Anke Specht

Semen-Derived Amyloid Fibrils Drastically Enhance HIV Infection

Sexual 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 show 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, Vpus from nonpandemic HIV-1 O strains are poor tetherin antagonists, whereas 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 global HIV/AIDS pandemic.

Role of Nef in primate lentiviral immunopathogenesis

Abstract.More than a decade ago it was established that intact nef genes are critical for efficient viral persistence and greatly accelerate disease progression in SIVmac-infected rhesus macaques and in HIV-1-infected humans. Subsequent studies established a striking number of Nef functions that evidently contribute to the maintenance of high viral loads associated with the development of immunodeficiency in the ‘evolutionary-recent’ human and the experimental macaque hosts. Recent data show that many Nef activities are conserved across different lineages of HIV and SIV. However, some differences also exist. For example, Nef alleles from most SIVs that do not cause disease in their natural monkey hosts, but not those of HIV-1 and its simian precursors, down-modulate TCR-CD3 to suppress T cell activation and programmed death. This evolutionary loss of a specific Nef function may contribute to the high virulence of HIV-1 in humans.

Vpu serine 52 dependent counteraction of tetherin is required for HIV-1 replication in macrophages, but not in ex vivo human lymphoid tissue

BackgroundThe human immunodeficiency virus type 1 (HIV-1) Vpu protein degrades CD4 and counteracts a restriction factor termed tetherin (CD317; Bst-2) to enhance virion release. It has been suggested that both functions can be genetically separated by mutation of a serine residue at position 52. However, recent data suggest that the S52 phosphorylation site is also important for the ability of Vpu to counteract tetherin. To clarify this issue, we performed a comprehensive analysis of HIV-1 with a mutated casein kinase-II phosphorylation site in Vpu in various cell lines, primary blood lymphocytes (PBL), monocyte-derived macrophages (MDM) and ex vivo human lymphoid tissue (HLT).ResultsWe show that mutation of serine 52 to alanine (S52A) entirely disrupts Vpu-mediated degradation of CD4 and strongly impairs its ability to antagonize tetherin. Furthermore, casein-kinase II inhibitors blocked the ability of Vpu to degrade tetherin. Overall, Vpu S52A could only overcome low levels of tetherin, and its activity decreased in a manner dependent on the amount of transiently or endogenously expressed tetherin. As a consequence, the S52A Vpu mutant virus was unable to replicate in macrophages, which express high levels of this restriction factor. In contrast, HIV-1 Vpu S52A caused CD4+ T-cell depletion and spread efficiently in ex vivo human lymphoid tissue and PBL, most likely because these cells express comparably low levels of tetherin.ConclusionOur data explain why the effect of the S52A mutation in Vpu on virus release is cell-type dependent and suggest that a reduced ability of Vpu to counteract tetherin impairs HIV-1 replication in macrophages, but not in tissue CD4+ T cells.

Nef-Mediated Enhancement of Virion Infectivity and Stimulation of Viral Replication Are Fundamental Properties of Primate Lentiviruses

ABSTRACT Nef is a multifunctional accessory protein of primate lentiviruses. Recently, it has been shown that the ability of Nef to downmodulate CD4, CD28, and class I major histocompatibility complex is highly conserved between most or all primate lentiviruses, whereas Nef-mediated downregulation of T-cell receptor-CD3 was lost in the lineage that gave rise to human immunodeficiency virus type 1 (HIV-1). Whether or not other Nef activities are preserved between different groups of primate lentiviruses remained to be determined. Here, we show that nef genes from a large variety of HIVs and simian immunodeficiency viruses (SIVs) enhance virion infectivity and stimulate viral replication in human cells and/or in ex vivo infected human lymphoid tissue (HLT). Notably, nef alleles from unpassaged SIVcpz and SIVsmm enhanced viral infectivity, replication, and cytopathicity in cell culture and in ex vivo infected HLT as efficiently as those from HIV-1 and HIV-2, their human counterparts. Furthermore, nef genes from several highly divergent SIVs that have not been found in humans were also highly active in human cells and/or tissues. Thus, most primate lentiviral Nefs enhance virion infectivity and stimulate viral replication. Moreover, our data show that SIVcpz and SIVsmm Nefs do not require adaptive changes to perform these functions in human cells or tissues and support the idea that nef alleles from other primate lentiviruses would also be capable of promoting efficient virus spread in humans.

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.

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

ABSTRACT 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.

Counteraction of HLA-C-mediated immune control of HIV-1 by Nef

ABSTRACT A host genetic variant (−35C/T) correlates with increased human leukocyte antigen C (HLA-C) expression and improved control of HIV-1. HLA-C-mediated immunity may be particularly protective because HIV-1 is unable to remove HLA-C from the cell surface, whereas it can avoid HLA-A- and HLA-B-mediated immunity by Nef-mediated down-modulation. However, some individuals with the protective −35CC genotype exhibit high viral loads. Here, we investigated whether the ability of HIV-1 to replicate efficiently in the “protective” high-HLA-C-expression host environment correlates with specific functional properties of Nef. We found that high set point viral loads (sVLs) were not associated with the emergence of Nef variants that had acquired the ability to down-modulate HLA-C or were more effective in removing HLA-A and HLA-B from the cell surface. However, in individuals with the protective −35CC genotype we found a significant association between sVLs and the efficiency of Nef-mediated enhancement of virion infectivity and modulation of CD4, CD28, and the major histocompatibility complex class II (MHC-II)-associated invariant chain (Ii), while this was not observed in subjects with the −35TT genotype. Since the latter Nef functions all influence the stimulation of CD4+ T helper cells by antigen-presenting cells, they may cooperate to affect both the activation status of infected T cells and the generation of an antiviral cytotoxic T-lymphocyte (CTL) response. In comparison, different levels of viremia in individuals with the common −35TT genotype were not associated with differences in Nef function but with differences in HLA-C mRNA expression levels. Thus, while high HLA-C expression may generally facilitate control of HIV-1, Nef may counteract HLA-C-mediated immune control in some individuals indirectly, by manipulating T-cell function and MHC-II antigen presentation.

Selective Downregulation of Rhesus Macaque and Sooty Mangabey Major Histocompatibility Complex Class I Molecules by Nef Alleles of Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 2

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Nef downregulates HLA-A and -B molecules, but not HLA-C or -E molecules, based on amino acid differences in their cytoplasmic domains to simultaneously evade cytotoxic T lymphocyte (CTL) and natural killer cell surveillance. Rhesus macaques and sooty mangabeys express orthologues of HLA-A, -B, and -E, but not HLA-C, and many of these molecules have unique amino acid differences in their cytoplasmic tails. We found that these differences also resulted in differential downregulation by primary simian immunodeficiency virus (SIV) SIVsmm/mac and HIV-2 Nef alleles. Thus, selective major histocompatibility complex class I downregulation is a conserved mechanism of immune evasion for pathogenic SIV infection of rhesus macaques and nonpathogenic SIV infection of sooty mangabeys.

Calcium-modulating cyclophilin ligand does not restrict retrovirus release

To the Editor: The HIV-1 accessory protein Vpu is required for efficient viral release from certain cell lines (termed nonpermissive) but not from others (termed permissive). Neil et al.1 showed that restricted viral release in nonpermissive cells is due to expression of the cellular protein tetherin (also called BST2, CD317 or HM1.24), which tethers viral particles to the cell surface by an unknown mechanism. Tetherindependent sequestration of viral particles is counteracted by Vpu1, in a process that is species specific2–6 and seems to encompass Vpumediated targeting of tetherin for degradation2,7. Therefore, a precise understanding of how tetherin inhibits viral release and how this release-restricting activity is counteracted by Vpu might identify new targets for antiviral intervention. Recently, Varthakavi et al.8 showed that calcium-modulating cyclophilin ligand (CAML) exerts antiretroviral effects largely identical to those reported for tetherin. Specifically, CAML was shown to restrict HIV-1 and murine leukemia virus (MLV) release in a Vpu-sensitive manner8. These results raise the intriguing possibility that CAML crucially contributes to the inhibition of retroviral release in nonpermissive cells, previously attributed exclusively to tetherin expression. To better understand the potential role of CAML in retrovirus release, we examined whether endogenous expression of CAML (encoded by CAMLG) in cell lines correlates with the nonpermissive phenotype for viral release (Fig. 1). We detected robust expression of tetherin messenger RNA and protein (encoded by BST2) (Fig. 1a–c) in HeLa (human cervix adenocarcinoma–derived cell line), CEMx174 (human T-B cell hybrid) and C8166-SEAP (human CD4+ lymphocyte cell line) cells, which we found to be nonpermissive (Fig. 1e), in agreement with previously reported results1,9. Conversely, we did not detect tetherin mRNA and protein in 293T cells (Fig. 1a–c), which were permissive (Fig. 1e), in concordance with published observations1,10. In stark contrast, CAML mRNA and protein (Fig. 1a,d) were ubiquitously expressed among all cell lines tested, and relative expression did not correlate with the requirement for Vpu for efficient release (Fig. 1a,d,e), indicating that expression of tetherin but not CAML is predictive of the virus release phenotype. In this context, it is noteworthy that for some cell lines the amount of tetherin expression at the cell surface did not reflect tetherin abundance in cell lysates (Fig. 1b,c), whereas the efficiency of tetherin surface expression generally correlated well with the efficiency of HIV-1 release restriction (Fig. 1b,e). Finally, we asked whether expression of CAML can be induced by interferon-α and might thus be upregulated during an antiviral response. Treatment of 293T and HeLa cells with interferon-α had no effect on CAML expression but induced tetherin expression in 293T cells (Supplementary Fig. 1), as expected1,10, suggesting that tetherin but not CAML is induced as a defense against viral infection. C8 16 6SE AP a b Unstained Isotype Tetherin