A comparison of HIV-1 and HIV-2 gag gene expression
Watkins, Gemma L. (2012) A comparison of HIV-1 and HIV-2 gag gene expression. PhD thesis, University of Warwick.
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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.
|Item Type:||Thesis or Dissertation (PhD)|
|Subjects:||Q Science > QR Microbiology|
|Library of Congress Subject Headings (LCSH):||HIV infections -- Genetic aspects|
|Official Date:||January 2012|
|Institution:||University of Warwick|
|Theses Department:||School of Life Sciences|
|Sponsors:||Medical Research Council (Great Britain) (MRC) ; University of Warwick|
|Extent:||x, 244 leaves : ill., charts|
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