SUMMARYNF-κB is essential for effective transcription of primate lentiviral genomes and also activates antiviral host genes. Here, we show that the early protein Nef of most primate lentiviruses enhances NF-κB activation. In contrast, the late protein Vpu of HIV-1 and its simian precursors inhibits activation of NF-κB, even in the presence of Nef. Although this effect of Vpu did not correlate with its ability to interact with β-TrCP, it involved the stabilization of IκB and reduced nuclear translocation of p65. Interestingly, however, Vpu did not affect casein kinase II-mediated phosphorylation of p65. Lack of Vpu was associated with increased NF-κB activation and induction of interferon and interferon-stimulated genes (ISGs) in HIV-1-infected T cells. Thus, HIV-1 and its simian precursors employ Nef to boost NF-κB activation early during the viral life cycle to initiate proviral transcription, while Vpu is used to downmodulate NF-κB-dependent expression of ISGs at later stages.

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Lentiviral Nefs recruit assembly polypeptide complexes and target sorting motifs in cellular receptors to induce their internalization. While Nef-mediated CD4 downmodulation is conserved, the ability to internalize CD3 was lost in HIV-1 and its precursors. Although both functions play key roles in lentiviral replication and pathogenicity, the underlying structural requirements are poorly defined. Here, we determine the structure of SIVmac239 Nef bound to the ExxxLM motif of another Nef molecule at 2.5 Å resolution. This provides a basis for a structural model, where a hydrophobic crevice in simian immunodeficiency virus (SIV) Nef targets a dileucine motif in CD4 and a tyrosine-based motif in CD3. Introducing key residues into this crevice of HIV-1 Nef enables CD3 binding but an additional N-terminal tyrosine motif is required for internalization. Our resolution of the CD4/Nef/AP2 complex and generation of HIV-1 Nefs capable of CD3 downregulation provide insights into sorting motif interactions and target discrimination of Nef.

As intracellular bacteria, chlamydiae block the apoptotic pathways of their host cells. However, the infection of epithelial cells causes the loss of cell membrane integrity and can result in nonapoptotic death. Normally, cells undergoing necrosis release high-mobility group box 1 protein (HMGB1) that acts as an important proinflammatory mediator. Here, we show that in Chlamydia trachomatis-infected HeLa cells HMGB1 is not translocated from the nucleus to the cytosol and not released from injured cells in increased amounts. At 48 h after infection, degradation of HMGB1 was observed. In infected cells, poly(ADP-ribose) polymerase 1 (PARP-1), a DNA repair enzyme that also regulates HMGB1 translocation, was found to be cleaved into fragments that correspond to a necrosislike pattern of PARP-1 degradation. Cell-free cleavage assays and immunoprecipitation using purified proteolytic fractions from infected cells demonstrated that the chlamydial-protease-like activity factor (CPAF) is responsible for the cleavage of both HMGB1 and PARP-1. Proteolytic cleavage of PARP-1 was accompanied by a significant decrease in the enzymatic activity in a time-dependent manner. The loss of PARP-1 function obviously affects the viability of Chlamydia-infected cells because silencing of PARP-1 in uninfected HeLa cells with specific small interfering RNA results in increased cell membrane permeability. Our findings suggest that the Chlamydia-specific protease CPAF interferes with necrotic cell death pathways. By the degradation of HMGB1 and PARP-1, the pathogen may have evolved a strategy to reduce the inflammatory response to membrane-damaged cells in vivo.

BackgroundThe presence of a vpx gene distinguishes HIV-2 from HIV-1, the main causative agent of AIDS. Vpx degrades the restriction factor SAMHD1 to boost HIV-2 infection of macrophages and dendritic cells and it has been suggested that the activation of antiviral innate immune responses after Vpx-dependent infection of myeloid cells may explain why most HIV-2-infected individuals efficiently control viral replication and become long-term survivors. However, the role of Vpx-mediated SAMHD1 antagonism in the virological and clinical outcome of HIV-2 infection remained to be investigated.ResultsHere, we analyzed the anti-SAMHD1 activity of vpx alleles derived from seven viremic and four long-term aviremic HIV-2-infected individuals. We found that effective Vpx-mediated SAMHD1 degradation and enhancement of myeloid cell infection was preserved in most HIV-2-infected individuals including all seven that failed to control the virus and developed AIDS. The only exception were vpx alleles from an aviremic individual that predicted a M68K change in a highly conserved nuclear localization signal which disrupted the ability of Vpx to counteract SAMHD1. We also found that HIV-2 is less effective than HIV-1 in inducing innate immune activation in dendritic cells.ConclusionsEffective immune control of viral replication in HIV-2-infected individuals is not associated with increased Vpx-mediated degradation of SAMHD1.

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Summary Simian immunodeficiency virus (SIVsmm) infection of sooty mangabeys (Cercocebus atys) is characterized by stable CD4+ T cell counts despite high plasma levels of CCR5-tropic viruses. However, in rare instances, SIVsmm acquires CXCR4 coreceptor tropism and causes severe CD4+ T cell depletion, albeit without clinical signs of immunodeficiency. Here, we show that CXCR4-tropic SIVsmm strains lost their ability to downmodulate TCR-CD3 by evolving unusual Nef mutations that initially reduced (I132V) and subsequently disrupted (I123L and L146F) interaction with the CD3 ζ chain. This coevolution of Env and Nef function suggests that CD3 downmodulation is advantageous for viral replication in activated CCR5+ memory T cells, but not in resting naive CXCR4+ T cells that have not yet undergone TCR-CD3-mediated stimulation. This may explain why HIV-1, which generally lacks the CD3 downmodulation function, commonly switches to CXCR4 usage, whereas this is extremely rare for SIV strains that have retained this Nef activity.

The role of the accessory viral Nef protein as a multifunctional manipulator of the host cell that is required for effective replication of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) in vivo is well established. It is unknown, however, whether Nef manipulates all or just specific subsets of CD4 ؉ T cells, which are the main targets of virus infection and differ substantially in their state of activation and importance for a functional immune system. Here, we analyzed the effect of Nef proteins differing in their T cell receptor (TCR)-CD3 downmodulation function in HIV-infected human lymphoid aggregate cultures and peripheral blood mononuclear cells. We found that Nef efficiently downmodulates TCR-CD3 in naive and memory CD4؉ T cells and protects the latter against apoptosis. In contrast, highly proliferative CD45RA ؉ CD45RO ؉ CD4 ؉ T cells were main producers of infectious virus but largely refractory to TCR-CD3 downmodulation. Such T cell subset-specific differences were also observed for Nef-mediated modulation of CD4 but not for enhancement of virion infectivity. Our results indicate that Nef predominantly modulates surface receptors on CD4 ؉ T cell subsets that are not already fully permissive for viral replication. As a consequence, Nef-mediated downmodulation of TCR-CD3, which distinguishes most primate lentiviruses from HIV type 1 (HIV-1) and its vpu-containing simian precursors, may promote a selective preservation of central memory CD4؉ T cells, which are critical for the maintenance of a functional immune system. IMPORTANCE The Nef proteins of human and simian immunodeficiency viruses manipulate infected CD4 ؉ T cells in multiple ways to promote viral replication and immune evasion in vivo.Here, we show that some effects of Nef are subset specific. Downmodulation of CD4 and TCR-CD3 is highly effective in central memory CD4؉ T cells, and the latter Nef function protects this T cell subset against apoptosis. In contrast, highly activated/proliferating CD4 ؉ T cells are largely refractory to receptor downmodulation but are main producers of infectious HIV-1. Nef-mediated enhancement of virion infectivity, however, was observed in all T cell subsets examined. Our results provide new insights into how primate lentiviruses manipulate their target cells and suggest that the TCR-CD3 downmodulation function of Nef may promote a selective preservation of memory CD4 ؉ T cells, which are critical for immune function, but has little effect on activated/proliferating CD4 ؉ T cells, which are the main targets for viral replication.