Sluis-Cremer, N. | Search bioRxiv

bioRxiv2022

Mechanism by which PF-3758309, a Pan Isoform Inhibitor of p21-Activated Kinases, Blocks Reactivation of HIV-1 Latency

The "block and lock" strategy is one approach that might elicit a sterilizing cure for HIV-1 infection. The "block" refers to a compounds ability to inhibit latent HIV-1 proviral transcription, while the "lock" refers to its capacity to induce permanent proviral silencing. We identified PF-3758309, a pan-isoform inhibitor of p21-activated kinases (PAKs), as a potent inhibitor of HIV-1 latency reversal (Vargas et al., Antimicrob Agents Chemother. 2019;63(2):e01744-18). The goal of this study was to define the mechanism(s) involved. We found that both 24ST1NLESG cells (a cell line model of HIV-1 latency) and purified CD4+ naive and central memory T cells express high levels of PAK2, and lower levels of PAK1 and PAK4. Knockdown of PAK1 or PAK2, but not PAK4, in 24ST1NLESG cells resulted in a modest, but statistically significant decrease in the magnitude of HIV-1 latency reversal. Overexpression of PAK1 significantly increased the magnitude of latency reversal. A phospho-protein array analysis revealed that PF-3758309 down-regulates the NF-{kappa}B signaling pathway, which provides the most likely mechanism by which PF-3758309 inhibits latency reversal. Finally, we used cellular thermal shift assays combined with liquid chromatography and mass spectrometry to ascertain whether PF-3758309 off-target binding contributed to its activity. In 24ST1NLESG cells and in peripheral blood mononuclear cells, PF-3758309 bound to mitogen-activated protein kinase 1 and protein kinase A; however, knockdown of either of these kinases did not impact HIV-1 latency reversal. Collectively, our study suggests that PAK1 and PAK2 play a key role in the maintenance of HIV-1 latency. IMPORTANCEThe persistence of latent, replication-competent HIV-1 proviruses in resting CD4+ T cells, and other cellular reservoirs, represents a major barrier to a cure. The "block and lock" strategy is one approach proposed to elicit a sterilizing cure for HIV-1 infection. In this study, we define the mechanism by which PF-3758309, a pan-isoform p21-activated kinase (PAK) inhibitor, blocks the reversal of HIV-1 latency. Our data show that PAK1 and PAK2 play a role in the maintenance of HIV-1 latency, and further suggest that PAK inhibitors, such as PF-3758309, could form part of "block and lock" therapeutic strategies.

microbiology↗

bioRxiv2020

B LYMPHOCYTES, BUT NOT DENDRITIC CELLS, EFFICIENTLY HIV-1 TRANS-INFECT NAÏVE CD4+ T CELLS: IMPLICATIONS FOR THE VIRAL RESERVOIR

Insight into the establishment and maintenance of HIV-1 infection in resting CD4+ T cell subsets is critical for the development of therapeutics targeting the HIV-1 reservoir. Although the frequency of HIV-1 infection, as quantified by the frequency of HIV-1 DNA, is lower in CD4+ naive T cells (TN) compared to the memory T cell subsets, recent studies have shown that TN cells harbor a large pool of replication-competent virus. Interestingly, however, TN cells are highly resistant to direct (cis) HIV-1 infection in vitro, in particular to R5-tropic HIV-1, as TN cells do not express CCR5. In this study, we investigated whether TN cells could be efficiently HIV-1 trans-infected by professional antigen-presenting B lymphocytes and myeloid dendritic cells (DC) in the absence of global T cell activation. We found that B cells, but not DC, have a unique ability to efficiently trans infect TN cells in vitro. In contrast, both B cells and DC mediated HIV-1 trans infection of memory and activated CD4+ T cells. Moreover, we found that TN isolated from HIV-1-infected nonprogressors (NP) harbor significantly disproportionately lower levels of HIV-1 DNA compared to TN isolated from progressors. This is consistent with our previous finding that APC derived from NP do not efficiently trans-infect CD4+ T cells due to alterations in APC cholesterol metabolism and cell membrane lipid raft organization. These findings support that B cell-mediated trans infection of TN cells with HIV-1 has a more profound role than previously considered in establishing the viral reservoir and control of HIV-1 disease progression. ImportanceThe latent human immunodeficiency virus type 1 (HIV-1) reservoir in persons on antiretroviral therapy represents a major barrier to a cure. Although most studies have focused on the HIV-1 reservoir in the memory T cell subset, replication competent HIV-1 has been isolated from naive T cells, and CCR5-tropic HIV-1 has been recovered from CCR5negTN cells from ART-suppressed HIV-1-infected individuals. In this study, we showed that CCR5negTN cells are efficiently trans infected with R-5 tropic HIV-1 by B lymphocytes, but not by myeloid dendritic cells. Furthermore, we found that TN isolated from NP harbor no or significantly less copies of HIV-1 DNA compared to ART-suppressed progressors. These findings support that B cell-mediated trans infection of TN cells with HIV-1 has a more profound role than previously considered in establishing the viral reservoir and control of HIV-1 disease progression. Understanding the establishment and maintenance of the HIV-1 latent reservoir is fundamental for the design of effective treatments for viral eradication.

microbiology↗