PLAN ESTRATÉGICO DE TURISMO COMUNITARIO PARA EL

To investigate the rate at which HIV-1/2 gag mRNA is synthesised and transitions from the nucleus to the cytoplasm, the level of HIV-1/2 gag mRNA in the nucleus and cytoplasm was compared by sub-cellular fractionation post-provirus transfection. At 6 h and 12 h post-transfection HIV-1 gag RNA levels in the nucleus were higher than HIV-2, reflecting the faster rate of transcription seen for HIV-1. Between 12 and 48 h, the level of HIV-1 gag RNA decreased. Expression of the viral Rev protein promotes increased export of unspliced HIV-1 gag mRNA by enhancing Rev/RRE dependent export of HIV RNA from the nucleus. Consequently, as more Rev is expressed during the viral life cycle, gag mRNA is more efficiently exported from the nucleus. Therefore the decrease in HIV-1 gag mRNA in the nucleus after 12 h likely reflects efficient export of full-length HIV-1 gag RNA to the

97 cytoplasm via the Rev-RRE pathway. To complement this, the level of HIV-1 viral RNA in the cytoplasm increased over the time course, reflecting the export of synthesised HIV-1 gag RNA from the nucleus.

The amount of HIV mRNA remaining in the nucleus is relative to both the rate of transcription (producing nascent mRNA) and the rate of mRNA export from the nucleus to the cytoplasm (removing mRNA from the nucleus). Assuming the rate of transcription and mRNA degradation are constant over time, if the rate of export is lower than the rate of mRNA transcription (T>E), then mRNA levels in the nucleus will increase, albeit at a lower rate than previous to export commencing. If the rate of mRNA synthesis is equivalent to the rate at which it is exported (T=E), the level of mRNA in the nucleus will remain relatively constant, whereas if the rate of mRNA export exceeds the rate of mRNA transcription (T<E), RNA levels in the nucleus will drop (summarised in figure 39).

Figure 39: The effect of differing export rates (E) on the level of RNA in the nucleus (blue line) assuming a constant level of transcription (T).

Thus HIV-1 export appears to have been occurring very efficiently as the level of overall RNA in the nucleus dropped after 12 h indicating that HIV-1 Gag RNA export was occurring at a greater rate than Gag RNA synthesis (T<E).

HIV-2 Gag mRNA accumulated much more slowly in the nucleus indicating less efficient transcription. After 12 h, the level of HIV-2 RNA in the nucleus only decreased slightly, rather than decreasing rapidly like HIV-1. This suggests that HIV-2 mRNA was undergoing export at a similar, or only slightly higher, rate to the rate at which it was being synthesised (T=E). The level of HIV-2 transcription was shown to be less efficient than HIV-1.

98 Therefore, as the export rate was not vastly different to the transcription rate for HIV-2, this suggests that export is equally inefficient for HIV-2 and, additionally, less efficient than for HIV-1.

The N:C ratios for HIV-1 and HIV-2 support these observations. The HIV-1 N:C ratio remained similar 6-12 h post-transfection. During this period the rate of mRNA increase in the nucleus and cytoplasm was the same i.e. mRNA synthesised matched the mRNA export rate (T=E). As HIV-1 transcription was shown to be very efficient, HIV-1 mRNA export must have been reasonably efficient to match the mRNA synthesis rate during this time. The N:C rate for HIV-1 dropped rapidly after 12 h indicating that HIV-1 mRNA was exported more efficiently from the nucleus after this time (T>E). As replication progressed, the presence of more Rev RNA in the nucleus may have served to accelerate the rate of HIV-1 gag mRNA export producing the observed drop in N:C ratio.

The HIV-2 N:C ratio increased initially from 6-12 h indicating that mRNA levels were increasing in the nucleus (via synthesis) but the rate of HIV-2 gag mRNA in the cytoplasm was not increasing by an equivalent amount. This suggests that HIV-2 export was slow to initiate as mRNA transcription levels were greater than the mRNA export rate (T>E). As overall HIV-2 transcription was shown to be inefficient, HIV-2 mRNA export must have been very inefficient (or not occurring) between 6-12 h to result in an increase in HIV-2 mRNA in the nucleus during this time. By 24 h, the HIV-2 N:C ratio started to decrease (T<E), but not to the same extent as for HIV-1, indicating that cytoplasmic HIV-2 RNA levels were not increasing as quickly for HIV-2 as for HIV-1. Consequently, these results suggest that the rate of gag RNA export appears to be much slower for HIV-2.

Increased HIV-2 mRNA instability in the cytoplasm could have resulted in the lower levels of HIV-2 gag mRNA observed. ActD was successfully used to shut down mRNA synthesis and thereby compare the rate at which existing HIV-1/2 mRNA in the cytoplasm was degraded. There was a detectable decrease in the level of both HIV-1/2 viral mRNA in the cytoplasm post-actD treatment indicating degradation. However, following the shutdown of mRNA

99 synthesis, HIV-1/2 cytoplasmic gag mRNA levels fell by an equivalent rate suggesting that the rate of gag mRNA degradation was the same for both viruses.

It is likely that depletion of nuclear viral RNA occurred via transport to the cytoplasm following transcription shutdown. This may account for the increase in cytoplasmic RNA levels for both HIV-1 and HIV-2 in the 2 h post- actD treatment. As HIV-1 transcription is more efficient than HIV-2, one can hypothesise that more HIV-1 RNA may have been present in the nucleus at the point of transcriptional shutdown. This is reflected by the greater peak in HIV-1 RNA appearing in the cytoplasm at T=2; potentially due to export of a larger quantity of HIV-1 RNA from the nucleus. If anything, these results therefore suggest that HIV-1 RNA may be degraded more quickly in the cytoplasm than HIV-2 to result in more similar levels by 8 h. Either way, these findings indicate that a greater rate of HIV-2 gag RNA degradation does not cause the lowered cytoplasmic levels of HIV-2 gag RNA seen in the cytoplasm. Consequently, we put forward the idea that HIV-2 export is much less efficient than HIV-1 export and, in conjunction with a lowered rate of HIV-2 transcription, accounts for the reduced levels of HIV-2 gag RNA found in the cytoplasm.

Overall, results indicate that both HIV gag mRNA transcription and export are less efficient for HIV-2 than for HIV-1. Inefficiencies within mRNA transcription and export may thus contribute to the lower rate of viral particle production observed for HIV-2 infected individuals by limiting the rate of gene expression and thus the availability of components required for virion assembly.