relevant; another hypothesis could be that these genes are currently in the course of acquisition as KSHV miRNAs targets. Some further investigations of their downregulation by KSHV miRNAs could thus be of interest, to check if they could, or not, be functionally relevant targets.
Concerning CASP3 targeting, it is worthy to note that during our experiments, we were not able to detect any downregulation of the protein or decrease of its activity upon transient expression of the miRNAs, but only in stable cell lines, or after a long induction of several days of KSHV miRNAs expression in our Flip-In system. Furthermore, when looking in the literature, there is no study that could perform any knock-down of CASP3 protein upon transient experiments, but only by the stable expression of small haipins RNAs (shRNAs).
This led us to hypothesise that CASP3 is a very stable protein with a very long half-life and not subjected to any turn-over (which seems consistent with its key role in the induction of apoptosis), and that the miRNAs could only exert an effect on the protein after a long period of expression. As such the levels of CASP3 would reduce in consequence of its dilution following cell division and through the alteration of its de novo synthesis by the miRNAs.
This would thus imply that during de novo infection, the viral miRNAs would not play any role through the targeting of CASP3, and that this repression would only exert its function in controlling apoptosis once the latent infection is well established. Indeed, during lytic infection, many of the KSHV proteins are known to play a role in the inhibition of apoptosis, and therefore the miRNAs’ role could be negligible in this function. The viral miRNAs would then act as a relay of the viral proteins functions, after the shut off of their expression induced by the establishment of the latency. This is in agreement with the hypothesis that viral miRNAs do not play any role (or if so a marginal role) during acute lytic infection, and that one of their principal functions is the control, during latency, of cellular genes (and naturally such cellular pathways) in a non-immunogenic manner, which would allow for a beneficial environment for the virus maintenance, permitting minimal viral protein expression and going
"undetected" by the cell (detailed in sections 6.2 and 6.3 of the introduction).
Repressing CASP3 expression makes perfect sense for the virus when considering the key effector role of this protein. Activation of CASP3 is the convergent step of all the major pathways of apoptosis induction, and which when activated, induces the events leading to programmed cell death in an irreversible manner. Nevertheless, as discussed in section 4 of the results, two other pro-apoptotic genes have been reported to be targets of KSHV miRNAs:
TWEAKR and BclAF1. While it remains unclear if BclAF1 plays a role in the inhibition of apoptosis during KSHV infection, the role of TWEAKR in the control of IFN-$-induced cell death has clearly been established. In addition, as detailed in section 5.6 of the introduction, the viral proteins expressed during KSHV latency have been shown for most of them (LANA, LANA-2, vCyc and vFLIP) to play a direct or indirect role in inhibiting programmed cell death at different levels. Furthermore, when studying the role of KSHV miRNAs during apoptosis, we have also observed through Annexin V assays on the staurosporine-treated DG- 75 cell line, an inhibition of caspase-independent apoptosis. This means that the viral miRNAs target at least another additional cellular factor, which is involved in the caspase- independent induction of apoptosis. Therefore, it is highly probable that KSHV miRNAs have evolved to target several cellular genes involved in programmed cell death to ensure the good control of its inhibition. In conclusion, as with other viral mechanisms of host interactions, KSHV miRNAs display redundancy or more so a combinatorial activity, by acting in concert with the viral proteins expressed during latency, in order to target cellular apoptotic pathways.
As a perspective, as most KSHV miRNAs bear viral-specific seed sequences with no homology to their cellular counterparts (such as for the three miRNAs that we have demonstrated to target CASP3 transcript), one can thus envisage as a novel therapeutic approach the delivery of specific inhibitors of these viral miRNAs in KSHV-infected patients, with no or minimal adverse effects, as the cellular miRNAs would thus not be affected.
Indeed, very promising results have been obtained in vivo and in preliminary clinical studies when inhibiting miR-122 in HCV-positive patients, effectively blocking viral replication and cure the patients from the infection (Haussecker and Kay, 2010; Lanford et al., 2010). We can thus expect that the specific inhibition in KSHV-patients of both the viral miRNAs targeting CASP3 and TWEAKR, could permit to restore apoptotic clearance induced by both the inner cellular mechanisms triggered by the viral infection and through the stimulation of the extrinsic pathway by the immune system.