Despite being closely related viruses with similar replication cycles, HIV-2 replicates more slowly than HIV-1 and produces fewer particles, resulting in a lower plasma viral load. Expression of the major structural gene, gag, from HIV-1 and HIV-2 proviruses was compared to investigate whether this could play a role in the difference in particle production observed between HIV-1 and HIV-2 infection. Using quantitative RT-PCR, significantly less full-length HIV-2 gag mRNA was found to be transcribed from its provirus than for HIV-1. Sub-cellular fractionation allowed us to determine HIV-1/2 gag mRNA levels in the nucleus and cytoplasm throughout a time course. RNA export of HIV-2 gag mRNA was shown to be slower than for HIV-1 gag mRNA. HIV-2 full-length gag RNA was shown to be translated much less efficiently than HIV-1 in a range of cell lines. Both HIV-1 and HIV-2 Gag have been proposed to be translated by internal ribosome entry. Shutting down capdependent translation (by poliovirus-mediated eIF4G cleavage) significantly reduced translation from both HIV-1/2 gag RNAs, with no evidence of compensatory IRES activity. This suggests that cap-dependent translation is the predominant mechanism for translation of both HIV-1 and HIV-2 RNA. Additional work explored HIV RNA-protein interactions by UV cross-linking experiments using cellular proteins. Several proteins differentially binding to HIV-1/2 5’ UTR RNAs were identified and, in particular, a 45 kDa protein binding only to the HIV-1 5’ UTR. Attempts were made to characterise the proteins binding with different affinities to HIV-1 and HIV-2 RNAs. Confocal microscopy was used to visualise HIV-1/2 Gag expression within the cell. Both HIV-1 and HIV-2 Gag expression was shown to be reduced when siRNA was used to inhibit the cellular clathrin adaptor protein AP-1. In conclusion, HIV-2 Gag gene expression was found to be less efficient than HIV-1 at the level of transcription, RNA export and translation. Future work will continue to investigate the mechanisms behind these differences.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:560311 |
| Date | January 2012 |
| Creators | Watkins, Gemma L. |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/45902/ |
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