Biology subjects

Sluis-Cremer, N.

Publications and source records attributed to Sluis-Cremer, N..

4 recordsLinked to original sources

bioRxiv2023

Structures of kinetic intermediate states of HIV-1 reverse transcriptase DNA synthesis

Reverse transcription of the retroviral single-stranded RNA into double-stranded DNA is an integral step during HIV-1 replication, and reverse transcriptase (RT) is a primary target for antiviral therapy. Despite a wealth of structural information on RT, we lack critical insight into the intermediate kinetic states of DNA synthesis. Using catalytically active substrates, and a novel blot/diffusion cryo-electron microscopy approach, we captured 11 structures that define the substrate binding, reactant, transition and product states of dATP addition by RT at 1.9 to 2.4 [A] resolution in the active site. Initial dATP binding to RT-template/primer complex involves a single Mg2+ (site B), and promotes partial closure of the active site pocket by a large conformational change in the {beta}3-{beta}4 loop in the Fingers domain, and formation of a negatively charged pocket where a second "drifting" Mg2+ can bind (site A). During the transition state, the -phosphate oxygen from a previously unobserved dATP conformer aligns with the site A Mg2+ and the primer 3'-OH for nucleophilic attack. In the product state, we captured two substrate conformations in the active site: 1) dATP that had yet to be incorporated into the nascent DNA, and 2) an incorporated dAMP with the pyrophosphate leaving group coordinated by metal B and stabilized through H- bonds in the active site of RT. This study provides insights into a fundamental chemical reaction that impacts polymerase fidelity, nucleoside inhibitor drug design, and mechanisms of drug resistance.

biophysics↗

bioRxiv2023

The HIV-1 capsid serves as a nanoscale reaction vessel for reverse transcription

The viral capsid performs critical functions during HIV-1 infection and is a validated target for antiviral therapy. Previous studies have established that the proper structure and stability of the capsid are required for efficient HIV-1 reverse transcription in target cells. Moreover, it has recently been demonstrated that permeabilized virions and purified HIV-1 cores undergo efficient reverse transcription in vitro when the capsid is stabilized by addition of the host cell metabolite inositol hexakisphosphate (IP6). However, the molecular mechanism by which the capsid promotes reverse transcription is undefined. Here we show that wild type HIV-1 particles can undergo efficient reverse transcription in vitro in the absence of a membrane-permeabilizing agent. This activity, originally termed "natural endogenous reverse transcription" (NERT), depends on expression of the viral envelope glycoprotein during virus assembly and its incorporation into virions. Truncation of the gp41 cytoplasmic tail markedly reduced NERT activity, indicating that gp41 permits the entry of nucleotides into virions. Protease treatment of virions markedly reduced NERT suggesting the presence of a proteinaceous membrane channel. By contrast to reverse transcription in permeabilized virions, NERT required neither the addition of IP6 nor a mature capsid, indicating that an intact viral membrane can substitute for the function of the viral capsid during reverse transcription in vitro. Collectively, these results demonstrate that the viral capsid functions as a nanoscale container for reverse transcription during HIV-1 infection.

microbiology↗

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↗