Semen-Derived Amyloid Fibrils Drastically Enhance HIV Infection

SummarySexual intercourse is the major route of HIV transmission. To identify endogenous factors that affect the efficiency of sexual viral transmission, we screened a complex peptide/protein library derived from human semen. We show that naturally occurring fragments of the abundant semen marker prostatic acidic phosphatase (PAP) form amyloid fibrils. These fibrils, termed Semen-derived Enhancer of Virus Infection (SEVI), capture HIV virions and promote their attachment to target cells, thereby enhancing the infectious virus titer by several orders of magnitude. Physiological concentrations of SEVI amplified HIV infection of T cells, macrophages, ex vivo human tonsillar tissues, and transgenic rats in vivo, as well as trans-HIV infection of T cells by dendritic or epithelial cells. Amyloidogenic PAP fragments are abundant in seminal fluid and boost semen-mediated enhancement of HIV infection. Thus, they may play an important role in sexual transmission of HIV and could represent new targets for its prevention

Tetherin-Driven Adaptation of Vpu and Nef Function and the Evolution of Pandemic and Nonpandemic HIV-1 Strains

Vpu proteins of pandemic HIV-1 M strains degrade the viral receptor CD4 and antagonize human tetherin to promote viral release and replication. We find that Vpus from SIVgsn, SIVmus and SIVmon infecting Cercopithecus primate species also degrade CD4 and antagonize tetherin. In contrast, SIVcpz, the immediate precursor of HIV-1, whose Vpu shares a common ancestry with SIVgsn/mus/mon Vpu, uses Nef rather than Vpu to counteract chimpanzee tetherin. Human tetherin, however, is resistant to Nef and thus poses a significant barrier to zoonotic transmission of SIVcpz to humans. Remarkably, Vpu from non-pandemic HIV-1 O strains are poor tetherin antagonists while those from the rare group N viruses do not degrade CD4. Thus, only HIV-1 M evolved a fully functional Vpu following the three independent cross-species transmissions that resulted in HIV-1 groups M, N, and O. This may explain why group M viruses are almost entirely responsible for the gobal HIV/AIDS pandemic

Inefficient Nef-Mediated Downmodulation of CD3 and MHC-I Correlates with Loss of CD4+ T Cells in Natural SIV Infection

Recent data suggest that Nef-mediated downmodulation of TCR-CD3 may protect SIVsmm-infected sooty mangabeys (SMs) against the loss of CD4+ T cells. However, the mechanisms underlying this protective effect remain unclear. To further assess the role of Nef in nonpathogenic SIV infection, we cloned nef alleles from 11 SIVsmm-infected SMs with high (>500) and 15 animals with low (<500) CD4+ T-cells/µl in bulk into proviral HIV-1 IRES/eGFP constructs and analyzed their effects on the phenotype, activation, and apoptosis of primary T cells. We found that not only efficient Nef-mediated downmodulation of TCR-CD3 but also of MHC-I correlated with preserved CD4+ T cell counts, as well as with high numbers of Ki67+CD4+ and CD8+CD28+ T cells and reduced CD95 expression by CD4+ T cells. Moreover, effective MHC-I downregulation correlated with low proportions of effector and high percentages of naïve and memory CD8+ T cells. We found that T cells infected with viruses expressing Nef alleles from the CD4low SM group expressed significantly higher levels of the CD69, interleukin (IL)-2 and programmed death (PD)-1 receptors than those expressing Nefs from the CD4high group. SIVsmm Nef alleles that were less active in downmodulating TCR-CD3 were also less potent in suppressing the activation of virally infected T cells and subsequent cell death. However, only nef alleles from a single animal with very low CD4+ T cell counts rendered T cells hyper-responsive to activation, similar to those of HIV-1. Our data suggest that Nef may protect the natural hosts of SIV against the loss of CD4+ T cells by at least two mechanisms: (i) downmodulation of TCR-CD3 to prevent activation-induced cell death and to suppress the induction of PD-1 that may impair T cell function and survival, and (ii) downmodulation of MHC-I to reduce CTL lysis of virally infected CD4+ T cells and/or bystander CD8+ T cell activation

Metabolic Turnover of Synaptic Proteins: Kinetics, Interdependencies and Implications for Synaptic Maintenance

Chemical synapses contain multitudes of proteins, which in common with all proteins, have finite lifetimes and therefore need to be continuously replaced. Given the huge numbers of synaptic connections typical neurons form, the demand to maintain the protein contents of these connections might be expected to place considerable metabolic demands on each neuron. Moreover, synaptic proteostasis might differ according to distance from global protein synthesis sites, the availability of distributed protein synthesis facilities, trafficking rates and synaptic protein dynamics. To date, the turnover kinetics of synaptic proteins have not been studied or analyzed systematically, and thus metabolic demands or the aforementioned relationships remain largely unknown. In the current study we used dynamic Stable Isotope Labeling with Amino acids in Cell culture (SILAC), mass spectrometry (MS), Fluorescent Non-Canonical Amino acid Tagging (FUNCAT), quantitative immunohistochemistry and bioinformatics to systematically measure the metabolic half-lives of hundreds of synaptic proteins, examine how these depend on their pre/postsynaptic affiliation or their association with particular molecular complexes, and assess the metabolic load of synaptic proteostasis. We found that nearly all synaptic proteins identified here exhibited half-lifetimes in the range of 2-5 days. Unexpectedly, metabolic turnover rates were not significantly different for presynaptic and postsynaptic proteins, or for proteins for which mRNAs are consistently found in dendrites. Some functionally or structurally related proteins exhibited very similar turnover rates, indicating that their biogenesis and degradation might be coupled, a possibility further supported by bioinformatics-based analyses. The relatively low turnover rates measured here (∼0.7% of synaptic protein content per hour) are in good agreement with imaging-based studies of synaptic protein trafficking, yet indicate that the metabolic load synaptic protein turnover places on individual neurons is very substantial

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

Nef-mediated primate lentiviral immune evasion mechanisms

One of the best established and conserved Nef activities between primate lentiviruses is the downmodulation of human major histocompatibility complex class I (MHC-I) molecules. HIV-1 Nef is capable of selectively downmodulating human leucocyte antigen (HLA)-A and -B, but not -C and -E molecules to evade both cytotoxic T lymphocyte (CTL) and natural killer (NK) cell surveillance. This selectivity is based on amino acid differences in cytoplasmic domains of MHC-I molecules. It remained elusive, however, whether the ability of specific MHC-I modulation is conserved between HIV and its simian precursors as well as in different lentiviral lineages and in recently, naturally or experimentally infected hosts. Previously it has been shown that genetic host factors, like a single-nucleotide polymorphism (SNP), rs9264942, which is located upstream of the HLA-C gene (HLA-C SNP) are associated with a better immune control, leading to low viral loads (VLs) in HIV-1-infected patients. HIV-1 is unable to remove HLA-C from the surface of infected cells to prevent HLA-C-mediated antigen presentation to CTLs. However, adaptive evolution of HIV-1 to its host allows the virus to "learn" how to efficiently persist in some HLA-C SNP individuals, causing high VLs. Thus, one goal of this study was to determine which adaptive changes in Nef occur in these patients. The present thesis shows that primate lentiviral Nefs efficiently manipulate the immune system in different hosts by the important and highly conserved mechanism of specific MHC-I downregulation to avoid CTL and NK cell lysis. Genetic host factors, like a HLA-C SNP, can disrupt the balance between both immune evasion mechanisms, leading to an improved immune control of the virus. However, high VLs are also observed in HLA-C SNP individuals and are associated with the emergence of Nef variants, which manipulate the antigen presenting process by indirect mechanisms rather than by downregulating HLA-C. Thus, the selective pressure in vivo to avoid the removal of HLA-C from the surface of infected cells, even in hosts where this might be beneficial, supports the potency of antiviral therapies aiming to induce HLA-C-mediated immune responses to achieve a better control of viral replication

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