particles, while cap3G RNAs remain in cells, enriched in polysomes and spliced viral RNAs.
Purpose of the Study:
- To investigate the replication properties of HIV-1 promoter mutants generating only cap1G or cap3G RNA isoforms.
- To assess the functional roles of each RNA isoform individually and in combination during viral replication and spreading infection.
Main Methods:
- Construction and analysis of HIV-1 promoter mutants producing single RNA 5' isoforms (cap1G-only or cap3G-only).
- Evaluation of RNA translation, splicing, and packaging efficiencies.
- Assessment of viral replication kinetics in cell lines (MT-4) and primary human CD4+ T cells, including during spreading infection.
Main Results:
- Both cap1G and cap3G RNAs can function as mRNA and genomic RNA when present as the sole form.
- cap3G RNA exhibits more efficient translation and splicing, whereas cap1G RNA shows slightly better packaging into nascent virions.
- cap3G-only virus demonstrates severe replication delays in both permissive and primary cells, while cap1G-only virus shows minor defects. Passage of cap3G-only virus leads to revertants with restored dual transcription start site use and replication efficiency.
Conclusions:
- While both RNA isoforms can support viral replication, the cap3G isoform is less efficient in vivo, leading to replication delays when exclusively produced.
- The preferential packaging of cap1G RNA and efficient translation/splicing of cap3G RNA contribute to the overall fitness of wild-type HIV-1.
- Restoration of dual transcription start site usage in revertants underscores the importance of both isoforms for optimal HIV-1 replication and pathogenesis.
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