We here present a novel method of generating latently infected, resting TCM
. This model has been used to analyze the main signaling pathways that lead to viral reactivation after TCR engagement (40
) and to begin characterize the methylation status of the viral promoter in primary cells (19
). This paradigm is suitable for a variety of analyses. For example, we propose to utilize ex vivo
latently infected TCM
cells to dissect the cellular events controlling the establishment of latency and the onset of reactivation. Specifically, we propose to examine how various modes of cellular activation (i.e.
, in response to TCR engagement, or cytokine stimulation) may affect latency. We envision that this model will also help us to understand how homeostatic proliferation (i.e., as induced by IL-7 or IL-15) and other microenvironmental cues affect the size and behavior of the latent reservoir. Finally, it will also be possible to investigate the virological determinants of latency, such as the potential roles of the HIV-1 accessory genes in latency induction and reactivation, and the contribution of various LTR cis
-acting elements to the transcriptional state of the virus in primary memory cells.
Viral tropism has been shown to be strongly linked to the latent reservoir. Specifically, it has been shown that the genotype of viruses in the latent reservoir predicts R5 tropism rather than X4 (51
). When cells differentiate into TCM
) or NP (ex vivo
) they are known to naturally downregulate CCR5, but they maintain CXCR4. This phenomenon has limited the scope of our experiments because, as one would expect, R5 tropic viruses infect NP cells inefficiently (Bosque and Planelles, unpublished observations). Therefore, only X4 or dual tropism viruses can be used in our primary cell model. Two possible explanations may satisfy the above conundrum. First, CCR5 could be expressed at certain times in the life of a central memory cell in vivo
, providing R5-tropic HIV-1 with a window of time for infection leading to latency. We have observed that R5 viruses do infect TCM
cells although with very low efficiency (2% compared with 40–60% infection by X4 viruses, using the same amounts of p24 in the inocula; unpublished data). This leads to the second possibility we would like to suggest. We do know that the size of the reservoir is small. Therefore, one may speculate that the inefficient infection of TCM
by CCR5 viruses may be a relevant factor determining the relatively small size of the latent pool. This issue should be examined in the near future.
The existence of, perhaps yet another reservoir, residing in the TTM
subset has recently been proposed (6
). It is plausible that TTM
may exhibit differences with respect to TCM
in terms of the types of stimuli that will induce cellular activation and/or viral reactivation. Therefore, when considering the design or discovery of anti-latency drugs, one should take into consideration the potential effects of drugs on both cell types.