Frank Kirchhoff

Two elements target SIV Nef to the AP-2 clathrin adaptor complex, but only one is required for the induction of CD4 endocytosis.

The simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV‐1) Nef proteins induce the endocytosis of CD4 and class I MHC molecules. Here we show that SIV Nef interacts with the AP‐2 adaptor complex via two elements located in the N‐terminal region of the Nef molecule, but only the N‐distal element is required to induce CD4 endocytosis. This N‐distal AP‐2 targeting element contains no canonical endocytic signals and probably contacts the AP‐2 complex via a novel interaction surface. The data support a model where SIV Nef induces CD4 endocytosis by promoting the normal interactions between the di‐leucine sorting signal in the CD4 cytoplasmic domain and AP‐2, but does not substitute for the CD4–AP‐2 adaptor interaction. Neither element is important for the induction of class I MHC endocytosis, thus indicating that different mechanisms underlie the induction of class I MHC and CD4 endocytosis by Nef. In contrast to SIV Nef, HIV‐1 Nef interacts with AP‐2 via a surface containing a di‐leucine endocytosis signal in the C‐terminal disordered loop of Nef. The fact that genetic selection maintains similar molecular interactions via different surfaces in SIV and HIV‐1 Nef proteins indicates that these interactions have critical roles for the viral life cycle in vivo.

Simian and Human Immunodeficiency Virus Nef Proteins Use Different Surfaces To Downregulate Class I Major Histocompatibility Complex Antigen Expression

ABSTRACT Simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) Nef proteins are related regulatory proteins that share several functions, including the ability to downregulate class I major histocompatibility complex (MHC) and CD4 expression on the cell surface and to alter T-cell-receptor-initiated signal transduction in T cells. We compared the mechanisms used by SIV mac239 Nef and HIV-1 Nef to downregulate class I MHC and found that the ability of SIV Nef to downregulate class I MHC requires a unique C-terminal region of the SIV mac239 Nef molecule which is not found in HIV-1 Nef. Interestingly, mutation of the PxxP motif in SIV Nef, unlike in HIV-1 Nef, does not affect class I MHC downregulation. We also found that downregulation of class I MHC by SIV Nef requires a conserved tyrosine in the cytoplasmic domain of the class I MHC heavy chain and involves accelerated endocytosis of class I complexes, as previously found with HIV-1 Nef. Thus, while SIV and HIV-1 Nef proteins use a similar mechanism to downregulate class I MHC expression, they have evolved different surfaces for molecular interactions with cell factors that regulate class I MHC traffic. Mutations in the C-terminal domain of SIV mac239 Nef selectively disrupt class I MHC downregulation, having no detectable effect on other functions of Nef, such as the downregulation of CD4 and CD3 surface expression, the stimulation of SIV virion infectivity, and the induction of SIV replication from T cells infected in the absence of stimulation. The resulting mutants will be useful reagents for studying the importance of class I MHC downregulation for SIV replication and AIDS pathogenesis in infected rhesus macaques.

Coreceptor usage of BOB/GPR15 and Bonzo/STRL33 by primary isolates of human immunodeficiency virus type 1.

Primary isolates of human and simian immunodeficiency viruses (HIV and SIV) use the chemokine receptor CCR5, in association with CD4, as coreceptor. During AIDS progression, HIV-1 and HIV-2 often adapt to use additional cofactors, particularly CXCR4. In contrast, SIV isolates do not use CXCR4, but other coreceptors such as BOB/GPR15 and Bonzo/STRL33. Only limited information is currently available on usage of BOB/GPR15 and Bonzo/STRL33 by HIV-1. Therefore, we investigated a panel of gp160 clones from 15 primary isolates, representing 5 different subtypes, for utilization of these cofactors. The majority of HIV-1 envelopes mediated entry into BOB/GPR15-expressing cells, albeit often with low efficiency. Usage of Bonzo/STRL33 was less common and usually inefficient. To investigate if HIV-1 entry via these orphan receptors is sufficient to allow virus replication, 15 uncloned primary HIV-1 isolates and 7 molecular clones were used to infect target cells expressing CD4 and Bonzo/STRL33 or BOB/GPR15. Three primary isolates and two molecular clones replicated efficiently in cells expressing BOB/GPR15. Two of these isolates were X4-tropic, two were R5X4-tropic and one was R5-tropic. In contrast, none of the HIV-1 variants showed significant levels of replication in Bonzo/STRL33-expressing cells. Our data show that some HIV-1 isolates of different genetic subtype and of different biological phenotype use BOB/GPR15 for productive infection and suggest that this cofactor may play a role in HIV-1 pathogenesis and transmission.

Long-Term Nonprogressive Infection with Human Immunodeficiency Virus Type 1 in a Hemophilia Cohort

Seven long-term nonprogressors (LTNPs) have been identified in a cohort of 128 human immunodeficiency virus (HIV)-1 infected individuals with hemophilia. Studies included quantitation of virus by polymerase chain reaction, characterization of primary virus isolates in vitro, analysis of lymphocyte surface markers, and measurement of virus-specific cytotoxic T lymphocytes (CTLs). Viruses of LTNPs exhibited slow growth in vivo and in vitro. LTNPs had expansion of CD8 T cells with increased expression of HLA-DR. Intermittent HIV-1-specific CTL effector activity was detected in freshly isolated peripheral blood mononuclear cells of most LTNPs. CTL precursor frequencies were higher in LTNPs than in patients with progressive disease. Virus antigen-specific lymphoproliferation was vigorous in some LTNPs. Thus, LTNPs in this cohort have maintained remarkably low virus burdens and vigorous HIV-1-specific cell-mediated immunity over a 15-year period. The presence of expanded, activated CD8 T cells with cytotoxic effector function in the peripheral blood suggests ongoing viral replication.

Co-receptor Usage of BOB/GPR15 in Addition to CCR5 Has No Significant Effect on Replication of Simian Immunodeficiency Virus In Vivo

Human immunodeficiency virus type 2 (HIV-2) and the closely related simian immunodeficiency viruses (SIVs) frequently use the orphan receptor BOB/GPR15 in addition to the chemokine receptor CCR5 for efficient entry and replication. However, the role of BOB/GPR15 in replication and pathogenesis of HIV-2 and SIV in vivo is unclear. This study shows that a single amino acid substitution in the V3 loop of the pathogenic SIVmac239 clone, 321P-->S, impaired the ability to use BOB/GPR15 for entry and replication but had little effect on the ability to use CCR5. This envelope variant replicated with an efficiency comparable with the parental SIVmac239 isolate in rhesus macaques. Furthermore, the mutant genotype and phenotype remained stable even after the onset of immunodeficiency. These results suggest that this cofactor plays only a minor role for the pathogenicity of the HIV-2/SIVmac/SIVsm group of primate lentiviruses.

Partial repair of defective NEF genes in a long-term nonprogressor with human immunodeficiency virus type 1 infection

A 36-bp deletion close to the 5 end of NEF that impaired Nef function was found in a long-term nonprogressor with human immunodeficiency virus type 1 (HIV-1) infection. Forms containing an adjacent duplication of 33 bp were also frequently observed. The duplication showed no homology to the deleted region but restored the overall length of the first variable loop of Nef. NEF alleles carrying the duplication were active in class I major histocompatibility complex (MHC-I) down-modulation and enhancement of virus infectivity. However, they showed little activity in CD4 down-regulation and were unable to stimulate viral replication in human peripheral blood mononuclear cells. Our study indicates that the enhancement of virion infectivity and the stimulation of HIV-1 replication in lymphocytes are distinct functions of Nef. Our findings also illustrate the capacity for repair of attenuating deletions in HIV-1 infection and suggest that a selective pressure for Nef-mediated MHC-I down-modulation and/or enhancement of virion infectivity exists.

Simian Immunodeficiency Virus Containing Mutations in N-Terminal Tyrosine Residues and in the PxxP Motif in Nef Replicates Efficiently in Rhesus Macaques

ABSTRACT SIVmac Nef contains two N-terminal tyrosines that were proposed to be part of an SH2-ligand domain and/or a tyrosine-based endocytosis signal and a putative SH3-ligand domain (P104xxP107). In the present study, we investigated the effects of combined mutations in these tyrosine and proline residues on simian immunodeficiency virus (SIV) Nef interactions with the cellular signal transduction and endocytic machinery. We found that mutation of Y28F, Y39F, P104A, and P107A (FFAA-Nef) had little effect on Nef functions such as the association with the cellular tyrosine kinase Src, downregulation of cell surface expression of CD4 and class I major histocompatibility complex, and enhancement of virion infectivity. However, mutations in the PxxP sequence reduced the ability of Nef to stimulate viral replication in primary lymphocytes. Three macaques infected with the SIVmac239 FFAA-Nef variant showed high viral loads during the acute phase of infection. Reversions in the mutated prolines were observed between 12 and 20 weeks postinfection. Importantly, reversion of A107→P, which restored the ability of Nef to coprecipitate a 62-kDa phosphoprotein in in vitro kinase assays, did not precede the development of a high viral load. The Y28/Y39→F28/F39substitutions did not revert. In conclusion, mutations in both the tyrosine residues and the putative SH3 ligand domain apparently do not disrupt major aspects of SIV Nef function in vivo.

Natural SIV Infection

Publisher Summary nThis chapter aims to provide a brief overview of the distribution and biological properties of primate lentiviruses and emergence of the human immunodeficiency viruses (HIV). The discovery that HIV and acquired immune deficiency syndrome (AIDS) are the result of relatively recent zoonotic transmissions of simian immunodeficiency virus (SIVs) from African nonhuman primates (NHPs) to humans, aroused a great deal of interest in natural primate lentiviral infections. Effective noninvasive methodologies for the detection and analysis of SIV-specific antibodies and virion RNA in fecal and urine samples of wild NHPs have been developed. These methods have allowed large-scale seroepidemiological and molecular studies of wild primate populations and yielded key insights into the genetic diversity and natural history of primate lentiviruses. SIVs or SIV-specific antibodies have been detected in more than 40 different African NHPs. HIV and SIV belong to the genus of lentiviruses and are generally referred to as “immunodeficiency” viruses.Publisher Summary This chapter aims to provide a brief overview of the distribution and biological properties of primate lentiviruses and emergence of the human immunodeficiency viruses (HIV). The discovery that HIV and acquired immune deficiency syndrome (AIDS) are the result of relatively recent zoonotic transmissions of simian immunodeficiency virus (SIVs) from African nonhuman primates (NHPs) to humans, aroused a great deal of interest in natural primate lentiviral infections. Effective noninvasive methodologies for the detection and analysis of SIV-specific antibodies and virion RNA in fecal and urine samples of wild NHPs have been developed. These methods have allowed large-scale seroepidemiological and molecular studies of wild primate populations and yielded key insights into the genetic diversity and natural history of primate lentiviruses. SIVs or SIV-specific antibodies have been detected in more than 40 different African NHPs. HIV and SIV belong to the genus of lentiviruses and are generally referred to as “immunodeficiency” viruses.

VIRIP: Ein natürlicher HIV-Hemmstoff mit einem neuartigen Wirkmechanismus

Es ist seit langem bekannt, dass eine Reihe von korpereigenen Verbindungen die Vermehrung von HIV — zumindest teilweise — unterdrucken kann. Beispielsweise fuhrte die Entdeckung, dass Chemokine die Korezeptoren von HIV binden und dadurch die virale Infektion verhindern, zur Entwicklung einer ganzen Reihe von neuen Inhibitoren, die verschiedene Schritte des Eintritts von HIV in die Wirtszelle blockieren (Moore u. Stevenson 2000; Ray u. Doms 2006). Haufig erwies es sich allerdings als schwierig, antivirale Verbindungen in menschlichen Geweben und Korperflussigkeiten aufzureinigen und zu charakterisieren. So ist auch 20 Jahre nach der Erstbeschreibung unklar, welcher antivirale Faktor von CD8+-T Zellen sekretiert wird (Walker et al. 1986), obwohl bei der Suche danach zahlreiche andere antivirale Verbindungen entdeckt wurden (Levy 2003). Wesentliche Hindernisse bei der Charakterisierung korpereigener antiviraler Substanzen sind die sehr begrenzten Mengen an menschlichem Untersuchungsmaterial und das Fehlen von standardisierten Methoden zur Aufreinigung der Inhibitoren.