Efficient Nef-mediated downmodulation of TCR-CD3 and CD28 is associated with high CD4+ T cell counts in viremic HIV-2 infection

The role of the multifunctional accessory Nef protein in the immunopathogenesis of HIV-2 infection is currently poorly understood. Here, we performed comprehensive functional analyses of 50 nef genes from 21 viremic (plasma viral load, >500 copies/ml) and 16 nonviremic (<500) HIV-2-infected individuals. On average, nef alleles from both groups were equally active in modulating CD4, TCR-CD3, CD28, MHC-I, and Ii cell surface expression and in enhancing virion infectivity. Thus, many HIV-2-infected individuals efficiently control the virus in spite of efficient Nef function. However, the potency of nef alleles in downmodulating TCR-CD3 and CD28 to suppress the activation and apoptosis of T cells correlated with high numbers of CD4(+) T cells in viremic patients. No such correlations were observed in HIV-2-infected individuals with undetectable viral load. Further functional analyses showed that the Nef-mediated downmodulation of TCR-CD3 suppressed the induction of Fas, Fas-L, PD-1, and CTLA-4 cell surface expression as well as the secretion of gamma interferon (IFN-γ) by primary CD4(+) T cells. Moreover, we identified a single naturally occurring amino acid variation (I132T) in the core domain of HIV-2 Nef that selectively disrupts its ability to downmodulate TCR-CD3 and results in functional properties highly reminiscent of HIV-1 Nef proteins. Taken together, our data suggest that the efficient Nef-mediated downmodulation of TCR-CD3 and CD28 help viremic HIV-2-infected individuals to maintain normal CD4(+) T cell homeostasis by preventing T cell activation and by suppressing the induction of death receptors that may affect the functionality and survival of both virally infected and uninfected bystander cells.     

AP-2 Is the Crucial Clathrin Adaptor Protein for CD4 Downmodulation by HIV-1 Nef in Infected Primary CD4+ T Cells

HIV-1 Nef-mediated CD4 downmodulation involves various host factors. We investigated the importance of AP-1, AP-2, AP-3, V1H-ATPase, β-COP, and ACOT8 for CD4 downmodulation in HIV-1-infected short hairpin RNA (shRNA)-expressing CD4⁺ T cells and characterized direct interaction with Nef by Förster resonance energy transfer (FRET). Binding of lentiviral Nefs to CD4 and AP-2 was conserved, and only AP-2 knockdown impaired Nef-mediated CD4 downmodulation from primary T cells. Altogether, among the factors tested, AP-2 is the most important player for Nef-mediated CD4 downmodulation.     

Two sorting motifs, a ubiquitination motif and a tyrosine motif, are involved in HIV-1 and simian immunodeficiency virus Nef-mediated receptor endocytosis

HIV-1 and SIV Nef proteins downregulate cell surface CD4 and MHC class I (MHC-I) molecules of infected cells, which are necessary for efficient viral replication and pathogenicity. We previously reported that K144 in HIV-1 Nef is di-ubiquitinated, and K144R substitution impairs Nef-mediated CD4 downregulation. In this report, we extend the role of ubiquitination at this lysine residue from Nef-mediated CD4 downregulation to Nef-mediated MHC-I downregulation and from HIV Nef to SIV Nef. All HIV-1 Nef mutants that contain K144R substitution are inactive in MHC-I downregulation. Tested MHC-I alleles include HLA-ABC endogenously expressed and HLA-A2 exogenously expressed in Jurkat T cells. CD4 downregulation by SIV Nef involves K176 that aligns with K144 in HIV-1 Nef, as well as an N-terminal tyrosine motif Y28Y39 not present in HIV-1 Nef. Dual mutation at K176 and Y28Y39 completely impaired SIV Nef-mediated CD4 and MHC-I downregulation, whereas a single mutation at K176 or Y28Y39 did not. The involvement of tyrosine motif in SIV Nef-mediated CD4 and MHC-I downregulation prompted us to investigate a putative tyrosine motif (Y202Y/F203) in HIV-1 Nef that is conserved among HIV-1 species. Single mutation at the tyrosine motif Y202F203 in HIV-1 Nef (NA7) greatly impaired Nef-mediated CD4 downregulation, which is similar to what we observed previously with the single mutation at lysine K144. Thus, our study demonstrated that Nef-mediated receptor endocytosis involves the ubiquitination motif and tyrosine motif.     

Nef-mediated disruption of HLA-A2 transport to the cell surface in T cells

Human immunodeficiency virus type 1 (HIV-1) Nef is a key pathogenic factor necessary for the development of AIDS. One important function of Nef is to reduce cell surface levels of major histocompatibility complex class I (MHC-I) molecules, thereby protecting HIV-infected cells from recognition by cytotoxic T lymphocytes. The mechanism of MHC-I downmodulation by Nef has not been clearly elucidated, and its reported effect on MHC-I steady-state levels ranges widely, from 2-fold in HeLa cells to 200-fold in HIV-infected primary T cells. Here, we directly compared downmodulation of HLA-A2 in HIV-infected HeLa cells to that in T cells. We found that similar amounts of Nef protein resulted in a much more dramatic downmodulation of HLA-A2 in T cells than in HeLa cells. A comparison of Nef's effects on HLA-A2 endocytosis, recycling, and transport rates indicated that the most prominent effect of Nef on HLA-A2 in T cells was to inhibit transport to the cell surface. The phosphatidylinositol 3-kinase inhibitor, LY294002, previously reported to inhibit Nef-mediated MHC-I downmodulation in astrocytic cells, did not directly affect Nef's ability to block transport of MHC-I to the cell surface in T cells.     

CD4 coexpression regulates DC-SIGN-mediated transmission of human immunodeficiency virus type 1

Dendritic cells (DCs) potently stimulate the cell-cell transmission of human immunodeficiency virus type 1 (HIV-1). However, the mechanisms that underlie DC transmission of HIV-1 to CD4⁺ T cells are not fully understood. DC-SIGN, a C-type lectin, efficiently promotes HIV-1 trans infection. DC-SIGN is expressed in monocyte-derived DCs (MDDCs), macrophage subsets, activated B lymphocytes, and various mucosal tissues. MDDC-mediated HIV-1 transmission to CD4⁺ T cells involves DC-SIGN-dependent and -independent mechanisms. DC-SIGN transmission of HIV-1 depends on the donor cell type. HIV-1 Nef can upregulate DC-SIGN expression and promote DC-T-cell clustering and HIV-1 spread. Nef also downregulates CD4 expression; however, the effect of the CD4 downmodulation on DC-mediated HIV-1 transmission has not been examined. Here, we report that CD4 expression levels correlate with inefficient HIV-1 transmission by monocytic cells expressing DC-SIGN. Expression of CD4 on Raji B cells strongly impaired DC-SIGN-mediated HIV-1 transmission to T cells. By contrast, enhanced HIV-1 transmission was observed when CD4 molecules on MDDCs and DC-SIGN-CD4-expressing cell lines were blocked with specific antibodies. Coexpression of CD4 and DC-SIGN in Raji cells promoted the internalization and intracellular retention of HIV-1. Interestingly, internalized HIV-1 particles were sorted and confined to late endosomal compartments that were positive for CD63 and CD81. Furthermore, in HIV-1-infected MDDCs, significant downregulation of CD4 by Nef expression correlated with enhanced viral transmission. These results suggest that CD4, which is present at various levels in DC-SIGN-positive primary cells, is a key regulator of HIV-1 transmission.     

IgE-Mediated Enhancement of CD4+ T Cell Responses in Mice Requires Antigen Presentation by CD11c+ Cells and Not by B Cells

IgE antibodies, administered to mice together with their specific antigen, enhance antibody and CD4⁺ T cell responses to this antigen. The effect is dependent on the low affinity receptor for IgE, CD23, and the receptor must be expressed on B cells. In vitro, IgE-antigen complexes are endocytosed via CD23 on B cells, which subsequently present the antigen to CD4⁺ T cells. This mechanism has been suggested to explain also IgE-mediated enhancement of immune responses in vivo. We recently found that CD23⁺ B cells capture IgE-antigen complexes in peripheral blood and rapidly transport them to B cell follicles in the spleen. This provides an alternative explanation for the requirement for CD23⁺ B cells. The aim of the present study was to determine whether B-cell mediated antigen presentation of IgE-antigen complexes explains the enhancing effect of IgE on immune responses in vivo. The ability of spleen cells, taken from mice 1–4 h after immunization with IgE-antigen, to present antigen to specific CD4⁺ T cells was analyzed. Antigen presentation was intact when spleens were depleted of CD19⁺ cells (i.e., primarily B cells) but was severely impaired after depletion of CD11c⁺ cells (i.e., primarily dendritic cells). In agreement with this, the ability of IgE to enhance proliferation of CD4⁺ T cells was abolished in CD11c-DTR mice conditionally depleted of CD11c⁺ cells. Finally, the lack of IgE-mediated enhancemen of CD4⁺ T cell responses in CD23^-/- mice could be rescued by transfer of MHC-II-compatible as well as by MHC-II-incompatible CD23⁺ B cells. These findings argue against the idea that IgE-mediated enhancement of specific CD4⁺ T cell responses in vivo is caused by increased antigen presentation by B cells. A model where CD23⁺ B cells act as antigen transporting cells, delivering antigen to CD11c⁺ cells for presentation to T cells is consistent with available experimental data.     

CD8+ T cells from SIV elite controller macaques recognize Mamu-B*08-bound epitopes and select for widespread viral variation

Background

It is generally accepted that CD8⁺ T cell responses play an important role in control of immunodeficiency virus replication. The association of HLA-B27 and -B57 with control of viremia supports this conclusion. However, specific correlates of viral control in individuals expressing these alleles have been difficult to define. We recently reported that transient in vivo CD8⁺ cell depletion in simian immunodeficiency virus (SIV)-infected elite controller (EC) macaques resulted in a brief period of viral recrudescence. SIV replication was rapidly controlled with the reappearance of CD8⁺ cells, implicating that these cells actively suppress viral replication in ECs.

Methods and Findings

Here we show that three ECs in that study made at least seven robust CD8⁺ T cell responses directed against novel epitopes in Vif, Rev, and Nef restricted by the MHC class I molecule Mamu-B*08. Two of these Mamu-B*08-positive animals subsequently lost control of SIV replication. Their breakthrough virus harbored substitutions in multiple Mamu-B*08-restricted epitopes. Indeed, we found evidence for selection pressure mediated by Mamu-B*08-restricted CD8⁺ T cells in all of the newly identified epitopes in a cohort of chronically infected macaques.

Conclusions

Together, our data suggest that Mamu-B*08-restricted CD8⁺ T cell responses effectively control replication of pathogenic SIV_mac239. All seven regions encoding Mamu-B*08-restricted CD8⁺ T cell epitopes also exhibit amino acid replacements typically seen only in the presence of Mamu-B*08, suggesting that the variation we observe is indeed selected by CD8⁺ T cell responses. SIV_mac239 infection of Indian rhesus macaques expressing Mamu-B*08 may therefore provide an animal model for understanding CD8⁺ T cell-mediated control of HIV replication in humans.     

Cord blood derived CD4+ CD25(high) T cells become functional regulatory T cells upon antigen encounter

Background: Upon antigen exposure, cord blood derived T cells respond to ubiquitous environmental antigens by high proliferation. To date it remains unclear whether these “excessive” responses relate to different regulatory properties of the putative T regulatory cell (Treg) compartment or even expansion of the Treg compartment itself.Methods: Cord blood (>37 week of gestation) and peripheral blood (healthy controls) were obtained and different Treg cell subsets were isolated. The suppressive potential of Treg populations after antigen exposure was evaluated via functional inhibition assays ([³H]thymidine incorporation assay and CFSE staining) with or without allergen stimulation. The frequency and markers of CD4⁺CD25^highFoxP3⁺ T cells were characterized by mRNA analysis and flow cytometry.Results: Cord blood derived CD4⁺CD25^high cells did not show substantial suppressor capacity upon TCR activation, in contrast to CD4⁺CD25^high cells freshly purified from adult blood. This could not be explained by a lower frequency of FoxP3⁺CD4⁺CD25^highcells or FOXP3 mRNA expression. However, after antigen-specific stimulation in vitro, these cells showed strong proliferation and expansion and gained potent suppressive properties. The efficiency of their suppressive capacity can be enhanced in the presence of endotoxins. If T-cells were sorted according to their CD127 expression, a tiny subset of Treg cells (CD4⁺CD25⁺CD127^low) is highly suppressive even without prior antigen exposure.Conclusion: Cord blood harbors a very small subset of CD4⁺CD25^high Treg cells that requires antigen-stimulation to show expansion and become functional suppressive Tregs.     

Resting regulatory CD4 T cells: a site of HIV persistence in patients on long-term effective antiretroviral therapy

Background

In HIV-infected patients on long-term HAART, virus persistence in resting long-lived CD4 T cells is a major barrier to curing the infection. Cell quiescence, by favouring HIV latency, reduces the risk of recognition and cell destruction by cytotoxic lymphocytes. Several cell-activation-based approaches have been proposed to disrupt cell quiescence and then virus latency, but these approaches have not eradicated the virus. CD4⁺CD25⁺ regulatory T cells (Tregs) are a CD4⁺ T-cell subset with particular activation properties. We investigated the role of these cells in virus persistence in patients on long-term HAART.

Methodology/Principal Findings

We found evidence of infection of resting Tregs (HLADR⁻CD69⁻CD25^hiFoxP3⁺CD4⁺ T cells) purified from patients on prolonged HAART. HIV DNA harbouring cells appear more abundant in the Treg subset than in non-Tregs. The half-life of the Treg reservoir was estimated at 20 months. Since Tregs from patients on prolonged HAART showed hyporesponsiveness to cell activation and inhibition of HIV-specific cytotoxic T lymphocyte-related functions upon activation, therapeutics targeting cell quiescence to induce virus expression may not be appropriate for purging the Treg reservoir.

Conclusions

Our results identify Tregs as a particular compartment within the latent reservoir that may require a specific approach for its purging.     

Impaired CD4 T cell memory response to Streptococcus pneumoniae precedes CD4 T cell depletion in HIV-infected Malawian adults

Objective

Invasive pneumococcal disease (IPD) is a leading cause of morbidity and mortality in HIV-infected African adults. CD4 T cell depletion may partially explain this high disease burden but those with relatively preserved T cell numbers are still at increased risk of IPD. This study evaluated the extent of pneumococcal-specific T cell memory dysfunction in asymptomatic HIV infection early on in the evolution of the disease.

Methods

Peripheral blood mononuclear cells were isolated from asymptomatic HIV-infected and HIV-uninfected Malawian adults and stained to characterize the underlying degree of CD4 T cell immune activation, senescence and regulation. Pneumococcal-specific T cell proliferation, IFN-γ, IL-17 production and CD154 expression was assessed using flow cytometry and ELISpot.

Results

We find that in asymptomatic HIV-infected Malawian adults, there is considerable immune disruption with an increase in activated and senescent CD4⁺CD38⁺PD-1⁺ and CD4⁺CD25^highFoxp3⁺ Treg cells. In the context of high pneumococcal exposure and therefore immune stimulation, show a failure in pneumococcal-specific memory T cell proliferation, skewing of T cell cytokine production with preservation of interleukin-17 but decreased interferon-gamma responses, and failure of activated T cells to express the co-stimulatory molecule CD154.

Conclusion

Asymptomatic HIV-infected Malawian adults show early signs of pneumococcal- specific immune dysregulation with a shift in the balance of CD4 memory, T helper 17 cells and Treg. Together these data offer a mechanistic understanding of how antigen-specific T cell dysfunction occurs prior to T cell depletion and may explain the early susceptibility to IPD in those with relatively preserved CD4 T cell numbers.     

Granulysin-expressing CD4+ T cells as candidate immune marker for tuberculosis during childhood and adolescence

Background

Granulysin produced by cytolytic T cells directly contributes to immune defense against tuberculosis (TB). We investigated granulysin as a candidate immune marker for childhood and adolescent TB.

Methods

Peripheral blood mononuclear cells (PBMC) from children and adolescents (1–17 years) with active TB, latent TB infection (LTBI), nontuberculous mycobacteria (NTM) infection and from uninfected controls were isolated and restimulated in a 7-day restimulation assay. Intracellular staining was then performed to analyze antigen-specific induction of activation markers and cytotoxic proteins, notably, granulysin in CD4⁺ CD45RO⁺ memory T cells.

Results

CD4⁺ CD45RO⁺ T cells co-expressing granulysin with specificity for Mycobacterium tuberculosis (Mtb) were present in high frequency in TB-experienced children and adolescents. Proliferating memory T cells (CFSE_lowCD4⁺CD45RO⁺) were identified as main source of granulysin and these cells expressed both central and effector memory phenotype. PBMC from study participants after TB drug therapy revealed that granulysin-expressing CD4⁺ T cells are long-lived, and express several activation and cytotoxicity markers with a proportion of cells being interferon-gamma-positive. In addition, granulysin-expressing T cell lines showed cytolytic activity against Mtb-infected target cells.

Conclusions

Our data suggest granulysin expression by CD4⁺ memory T cells as candidate immune marker for TB infection, notably, in childhood and adolescence.     

Caspase-3 is transiently activated without cell death during early antigen driven expansion of CD8(+) T cells in vivo

Background

CD8⁺ T cell responses develop rapidly during infection and are swiftly reduced during contraction, wherein >90% of primed CD8⁺ T cells are eliminated. The role of apoptotic mechanisms in controlling this rapid proliferation and contraction of CD8⁺ T cells remains unclear. Surprisingly, evidence has shown non-apoptotic activation of caspase-3 to occur during in vitro T-cell proliferation, but the relevance of these mechanisms to in vivo CD8⁺ T cell responses has yet to be examined.

Methods and Findings

We have evaluated the activity of caspase-3, a key downstream inducer of apoptosis, throughout the entirety of a CD8⁺ T cell response. We utilized two infection models that differ in the intensity, onset and duration of antigen-presentation and inflammation. Expression of cleaved caspase-3 in antigen specific CD8⁺ T cells was coupled to the timing and strength of antigen presentation in lymphoid organs. We also observed coordinated activation of additional canonical apoptotic markers, including phosphatidylserine exposure. Limiting dilution analysis directly showed that in the presence of IL7, very little cell death occurred in both caspase-3^hi and caspase-3^low CD8⁺ T cells. The expression of active caspase-3 peaked before effector phenotype (CD62L^low) CD8⁺ T cells emerged, and was undetectable in effector-phenotype cells. In addition, OVA-specific CD8⁺ cells remained active caspase-3^low throughout the contraction phase.

Conclusions

Our results specifically implicate antigen and not inflammation in driving activation of apoptotic mechanisms without cell death in proliferating CD8⁺ T cells. Furthermore, the contraction of CD8⁺ T cell response following expansion is likely not mediated by the key downstream apoptosis inducer, caspase-3.     

Phenotypic characterization of HIV-specific CD8+ T cells during early and chronic infant HIV-1 infection

Although CD8⁺ T cells play an important role in the containment of adult HIV-1 replication, their role in infant HIV-1 infection is not as well understood. Impaired HIV-specific CD8⁺ T cell responses may underlie the persistently high viral loads observed in infants. We examined the frequency and phenotype of infant HIV-specific CD8⁺ T cells in 7 HIV-infected antiretroviral therapy-naïve infants during the first 2 years of life, using class I HLA tetramers and IFN-γ-ELISPOT. The frequency (0.088–3.9% of CD3⁺CD8⁺ cells) and phenotype (CD27⁺CD28⁻, CD45RA^+/−, CD57^+/−, HLA-DR⁺, CD95⁺) of infant HIV-specific CD8⁺ T cells were similar to reports in adults undergoing early infection. Unlike adults, at 23–24 months post-infection a high frequency of HIV-specific CD8⁺ T cells expressed HLA-DR (mean 80%, range 68–85%) and CD95 (mean 88%, range 79–96%), suggesting sustained activation and vulnerability to apoptosis. Despite comparable expansion of HIV-specific CD8⁺ T cells of a similar phenotype to adults during early infection, infant T cells failed to contain HIV-1 replication, and remained persistently activated and vulnerable to apoptosis during chronic infection.     

"Negative vaccination" by specific CD4 T cell tolerisation enhances virus-specific protective antibody responses

Background

Cooperation of CD4⁺ T helper cells with specific B cells is crucial for protective vaccination against pathogens by inducing long-lived neutralizing antibody responses. During infection with persistence-prone viruses, prolonged virus replication correlates with low neutralizing antibody responses. We recently described that a viral mutant of lymphocytic choriomeningitis virus (LCMV), which lacks a T helper epitope, counterintuitively induced an enhanced protective antibody response. Likewise, partial depletion of the CD4⁺ T cell compartment by using anti-CD4 antibodies enhanced protective antibodies.

Principal Findings

Here we have developed a protocol to selectively reduce the CD4⁺ T cell response against viral CD4⁺ T cell epitopes. We demonstrate that in vivo treatment with LCMV-derived MHC-II peptides induced non-responsiveness of specific CD4⁺ T cells without affecting CD4⁺ T cell reactivity towards other antigens. This was associated with accelerated virus-specific neutralizing IgG-antibody responses. In contrast to a complete absence of CD4⁺ T cell help, tolerisation did not impair CD8⁺ T cell responses.

Conclusions

This result reveals a novel “negative vaccination” strategy where specific CD4⁺ T cell unresponsiveness may be used to enhance the delayed protective antibody responses in chronic virus infections.     

Local induction of immunosuppressive CD8+ T cells in the gut-associated lymphoid tissues

Background

In contrast to intestinal CD4⁺ regulatory T cells (T_regs), the generation and function of immunomodulatory intestinal CD8⁺ T cells is less well defined. To dissect the immunologic mechanisms of CD8⁺ T cell function in the mucosa, reactivity against hemagglutinin (HA) expressed in intestinal epithelial cells of mice bearing a MHC class-I-restricted T-cell-receptor specific for HA was studied.

Methodology and Principal Findings

HA-specific CD8⁺ T cells were isolated from gut-associated tissues and phenotypically and functionally characterized for the expression of Foxp3⁺ and their suppressive capacity. We demonstrate that intestinal HA expression led to peripheral induction of HA-specific CD8⁺Foxp3⁺ T cells. Antigen-experienced CD8⁺ T cells in this transgenic mouse model suppressed the proliferation of CD8⁺ and CD4⁺ T cells in vitro. Gene expression analysis of suppressive HA-specific CD8⁺ T cells revealed a specific up-regulation of CD103, Nrp1, Tnfrsf9 and Pdcd1, molecules also expressed on CD4⁺ T_reg subsets. Finally, gut-associated dendritic cells were able to induce HA-specific CD8⁺Foxp3⁺ T cells.

Conclusion and Significance

We demonstrate that gut specific antigen presentation is sufficient to induce CD8⁺ T_regs in vivo which may maintain intestinal homeostasis by down-modulating effector functions of T cells.     

Cell-to-cell interactions and signals involved in the reconstitution of peripheral CD8 T(CM) and T(EM) cell pools

We here describe novel aspects of CD8⁺ and CD4⁺ T cell subset interactions that may be clinically relevant and provide new tools for regulating the reconstitution of the peripheral CD8⁺ T cell pools in immune-deficient states. We show that the reconstitution capacity of transferred isolated naïve CD8⁺ T cells and their differentiation of effector functions is limited, but both dramatically increase upon the co-transfer of CD4⁺ T cells. This helper effect is complex and determined by multiple factors. It was directly correlated to the number of helper cells, required the continuous presence of the CD4⁺ T cells, dependent on host antigen-presenting cells (APCs) expressing CD40 and on the formation of CD4/CD8/APC cell clusters. By comparing the recovery of (CD44⁺CD62L^high) T_CM and (CD44⁺CD62L^low) T_EM CD8⁺ T cells, we found that the accumulation of T_CM and T_EM subsets is differentially regulated. T_CM-cell accumulation depended mainly on type I interferons, interleukin (IL)-6, and IL-15, but was independent of CD4⁺ T-cell help. In contrast, T_EM-cell expansion was mainly determined by CD4⁺ T-cell help and dependent on the expression of IL-2Rβ by CD8 cells, on IL-2 produced by CD4⁺ T-cells, on IL-15 and to a minor extent on IL-6.     

Expression of Nef downregulates CXCR4, the major coreceptor of human immunodeficiency virus, from the surfaces of target cells and thereby enhances resistance to superinfection

Lentiviral Nef proteins are key factors for pathogenesis and are known to downregulate functionally important molecules, including CD4 and major histocompatibility complex class I (MHC-I), from the surfaces of infected cells. Recently, we demonstrated that Nef reduces cell surface levels of the human immunodeficiency virus type 1 (HIV-1) entry coreceptor CCR5 (N. Michel, I. Allespach, S. Venzke, O. T. Fackler, and O. T. Keppler, Curr. Biol. 15:714-723, 2005). Here, we report that Nef downregulates the second major HIV-1 coreceptor, CXCR4, from the surfaces of HIV-infected primary CD4 T lymphocytes with efficiencies comparable to those of the natural CXCR4 ligand, stromal cell-derived factor-1 alpha. Analysis of a panel of mutants of HIV-1(SF2) Nef revealed that the viral protein utilized the same signature motifs for downmodulation of CXCR4 and MHC-I, including the proline-rich motif P(73)P(76)P(79)P(82) and the acidic cluster motif E(66)E(67)E(68)E(69.) Expression of wild-type Nef, but not of specific Nef mutants, resulted in a perinuclear accumulation of the coreceptor. Remarkably, the carboxy terminus of CXCR4, which harbors the classical motifs critical for basal and ligand-induced receptor endocytosis, was dispensable for the Nef-mediated reduction of surface exposure. Functionally, the ability of Nef to simultaneously downmodulate CXCR4 and CD4 correlated with maximum-level protection of Nef-expressing target cells from fusion with cells exposing X4 HIV-1 envelopes. Furthermore, the Nef-mediated downregulation of CXCR4 alone on target T lymphocytes was sufficient to diminish cells' susceptibility to X4 HIV-1 virions at the entry step. The downregulation of chemokine coreceptors is a conserved activity of Nef to modulate infected cells, an important functional consequence of which is an enhanced resistance to HIV superinfection.     

Human immunodeficiency virus type 1 inhibits DNA damage-triggered apoptosis by a Nef-independent mechanism

It is controversial whether the accessory human immunodeficiency virus type 1 (HIV-1) Nef protein inhibits or enhances apoptosis. To address this issue, we investigated the effect of Nef on programmed cell death with vectors or proviral HIV-1 constructs coexpressing Nef and green fluorescent protein from single bicistronic RNAs. This approach allows us to readily identify transfected or infected cells and to correlate cell death directly with Nef expression levels. We demonstrate that Nef does not significantly affect apoptosis in transfected or HIV-1-infected Jurkat T cells or primary human peripheral blood mononuclear cells. Unexpectedly, however, both nef⁺ and nef-defective HIV-1 infection blocked apoptosis in cells treated with UV light or etoposide but not cell death induced by CD95 antibody, TRAIL, Ly294002, or serum starvation. Our results show that HIV-1 infection inhibits DNA damage-induced but not death receptor-dependent cell death by a Nef-independent mechanism.     

Association of Nef with p21-activated kinase 2 is dispensable for efficient human immunodeficiency virus type 1 replication and cytopathicity in ex vivo-infected human lymphoid tissue

Interaction of the human immunodeficiency virus type 1 (HIV-1) Nef protein with p21-activated kinase 2 (PAK2) has been proposed to play a role in T-cell activation, viral replication, apoptosis, and progression to AIDS. However, these hypotheses were based on results obtained using Nef mutants impaired in multiple functions. Recently, it was reported that Nef residue F191 is specifically involved in PAK2 binding. However, only a limited number of Nef activities were investigated in these studies. To further evaluate the role of F191 in Nef function and to elucidate the biological relevance of Nef-PAK2 interaction, we performed a comprehensive analysis of HIV-1 Nef mutants carrying F191H and F191R mutations. We found that the F191H mutation reduces and the F191R mutation disrupts the association of Nef with PAK2. Both mutants upregulated the major histocompatibility complex II (MHC-II)-associated invariant chain and downregulated CD4, MHC-I, and CD28, although with reduced efficiency for the latter. Furthermore, the F191H/R changes neither affected the levels of interleukin-2 receptor expression and apoptosis of HIV-1-infected primary T cells nor reduced Nef-mediated induction of NFAT. Unexpectedly, the F191H change markedly reduced and the F191R mutation disrupted the ability of Nef to enhance virion infectivity in P4-CCR5 indicator cells but not in TZM-bl cells or peripheral blood mononuclear cells. Most importantly, all HIV-1 Nef mutants replicated efficiently and caused CD4⁺ T-cell depletion in ex vivo-infected human lymphoid tissue. Altogether, our data show that the interaction of Nef with PAK2 does not play a major role in T-cell activation, viral replication, and apoptosis.     

miR‐142‐3p restricts cAMP production in CD4+CD25− T cells and CD4+CD25+ TREG cells by targeting AC9 mRNA

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates diverse cellular functions. It has been found that CD4⁺CD25⁺ regulatory T (T_REG) cells exert their suppressor function by transferring cAMP to responder T cells. Here, we show that miR-142-3p regulates the production of cAMP by targeting adenylyl cyclase (AC) 9 messenger RNA in CD4⁺CD25⁻ T cells and CD4⁺CD25⁺ T_REG cells. miR-142-3p limits the level of cAMP in CD4⁺CD25⁻ T cells by inhibiting AC9 production, whereas forkhead box P3 (FOXP3) downregulates miR-142-3p to keep the AC9/cAMP pathway active in CD4⁺CD25⁺ T_REG cells. These findings reveal a new molecular mechanism through which CD4⁺CD25⁺ T_REG cells contain a high level of cAMP for their suppressor function, and also suggest that the microRNA controlling AC expression might restrict the final level of cAMP in various types of cells.