Erica Giuliani

HIV-1 NEF AND VPU INTERFERE WITH L-SELECTIN (CD62L) CELL SURFACE EXPRESSION TO INHIBIT ADHESION AND SIGNALING IN INFECTED CD4+ T LYMPHOCYTES

ABSTRACT Leukocyte recirculation between blood and lymphoid tissues is required for the generation and maintenance of immune responses against pathogens and is crucially controlled by the L-selectin (CD62L) leukocyte homing receptor. CD62L has adhesion and signaling functions and initiates the capture and rolling on the vascular endothelium of cells entering peripheral lymph nodes. This study reveals that CD62L is strongly downregulated on primary CD4+ T lymphocytes upon infection with human immunodeficiency virus type 1 (HIV-1). Reduced cell surface CD62L expression was attributable to the Nef and Vpu viral proteins and not due to increased shedding via matrix metalloproteases. Both Nef and Vpu associated with and sequestered CD62L in perinuclear compartments, thereby impeding CD62L transport to the plasma membrane. In addition, Nef decreased total CD62L protein levels. Importantly, infection with wild-type, but not Nef- and Vpu-deficient, HIV-1 inhibited the capacity of primary CD4+ T lymphocytes to adhere to immobilized fibronectin in response to CD62L ligation. Moreover, HIV-1 infection impaired the signaling pathways and costimulatory signals triggered in primary CD4+ T cells by CD62L ligation. We propose that HIV-1 dysregulates CD62L expression to interfere with the trafficking and activation of infected T cells. Altogether, this novel HIV-1 function could contribute to virus dissemination and evasion of host immune responses. IMPORTANCE L-selectin (CD62L) is an adhesion molecule that mediates the first steps of leukocyte homing to peripheral lymph nodes, thus crucially controlling the initiation and maintenance of immune responses to pathogens. Here, we report that CD62L is downmodulated on the surfaces of HIV-1-infected T cells through the activities of two viral proteins, Nef and Vpu, that prevent newly synthesized CD62L molecules from reaching the plasma membrane. We provide evidence that CD62L downregulation on HIV-1-infected primary T cells results in impaired adhesion and signaling functions upon CD62L triggering. Removal of cell surface CD62L may predictably keep HIV-1-infected cells away from lymph nodes, the privileged sites of both viral replication and immune response activation, with important consequences, such as systemic viral spread and evasion of host immune surveillance. Altogether, we propose that Nef- and Vpu-mediated subversion of CD62L function could represent a novel determinant of HIV-1 pathogenesis.

The human immunodeficiency virus type 1 Vpr protein upregulates PVR via activation of the ATR-mediated DNA damage response pathway.

Viral infection may induce the cell-surface expression of PVR (CD155) that, upon recognition by its cognate activating DNAM-1 receptor present on cytotoxic lymphocytes, may promote antiviral immune responses. Here we show that expression of the human immunodeficiency virus type 1 (HIV-1) Vpr protein in Jurkat T cells increases cell-surface and total PVR levels. Analysis of mutated Vpr variants indicated that Vpr uses the same protein surfaces, and hence probably the same mechanisms, to upregulate PVR and arrest the cell cycle in the G2 phase. Moreover, we found that PVR upregulation by Vpr relied on the ability of the protein to activate the ATR kinase that triggers the DNA damage response pathway and G2 arrest. Finally, we showed that Vpr contributes to PVR up-modulation in HIV-infected CD4(+) T lymphocytes and inhibits the PVR downregulating activity of the viral Nef protein.

The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222

Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells.

The ADAR1 editing enzyme is encapsidated into HIV-1 virions

Adenosine deaminase acting on RNA1 (ADAR1) was previously reported to affect HIV-1 replication. We report data showing that ADAR1 interacts with the HIV-1 p55 Gag protein, the major structural protein of the immature virus capsid. Furthermore, we found that the endogenous ADAR1 is incorporated into virions purified from the supernatant of primary HIV-1-infected CD4(+) T lymphocytes. Additional experiments demonstrated that the expression of the p55 Gag protein is sufficient for ADAR1 incorporation into virus-like particles (VLPs). Overall, our data originally support the evidence that ADAR1 can be part of the cell protein array uploaded in HIV-1 particles.

HIV-1 Infection Causes a Down-Regulation of Genes Involved in Ribosome Biogenesis

HIV-1 preferentially infects CD4+ T cells, causing fundamental changes that eventually lead to the release of new viral particles and cell death. To investigate in detail alterations in the transcriptome of the CD4+ T cells upon viral infection, we sequenced polyadenylated RNA isolated from Jurkat cells infected or not with HIV-1. We found a marked global alteration of gene expression following infection, with an overall trend toward induction of genes, indicating widespread modification of the host biology. Annotation and pathway analysis of the most deregulated genes showed that viral infection produces a down-regulation of genes associated with the nucleolus, in particular those implicated in regulating the different steps of ribosome biogenesis, such as ribosomal RNA (rRNA) transcription, pre-rRNA processing, and ribosome maturation. The impact of HIV-1 infection on genes involved in ribosome biogenesis was further validated in primary CD4+ T cells. Moreover, we provided evidence by Northern Blot experiments, that host pre-rRNA processing in Jurkat cells might be perturbed during HIV-1 infection, thus strengthening the hypothesis of a crosstalk between nucleolar functions and viral pathogenesis.

Expression and Function of NKG2D Is Impaired in CD8+ T Cells of Chronically HIV-1-Infected Patients Without ART.

Objectives:Increasing line of evidence indicates that the NKG2D-activating receptor plays a relevant role in the effector functions of cytotoxic lymphocytes. In this study, we investigated the expression and function of NKG2D in CD8+ T cells from chronically HIV-1–infected patients with or without antiretroviral therapy (ART). Methods:We measured by flow cytometry the expression of NKG2D on CD8+ T-cell subsets of ART-naive and ART patients as well as seronegative healthy subjects (HIV-neg). An intrapatient analysis before and after ART initiation was also performed. Results were correlated with viral load, CD4+ T-cell counts, markers of immune activation (CD38, sCD14), and soluble NKG2D ligands (sMICA and sULBP2). The function of NKG2D on CD8+ T cell cytotoxicity was tested by ex vivo degranulation assays. Results:We showed that NKG2D was downregulated on all CD8+ T-cell subsets of ART-naive patients. The expression of NKG2D on CD8+ T cells inversely correlated with viral load and CD38 expression but not with plasma levels of sMICA and sULBP2. Importantly, we found that NKG2D-mediated costimulation of CD8+ T-cell lytic activity was strongly reduced in ART-naive patients if compared with HIV-neg and ART subjects. Finally, intrapatient analysis demonstrated that effective anti-HIV-1 therapy restores NKG2D expression and NKG2D-induced cytotoxicity by CD8+ T cells. Conclusions:These data underscore that NKG2D downregulation contributes to impaired CD8+ T-cell responses in untreated HIV-1 infection and have implications for monitoring immune functions and response to treatments, and for the development of novel anti-HIV-1 strategies combining ART with drugs that stimulate NKG2D expression and function.

NK cells of HIV-1-infected patients with poor CD4+ T-cell reconstitution despite suppressive HAART show reduced IFN-γ production and high frequency of autoreactive CD56bright cells

HIV-1-infected patients failing to recover CD4+ T-cell count despite HAART (immunological non-responders, NRs), are at increased risk of disease progression and death. To better understand the NR status, we performed a comprehensive assessment of NK cells in NR patients as compared to immunologic responders. NRs exhibited an accumulation of CD56bright NK cells inversely correlated with CD4+ counts. Both CD56bright and CD56dim NK cells of NRs displayed unimpaired degranulation ability, but poorly responded to cytokine stimulation in terms of NKp44 up-regulation and IFN-γ production that may explain the susceptibility of NRs to infections and tumors. Notably, CD56bright NK cells from NRs showed higher cytotoxicity against autologous activated CD4+ T cells. Moreover, NRs had reduced Treg cell counts that showed an inverse correlation with autoreactive CD56bright cells. These data suggest that accumulation of CD56bright NK cells, possibly linked to decreased homeostatic control by Tregs, contributes to poor immune reconstitution in NRs.

Brief Report: L-Selectin (CD62L) Is Downregulated on CD4+ and CD8+ T Lymphocytes of HIV-1-Infected Individuals Naive for ART.

Abstract:The expression of L-selectin (CD62L) in HIV-1 infection has not been extensively investigated. Here, we measured CD62L expression on T-cell subsets of HIV-1–infected individuals naive for antiretroviral therapy (ART-naive) or receiving therapy (ART), and seronegative control subjects (HIV-neg). We found reduced frequencies of CD62L+ cells among CD4+ and CD8+ T cells from ART-naive as compared with ART and HIV-neg groups, particularly within naive and central memory subsets. CD62L expression on T cells inversely correlated with viral load and rapidly increased after ART initiation. Plasma sCD62L levels did not correlate with CD62L expression, being higher in all HIV-1–infected individuals as compared with HIV-neg subjects. Finally, CD62L downregulation was found associated with the expression of the CD38 activation marker in CD8+ T cells, but not in CD4+ T cells. We suggest that CD62L downregulation due to unconstrained HIV-1 replication may have important consequences for T-cell circulation and function and for disease progression.

Dual regulation of L-selectin (CD62L) by HIV-1: Enhanced expression by Vpr in contrast with cell-surface down-modulation by Nef and Vpu

The HIV-1 accessory protein Vpr displays various activities that can favor viral replication such as G2 cell cycle arrest. Vpr also modulates host gene expression, although this property is poorly characterized. Here, we investigated the effect of Vpr on L-selectin (CD62L), which crucially controls leukocytes circulation and generation of immune responses against pathogens. We report that Vpr up-regulates CD62L mRNA level when individually expressed in Jurkat T cells as well as during HIV-1 infection of primary CD4+ T cells. Vpr mutant analysis and use of inhibitors suggest that the effect of Vpr on CD62L occurs independently of G2 arrest but requires activation of the ATR kinase. Yet, induction of CD62L expression by Vpr is contrasted by down-regulation of CD62L protein by Nef that, together with Vpu, induces a net reduction of cell-surface CD62L on HIV-1-infected cells, which may impact viral spread and evasion of immune responses.

In Vitro Exposure to Prostratin but Not Bryostatin-1 Improves Natural Killer Cell Functions Including Killing of CD4+ T Cells Harboring Reactivated Human Immunodeficiency Virus

In the attempt of purging the HIV-1 reservoir through the “shock-and-kill” strategy, it is important to select latency-reversing agents (LRAs) devoid of deleterious effects on the antiviral function of immune effector cells. Here, we investigated two LRAs with PKC agonist activity, prostratin (PRO) and bryostatin-1 (BRY), for their impact on the function of natural killer (NK) cells, the major effectors of innate immunity whose potential in HIV-1 eradication has emerged in recent clinical trials. Using NK cells of healthy donors, we found that exposure to either PRO or BRY potently activated NK cells, resulting in upmodulation of NKG2D and NKp44 activating receptors and matrix metalloprotease-mediated shedding of CD16 receptor. Despite PRO and BRY affected NK cell phenotype in the same manner, their impact on NK cell function was diverse and showed considerable donor-to-donor variation. Altogether, in most tested donors, the natural cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) of NK cells were either improved or maintained by PRO, while both activities were impaired by BRY. Moreover, we analyzed the effect of these drugs on the capacity of treated NK cells to kill autologous latently infected CD4+ T cells reactivated via the same treatment. First, we found that PRO but not BRY increased upmodulation of the ULBP2 ligand for NKG2D on reactivated p24+ cells. Importantly, we showed that clearance of reactivated p24+ cells by NK cells was enhanced when both targets and effectors were exposed to PRO but not to BRY. Overall, PRO had a superior potential compared with BRY as to the impact on key NK cell functions and on NK-cell-mediated clearance of the HIV-1 reservoir. Our results emphasize the importance of considering the effects on NK cells of candidate “shock-and-kill” interventions. With respect to combinative approaches, the impact on NK cells of each LRA should be re-evaluated upon combination with a second LRA, which may have analogous or opposite effects, or with immunotherapy targeting NK cells. In addition, avoiding co-administration of LRAs that negatively impact ADCC activity by NK cells might be essential for successful application of antibodies or vaccination to “shock-and-kill” strategies.