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

Selektiver Verlust der Nef-vermittelten CD3-Modulation in SIVsmm-infizierten Rauchgrauen Mangaben mit niedrigen CD4+ T-Zellzahlen

Das Nef-Protein der Immundefizienzviren ermöglicht effektive Persistenz und trägt dadurch entscheidend zur Progression zu AIDS im Menschen bei. Ein fundamentaler Unterschied zwischen der HIV-1 Infektion im Menschen und der apathogenen SIV-Infektion ist das Fehlen einer chronisch erhöhten Immunaktivierung. SIVsmm-infizierte Rauchgraue Mangaben haben trotz hoher Viruslast meist eine stabile Anzahl CD4+ T-Zellen. Etwa 5-10 % dieser Affen zeigen signifikante Verluste der CD4+ T-Zellen. Wir zeigten bereits, dass die niedrige Zahl CD4+ T-Zellen in SIVsmm infizierten Rauchgrauen Mangaben mit ineffektiver CD3-Herabregulierung von Nef korreliert. Somit könnte diese Nef-Funktion und die damit verbundene T-Zellaktivierung, die Hyperaktivierung des Immunsystems verhindern. In einer anderen Studie wurde gezeigt, dass der Verlust von CD4+ T-Zellen auch als Folge eines erweiterten viralen Korezeptortropismus möglich ist. Hier wurde untersucht, ob auch Nef-Funktionen als Ursache für die extrem niedrigen CD4+ T-Zellzahlen der untersuchten Rauchgrauen Mangaben verantwortlich waren. Die Ergebnisse zeigen, dass Nef die Fähigkeit CD3 herabzuregulieren erst nach dem Absinken der CD4+ T-Zellzahlen und der Erweiterung des viralen Korezeptortropismus verlor. Somit war der Verlust der Nef-vermittelten CD3-Modulation nicht die Ursache, sondern die Folge der niedrigen CD4+ T-Zellzahlen. Als spezifische Determinanten für die Modulation von CD3 wurden die Aminosäuren 123 und 146 in SIVsmm Nef identifiziert. Somit ist die CD3-Modulation von anderen Nef-Funktionen genetisch trennbar. Mutationsanalysen zeigten eine Beschleunigung der Endozytose und eine erhöhte Bindefähigkeit zu CD3-zeta als Mechanismus der Nef-vermittelten Herunterregulierung von CD3. Erstmals wurden SIVmac239 Nef-Mutanten hergestellt, die selektiv defekt für die CD3-Herabregulierung sind. Dadurch kann jetzt im Rhesusaffen-Modell geklärt werden, ob der Verlust der CD3-Modulation von Nef die Pathogenität des Virus weiter erhöht

Incorporation of podoplanin into HIV released from HEK-293T cells, but not PBMC, is required for efficient binding to the attachment factor CLEC-2

Abstract Background Platelets are associated with HIV in the blood of infected individuals and might modulate viral dissemination, particularly if the virus is directly transmitted into the bloodstream. The C-type lectin DC-SIGN and the novel HIV attachment factor CLEC-2 are expressed by platelets and facilitate HIV transmission from platelets to T-cells. Here, we studied the molecular mechanisms behind CLEC-2-mediated HIV-1 transmission. Results Binding studies with soluble proteins indicated that CLEC-2, in contrast to DC-SIGN, does not recognize the viral envelope protein, but a cellular factor expressed on kidney-derived 293T cells. Subsequent analyses revealed that the cellular mucin-like membranous glycoprotein podoplanin, a CLEC-2 ligand, was expressed on 293T cells and incorporated into virions released from these cells. Knock-down of podoplanin in 293T cells by shRNA showed that virion incorporation of podoplanin was required for efficient CLEC-2-dependent HIV-1 interactions with cell lines and platelets. Flow cytometry revealed no evidence for podoplanin expression on viable T-cells and peripheral blood mononuclear cells (PBMC). Podoplanin was also not detected on HIV-1 infected T-cells. However, apoptotic bystander cells in HIV-1 infected cultures reacted with anti-podoplanin antibodies, and similar results were obtained upon induction of apoptosis in a cell line and in PBMCs suggesting an unexpected link between apoptosis and podoplanin expression. Despite the absence of detectable podoplanin expression, HIV-1 produced in PBMC was transmitted to T-cells in a CLEC-2-dependent manner, indicating that T-cells might express an as yet unidentified CLEC-2 ligand. Conclusions Virion incorporation of podoplanin mediates CLEC-2 interactions of HIV-1 derived from 293T cells, while incorporation of a different cellular factor seems to be responsible for CLEC-2-dependent capture of PBMC-derived viruses. Furthermore, evidence was obtained that podoplanin expression is connected to apoptosis, a finding that deserves further investigation.</p

Link between Primate Lentiviral Coreceptor Usage and Nef Function

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

Development and Adjustment of Tools for Superconducting RF Gun Cavities

For the superconducting radio frequency (SRF), 1.6-cell gun cavities (CV) developed at DESY, a similar fabrication andtreatment process, as for the European XFEL 9-cell cavities is foreseen. The different length and geometry of thesecavities lead to a number of adjustments to existing and the development of new tools. This paper covers the newdesigns and adaptations of a tuning tool, chemistry flanges, a wall thickness measurement device, as well as a new high pressure rinsing spray head and an optical inspection camera for the 1.6-cell, 1.3 GHz DESY SRF gun cavities

The Presence of a vpu Gene and the Lack of Nef-Mediated Downmodulation of T Cell Receptor-CD3 Are Not Always Linked in Primate Lentiviruses ▿ †

Nef is an accessory protein critical for the ability of human and simian immunodeficiency viruses (HIV and SIV) to replicate efficiently in their respective hosts. Previous analyses of members of 15 different primate lentivirus lineages revealed a link between Nef function and the presence of a vpu gene. In particular, Nef proteins of all vpu-containing viruses had lost their ability to downmodulate the T cell (TCR-CD3) receptor. Here we examined Nef proteins from eight additional SIV lineages, including SIVgor, SIVwrc, SIVolc, SIVgri, SIVdrl, SIVlho, SIVden, and SIVasc, from western lowland gorillas, western red colobus monkeys, olive colobus monkeys, grivet monkeys, drills, L'Hoest's monkeys, Dent's mona monkeys, and red-tailed monkeys, respectively. We found that except for the nef gene of SIVdrl, all of them were efficiently expressed and modulated CD4, major histocompatibility complex class I (MHC-I), CD28, CXCR4, and Ii cell surface expression and/or enhanced viral infectivity and replication. Furthermore, the Nef proteins of SIVgri, SIVlho, SIVwrc, SIVolc, and SIVgor antagonized tetherin. As expected, the Nef protein of SIVgor, which carries vpu, failed to downmodulate CD3, whereas those of SIVwrc, SIVgri, SIVlho, and SIVasc, which lack vpu, were capable of performing this function. Surprisingly, however, the Nef protein of the vpu-containing SIVden strain retained the ability to downmodulate TCR-CD3, whereas that of SIVolc, which does not contain vpu, was unable to perform this function. Although the SIVden Vpu is about 20 amino acids shorter than other Vpu proteins, it degrades CD4 and antagonizes tetherin. Our data show that there are exceptions to the link between the presence of a vpu gene and nef alleles deficient in CD3 modulation, indicating that host properties also affect the selective pressure for Nef-mediated disruption of TCR-CD3 signaling. Our results are also further evidence that tetherin antagonism is a common function of primate lentivirus Nef proteins and that the resistance of human tetherin to Nef represents a relevant barrier to cross-species transmission of SIVs to humans

SRF Gun Development at DESY

A future upgrade of the European XFEL (E-XFEL) foresees an additional CW operation mode, which will increase the flexibility in the photon beam time structure [1–3]. One of the challenges of this operational mode is the need fora CW operating photo injector. We believe that using an SRF gun is the preferred approach as the beam parameters of normal conducting pulsed guns can be potentially met by SRF guns operating CW. For more than a decade DESY, incollaboration with TJNAF, NCBJ, BNL, HZB and HZDR, has performed R&D to develop an all superconducting RFgun with a lead cathode. In the frame of E-XFEL CW upgrade feasibility studies, the SRF-gun R&D program gained more attention and support. Within the next few years we would like to demonstrate the performance of the all superconducting injector required for the E-XFEL upgrade. The selected approach offers advantages w.r.t. the cleanliness of the superconducting surface, but requires a complete disassembly of a cryostat and stripping the gun cavity in a cleanroom to exchange the cathode. Thus it is practical only when the life time of the cathode is at least several months. In this paper we present the actual status of the R&D program, next steps and the longer term plans