Summary auto-generated
This study examined HIV-1 mRNA expression kinetics following cell-to-cell transmission using synchronized infection of HUT-78 cells with H3B cells persistently infected with HIV-1. Researchers used quantitative RT-PCR to measure cytoplasmic levels of tat, rev, nef, env, and gag mRNAs at multiple timepoints (0-24 hours). Results showed that despite dramatic increases in env and gag mRNAs late in infection (20-fold and 7-fold increases by 24 hours), levels of the regulatory mRNAs (tat, rev, nef) never exceeded those present in the chronically infected donor H3B cells. This contrasts sharply with cell-free virus infections, where tat, rev, and nef mRNAs are induced early. Nuclear run-off assays confirmed that overall HIV transcription increased 5-15 fold between 8-24 hours post-infection. The findings suggest that tat and rev proteins pre-existing in donor cells are sufficient to support highly productive infection following cell-to-cell transmission, and that increased production of these regulatory proteins is not required for the enhanced virus yield observed with this transmission route.
Key findings
- Env and gag mRNAs increased dramatically (20-fold and 7-fold respectively) by 24 hours post-infection following cell-to-cell transmission.
- Tat, rev, and nef mRNA levels remained at or below levels present in persistently infected donor cells throughout the infection period, unlike in cell-free infections where these regulatory transcripts are induced early.
- Pre-existing tat and rev mRNAs in donor cells are sufficient to establish and maintain highly productive infection through cell-to-cell transmission.
- Total HIV transcription in infected nuclei increased 5-15 fold between 8-24 hours, correlating with late increases in structural protein mRNAs.
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Abstract
The temporal appearance and levels of human immunodeficiency virus type 1 (HIV-1) tat, rev, nef, env and gag mRNA species were examined using a synchronized, one-step, cell-to-cell HIV-1 infection model involving HUT-78 cells and HIV- 1 persistently infected H3B cells. Individual mRNAs were quantified by RT-PCR using RNA standards transcribed in vitro from cDNA clones. Consistent with an infection that produces high yields of virus, significant levels of env and gag mRNAs were detected in the cytoplasm of infected cells late in the infection cycle. However, at no time after infection did levels of tat, rev and nef mRNA, which encode the regulatory proteins of HIV-1, exceed their levels present in the persistently infected virus donor H3B cells. The absence of early phase induction of these mRNAs is in contrast to what is observed in cell-free HIV-1 infections or in PMA-stimulated HIV-1 chronically infected cell lines. Our results suggest that tat and rev mRNAs are already present in the cytoplasm of the persistently infected virus donor cells at levels sufficient for initiation and establishment of a highly productive infection in HIV-1 fusion-mediated infected cells. Thus, lack of sufficient Tat and Rev proteins is not likely to be the limiting factor for virus production in H3B cells, nor is increased production of these proteins likely to be the cause of the increased virus production seen following cell-to-cell transmission.