Marc Permanyer

Cell Cycle Control and HIV-1 Susceptibility Are Linked by CDK6-Dependent CDK2 Phosphorylation of SAMHD1 in Myeloid and Lymphoid Cells

Proliferating cells are preferentially susceptible to infection by retroviruses. Sterile α motif and HD domain–containing protein-1 (SAMHD1) is a recently described deoxynucleotide phosphohydrolase controlling the size of the intracellular deoxynucleotide triphosphate (dNTP) pool, a limiting factor for retroviral reverse transcription in noncycling cells. Proliferating (Ki67+) primary CD4+ T cells or macrophages express a phosphorylated form of SAMHD1 that corresponds with susceptibility to infection in cell culture. We identified cyclin-dependent kinase (CDK) 6 as an upstream regulator of CDK2 controlling SAMHD1 phosphorylation in primary T cells and macrophages susceptible to infection by HIV-1. In turn, CDK2 was strongly linked to cell cycle progression and coordinated SAMHD1 phosphorylation and inactivation. CDK inhibitors specifically blocked HIV-1 infection at the reverse transcription step in a SAMHD1-dependent manner, reducing the intracellular dNTP pool. Our findings identify a direct relationship between control of the cell cycle by CDK6 and SAMHD1 activity, which is important for replication of lentiviruses, as well as other viruses whose replication may be regulated by intracellular dNTP availability.

Endocytosis of HIV: anything goes

The major pathway for HIV internalization in CD4+ T cells has been thought to be the direct fusion of virus and cell membranes, because the cell surface is the point of entry of infectious particles. However, the exact contribution of endocytic pathways to the infection of CD4+ T lymphocytes is unknown, and the mechanisms involved in endocytosis of HIV particles are unclear. Recent evidence suggests that endocytosis of cell-free and cell-associated virus particles could lead to effective virus entry and productive infections. Such observations have, in turn, spurred a debate on the relevance of endosomal entry as a mechanism of escape from the immune system and HIV entry inhibitors. In this paper, we review the endocytosis of HIV and discuss its role in HIV infection and pathogenesis.

p21 regulates the HIV-1 restriction factor SAMHD1

Allouch et al. (1) have shown that CDKN1A (p21) restricts HIV-1 replication in monocyte-derived macrophages (MDM) by controlling the expression of the ribonucleotide reductase subunit R2 (RNR2) of the ribonucleotide reductase enzyme that, in turn, controls the intracellular deoxynucleotide (dNTP) pool required for HIV-1 reverse transcription. dNTP levels are also tightly controlled by the dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1), which is constitutively expressed in myeloid and lymphoid cells and is counteracted by the lentiviral virus protein x (Vpx) (reviewed in ref. 2). SAMHD1 is deactivated in proliferating cells by a mechanism that requires phosphorylation of SAMHD1 (3). Allouch et al. (1) conclude that p21-driven HIV-1 restriction in macrophages is independent of SAMHD1 because ( i ) p21 did not affect SAMHD1 expression and ( ii ) Vpx did not affect p21 expression. Here, we show that M-CSF induces monocyte differentiation into macrophages and cell proliferation (Fig. 1 A ), and RNA interference of p21 leads to an increase in the number of proliferating cells (Fig. 1 B ). Macrophages become susceptible to HIV-1 replication (Fig. 1 C ) because SAMHD1 is inactivated as measured by specific SAMHD1 phosphorylation at residue T592 (Fig. 1 D ). Delivery of simian immunodeficiency virus (SIV) mac Vpx-induced SAMHD1 degradation (Fig. 1 E ) and subsequently increased virus infection (Fig. 1 F ) (2, 4, 5). Of importance, siRNA-induced down-regulation of p21 (Fig. 2 A ) strongly enhanced the phosphorylation of SAMHD1 (Fig. 2 B and C ), followed by an increase in … [↵][1]2To whom correspondence should be addressed. E-mail: jaeste{at}irsicaixa.es. [1]: #xref-corresp-1-1

Restriction of HIV-1 Replication in Primary Macrophages by IL-12 and IL-18 through the Upregulation of SAMHD1

Monocyte-derived macrophages (MDM) can polarize into different subsets depending on the environment and the activation signal to which they are submitted. Differentiation into macrophages allows HIV-1 strains to infect cells of the monocytic lineage. In this study, we show that culture of monocytes with a combination of IL-12 and IL-18 led to macrophage differentiation that was resistant to HIV-1 infection. In contrast, M-CSF–derived MDM were readily infected by HIV-1. When monocytes were differentiated in the presence of M-CSF and then further treated with IL-12/IL-18, cells became resistant to infection. The restriction on HIV-1 replication was not dependent on virus entry or coreceptor expression, as vesicular stomatitis virus-pseudotyped HIV-1 replication was also blocked by IL-12/IL-18. The HIV-1 restriction factor sterile α motif and HD domain–containing protein-1 (SAMHD1) was significantly overexpressed in IL-12/IL-18 MDM compared with M-CSF MDM, and degradation of SAMHD1 by RNA interference or viral-like particles carrying the lentiviral protein Vpx restored HIV-1 infectivity of IL-12/IL-18 MDM. SAMHD1 overexpression induced by IL-12/IL-18 was not dependent on IFN-γ. Thus, we conclude that IL-12 and IL-18 may contribute to the response against HIV-1 infection through the induction of restriction factors such as SAMHD1.

Antiretroviral Agents Effectively Block HIV Replication after Cell-to-Cell Transfer

ABSTRACT Cell-to-cell transmission of HIV has been proposed as a mechanism contributing to virus escape to the action of antiretrovirals and a mode of HIV persistence during antiretroviral therapy. Here, cocultures of infected HIV-1 cells with primary CD4+ T cells or lymphoid cells were used to evaluate virus transmission and the effect of known antiretrovirals. Transfer of HIV antigen from infected to uninfected cells was resistant to the reverse transcriptase inhibitors (RTIs) zidovudine (AZT) and tenofovir, but was blocked by the attachment inhibitor IgGb12. However, quantitative measurement of viral DNA production demonstrated that all anti-HIV agents blocked virus replication with similar potency to cell-free virus infections. Cell-free and cell-associated infections were equally sensitive to inhibition of viral replication when HIV-1 long terminal repeat (LTR)-driven green fluorescent protein (GFP) expression in target cells was measured. However, detection of GFP by flow cytometry may incorrectly estimate the efficacy of antiretrovirals in cell-associated virus transmission, due to replication-independent Tat-mediated LTR transactivation as a consequence of cell-to-cell events that did not occur in short-term (48-h) cell-free virus infections. In conclusion, common markers of virus replication may not accurately correlate and measure infectivity or drug efficacy in cell-to-cell virus transmission. When accurately quantified, active drugs blocked proviral DNA and virus replication in cell-to-cell transmission, recapitulating the efficacy of antiretrovirals in cell-free virus infections and in vivo.

Palbociclib, a selective inhibitor of cyclin-dependent kinase4/6, blocks HIV-1 reverse transcription through the control of sterile α motif and HD domain-containing protein-1 (SAMHD1) activity.

Background:Sterile &agr; motif and HD domain-containing protein-1 (SAMHD1) inhibits HIV-1 reverse transcription by decreasing the pool of intracellular deoxynucleotides. SAMHD1 is controlled by cyclin-dependent kinase (CDK)-mediated phosphorylation. However, the exact mechanism of SAMHD1 regulation in primary cells is unclear. We explore the effect of palbociclib, a CDK6 inhibitor, in HIV-1 replication. Methods:Human primary monocytes were differentiated into macrophages with monocyte-colony stimulating factor and CD4+ T lymphocytes stimulated with phytohaemagglutinin (PHA)/interleukin-2. Cells were treated with palbociclib and then infected with a Green fluorescent protein-expressing HIV-1 or R5 HIV-1 BaL. Viral DNA was measured by quantitative PCR and infection assessed by flow cytometry. Deoxynucleotide triphosphate (dNTP) content was determined using a polymerase-based method. Results:Pan-CDK inhibitors AT7519, roscovitine and purvalanol A reduced SAMHD1 phosphorylation. HIV-1 replication was blocked by AT7519 (66.4 ± 3.8%; n = 4), roscovitine (47.3 ± 3.9%; n = 4) and purvalanol A (55.7 ± 15.7%; n = 4) at subtoxic concentrations. Palbociclib, a potent and selective CDK6 inhibitor, blocked SAMHD1 phosphorylation, intracellular dNTP levels, HIV-1 reverse transcription and HIV-1 replication in primary macrophages and CD4+ T lymphocytes. Notably, treatment of macrophages with palbociclib led to reduced CDK2 activation, measured as the phosphorylation of the T-loop at the Thr160. The antiviral effect was lost when SAMHD1 was degraded by Vpx, providing further evidence for a role of SAMHD1 in mediating the antiretroviral effect. Conclusions:Our results indicate that SAMHD1-mediated HIV-1 restriction is controlled by CDK as previously suggested but point to a preferential role for CDK2 and CDK6 as mediators of SAMHD1 activation. Our study provides a new signaling pathway susceptible for the development of new therapeutic approaches against HIV-1 infection.

Increased expression of SAMHD1 in a subset of HIV-1 elite controllers

OBJECTIVES SAMHD1 and the CDKN1A (p21) cyclin-dependent kinase inhibitor have been postulated to mediate HIV-1 restriction in CD4+ cells. We have shown that p21 affects HIV replication through its effect on SAMHD1. Thus, we aimed at evaluating the expression of SAMHD1 and p21 in different HIV+ phenotypic groups. PATIENTS AND METHODS We evaluated SAMHD1 and CDKN1A mRNA expression in CD4+ T cells from HIV+ individuals including elite controllers (n = 12), individuals who control HIV without the need for antiretroviral treatment, viraemic progressors (n = 10) and HIV-1 seronegative healthy donors (n = 14). Immunological variables were measured by flow cytometry. RESULTS We show that a subset of HIV+ elite controllers with lower T cell proliferation levels (Ki67+ cells) expressed higher SAMHD1 compared with healthy donors or viraemic progressors. Conversely, there was no difference in p21 expression before or after T cell activation with a bispecific CD3/CD8 antibody. CONCLUSIONS Our results suggest that SAMHD1 may play a role in controlling virus replication in HIV+ individuals and slow the rate of disease progression.

ADS-J1 Inhibits HIV-1 Entry by Interacting with gp120 and Does Not Block Fusion-Active gp41 Core Formation

ABSTRACT We had shown that virus resistance to ADS-J1 was associated with amino acid changes in the envelope glycoprotein, mostly located in the gp120 coding region. Time-of-addition and endocytic virus transfer assays clearly demonstrated that ADS-J1 behaved as a gp120 inhibitor. ADS-J1-resistant virus was cross-resistant to the polyanion dextran sulfate, and recombination of gp120 recovered only the ADS-J1-resistant phenotype. In summary, ADS-J1 blocks an early step of virus entry that appears to be driven by gp120 alone.

Trans-infection but Not Infection from within Endosomal Compartments after Cell-to-cell HIV-1 Transfer to CD4+ T Cells

Background: Endocytosis has been postulated as a route of HIV entry into cells. Results: Endocytosed virus led to productive infection of cells, except when cells were cultured with the anti-gp120 antibody IgGb12. Conclusion: Endocytosis may serve as a virus reservoir capable of inducing trans-infection. Significance: The route of HIV transmission is important for understanding pathogenesis and drug therapy. Cellular contacts between HIV-1-infected donor cells and uninfected primary CD4+ T lymphocytes lead to virus transfer into endosomes. Recent evidence suggests that HIV particles may fuse with endosomal membranes to initiate a productive infection. To explore the role of endocytosis in the entry and replication of HIV, we evaluated the infectivity of transferred HIV particles in a cell-to-cell culture model of virus transmission. Endocytosed virus led to productive infection of cells, except when cells were cultured in the presence of the anti-gp120 mAb IgGb12, an agent that blocks virus attachment to CD4, suggesting that endocytosed virus was recycled to the outer cell surface. Confocal microscopy confirmed the colocalization of internalized virus antigen and the endosomal marker dynamin. Additionally, virus transfer, fusion, or productive infection was not blocked by dynasore, dynamin-dependent endosome-scission inhibitor, at subtoxic concentrations, suggesting that the early capture of virus into intracellular compartments did not depend on endosomal maturation. Our results suggest that endocytosis is not a mechanism of infection of primary CD4 T cells, but may serve as a reservoir capable of inducing trans-infection of cells after the release of HIV particles to the extracellular environment.

The Cortical Actin Determines Different Susceptibility of Naïve and Memory CD4+ T Cells to HIV-1 Cell-to-Cell Transmission and Infection

Memory CD4+ T cells are preferentially infected by HIV-1 compared to naïve cells. HIV-1 fusion and entry is a dynamic process in which the cytoskeleton plays an important role by allowing virion internalization and uncoating. Here, we evaluate the role of the cortical actin in cell-to-cell transfer of virus antigens and infection of target CD4+ T cells. Using different actin remodeling compounds we demonstrate that efficiency of HIV-internalization was proportional to the actin polymerization of the target cell. Naïve (CD45RA+) and memory (CD45RA−) CD4+ T cells could be phenotypically differentiated by the degree of cortical actin density and their capacity to capture virus. Thus, the higher cortical actin density of memory CD4+ T cells was associated to increased efficiency of HIV-antigen internalization and the establishment of a productive infection. Conversely, the lower cortical actin density in naïve CD4+ T cells restricted viral antigen transfer and consequently HIV-1 infection. In conclusion, the cortical actin density differentially affects the susceptibility to HIV-1 infection in naïve and memory CD4+ T cells by modulating the efficiency of HIV antigen internalization.