Elite controllers represent a model for successful immune activity against HIV-1, and a widespread consensus has emerged that the identification of specific host defense mechanisms in these patients may provide critical information for the design of HIV-1 vaccines and for the development of clinical strategies to induce a functional cure of this disease. Here, we shed light on these mechanisms by demonstrating that p21, a cyclin-dependent kinase inhibitor that has previously been recognized mainly for its role as a tumor suppressor (29
), was strongly upregulated in CD4+
T cells from elite controllers and reduced the susceptibility of these cells to HIV-1 by inhibiting viral reverse transcription and mRNA transcription. Moreover, we showed that the antiretroviral effects of p21 on HIV-1 mRNA transcription were, at least in part, indirectly mediated by the ability of p21 to inhibit the enzymatic activity of CDK9, a host protein essential for proper elongation of HIV-1 mRNA (27
). Overall, these results suggest that p21 represents a previously unrecognized, CD4+
T cell–intrinsic HIV-1 immune defense mechanism that contributes to the ability of elite controllers to maintain undetectable VLs and resist HIV-1–associated disease manifestations.
To our knowledge, the studies presented here are the first to show that a specific host protein is directly involved in reducing the susceptibility of CD4+
T cells to HIV-1 in vivo in elite controllers. Whether upregulation of p21 expression in T cells is a constitutive characteristic of elite controllers or evolves as part of a specific immune response to HIV-1 infection in these patients is currently unclear, but studies involving genetic relatives of elite controllers and preinfection analysis of HIV-1 patients who develop an elite controller phenotype will be important in this context. Furthermore, it is noteworthy that the CDKN1A
gene is located on chromosome 6 in relative proximity to MHC class I genes, some of which are significantly associated with the elite controller phenotype (10
). Although we did not observe any relationship between p21 expression intensity in CD4+
T cells and protective HLA alleles, such as HLA-B57 and HLA-B27, it remains to be determined in larger studies whether MHC class I gene polymorphisms associated with better HIV-1 disease outcomes are linked to specific mutations in the CDKN1A
) that may affect its expression. Moreover, the transcription factor Foxo3A, which may be involved in the regulation of CDKN1A
gene expression, is uniquely expressed in CD4+
T cells from elite controllers (33
), and it will be important to analyze whether it participates in the epigenetic control of high-level p21 expression in such cells.
The ability of p21 to modulate the susceptibility of human cells to HIV-1 infection by interfering with individual viral replication steps is increasingly being recognized. In hematopoietic stem cells, p21 was shown to selectively inhibit HIV-1 integration, while leaving reverse transcription unaffected (20
); this might represent a stem cell–specific mode of protection against HIV-1 infection (34
). In macrophages, p21 can reduce the efficacy of reverse transcription and integration (19
), and prior studies in lymphocytic cell lines suggested that pharmaceutical cyclin-dependent kinase inhibitors can suppress HIV-1 gene expression from proviral HIV-1 DNA (35
). Our data in activated primary CD4+
T cells suggest that p21 can independently inhibit HIV-1 reverse transcription and mRNA transcription; an independent effect on HIV-1 integration was less evident, given that ratios between LRT transcripts and integrated HIV-1 copy levels were almost identical between elite controllers and HIV-1–negative persons and were unaffected by p21 inhibition. Overall, these data suggest that p21 can affect multiple early viral replication steps and that the specific influence of p21 on the fate of viral transcripts critically depends on the infected cell type.
As a molecular mechanism underlying the ability of p21 to inhibit viral replication, we show here that p21 blocked the enzymatic activity of CDK9 and its known function to enhance transcriptional elongation of HIV-1 mRNA transcripts through phosphorylation of RNA polymerase II (27
). This suggests that p21 affects HIV-1 mRNA transcription by a targeted blockade of a host protein that is essential for effective HIV-1 gene expression. A similar inhibitory effect on CDK9 was recently demonstrated for p57, a cyclin-dependent kinase inhibitor closely related to p21 (37
); however, p57 expression in CD4+
T cells did not differ significantly between elite controllers and HIV-1–negative persons and is therefore unlikely to contribute substantially to restriction of HIV-1 infection in elite controllers. It is also important to recognize that pharmaceutical inhibitors of cyclin-dependent kinases can potently suppress HIV-1 replication in vitro by mechanisms that involve CDK9 inhibition (17
). The ability of p21 to block the host protein CDK9, instead of interfering directly with viral replication enzymes, may also explain why HIV-1 does not appear to be capable to escape from p21-mediated restriction of HIV-1 replication in a similar manner as it evades alternative host restriction factors. In line with this, no selection of viral escape variants by pharmaceutical cyclin-dependent kinase inhibitors was observed, even when generation of viral escape variants was aggressively attempted by long-term viral cultures in the presence of cyclin-dependent kinase inhibitor monotherapy in suboptimal concentrations (35
An unresolved issue here is how p21 can affect HIV-1 reverse transcription during the viral preintegration phase. It is possible that p21 indirectly affects reverse transcription through interactions with host proteins that activate different steps of the reverse transcription process, although to our knowledge no such interactions have been documented in prior work. Alternatively, p21 may influence reverse transcription by nonspecifically altering the activation status of the cells. Indeed, p21 has been shown to play a role in regulating activation and proliferation of T lymphocytes in vivo (38
). Although we were unable to detect a direct effect of p21 inhibition on CD4+
T cell activation in our short-term culture system of ex vivo activated CD4+
T cells, our experiments do not exclude the possibility that such unspecific effects on cellular physiology may indirectly affect the susceptibility of CD4+
T cells to HIV-1.
Increasing evidence suggests that control of HIV-1 infection in elite controllers is likely to involve multiple different components of the immune system. For instance, a series of prior studies has emphasized the strong cytolytic properties of HIV-1–specific CD8+
T cells in elite controllers (3
), which at least in in vitro assays are capable of effectively restricting HIV-1 replication (7
). Furthermore, T cells from elite controllers also appear to have unique abilities to proliferate and survive, which may be mediated by a distinct phosphorylation profile of the transcription factor Foxo3a (33
). More recently, specific functional properties of myeloid dendritic cells in elite controllers were identified, which were mediated and maintained by a selective expression of immunomodulatory receptors from the leukocyte immunoglobulin-like receptor group (39
). It is possible that these different mechanisms all synergize in providing effective HIV-1 immune defense in elite controllers and that, for example, a highly functional CD8+
T cell response against HIV-1 may only develop in the setting of a CD4+
T cell compartment that is less capable of supporting highly replicative HIV-1 infection. Overall, our finding of a cell-intrinsic restriction of HIV-1 replication in CD4+
T cells by upregulation of p21 adds what we believe to be a previously unrecognized component to the understanding of mechanisms contributing to undetectable viremia in elite controllers and might be helpful for future clinical approaches to enhance host resistance to HIV-1 infection.