conformational-evaluation-hiv-1-trimeric-envelope-glycoproteins-using

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Increasing evidence suggests that extracellular Vpr could contribute to HIV pathogenesis through its effect on bystander cells. Soluble forms of Vpr have been detected in the sera and cerebrospinal fluids of HIV-1-infected patients, and in vitro studies have implicated extracellular Vpr as an effector of cellular responses, including G2 arrest, apoptosis and induction of cytokines and chemokines production, presumably through its ability to transduce into multiple cell types. However, the mechanism underlying Vpr release from HIV-1-producing cells remains undefined and the biological modifications that the extracellular protein may undergo are largely unknown. We provide evidence indicating that soluble forms of Vpr are present in the extracellular medium of HIV-1-producing cells. Release of Vpr in the extracellular medium did not originate from decaying or disrupted HIV-1 virions that package Vpr but rather appeared associated with HIV-1-mediated cytopathicity. Interestingly, Vpr was found to undergo proteolytic processing at a very well conserved proprotein convertase (PC) cleavage site, R(85)QRR(88) downward arrow, located within the functionally important C-terminal arginine-rich domain of the protein. Vpr processing occurred extracellularly upon close contact to cells and most likely involved a cell surface-associated PC. Consistently, PC inhibitors suppressed Vpr processing, while expression of extracellular matrix-associated PC5 and PACE4 enhanced Vpr cleavage. PC-mediated processing of extracellular Vpr led to the production of a truncated Vpr product that was defective for the induction of cell cycle arrest and apoptosis when expressed in human cells. Collectively, these results suggest that cell surface processing of extracellular Vpr by PCs might regulate the levels of active soluble Vpr.

Cell-surface processing of extracellular human immunodeficiency virus type 1 Vpr by proprotein convertases.

Zunehmender Beweis schl&auml;gt vor, dass extrazellul&auml;re Vpr zu HIV Pathogenese durch seine Folgen f&uuml;r Zuschauer Zellen beitragen k&ouml;nnte. L&ouml;sliche Formen der Vpr im Serum und Liquor Fl&uuml;ssigkeiten von HIV-1-infizierten Patienten erkannt wurden, und in-vitro-Studien haben extrazellul&auml;re Vpr verwickelt, als ein Effektor der zellul&auml;ren Reaktionen, einschlie&szlig;lich G2 Verhaftung, Apoptosis und Induktion der Zytokine und Chemokine, vermutlich durch seine F&auml;higkeit, in mehreren Zelltypen zur&uuml;ckwirken. Jedoch der Mechanismus zugrunde liegenden Vpr Freisetzung von HIV-1-produzierenden Zellen bleibt undefiniert und die biologischen Ver&auml;nderungen, die das extrazellul&auml;re Protein unterziehen kann, sind weitgehend unbekannt. Wir belegen, dass l&ouml;sliche Formen der Vpr im extrazellul&auml;ren Medium der HIV-1-produzierenden Zellen vorhanden sind. Ver&ouml;ffentlichung von Vpr im extrazellul&auml;ren Medium Ursprung nicht aus zerfallenden oder gest&ouml;rt von HIV-1-Virionen, dass Paket Vpr aber erschien eher verbunden mit HIV-1-vermittelte Cytopathicity. Interessanterweise fand sich Vpr proteolytische Verarbeitung auf ein sehr gut erhaltene proprotein Konvertase (PC) Spaltung gelegen, R(85)QRR(88) des Pfeils, innerhalb der funktionell wichtigen C-terminalen Arginin-reiche Dom&auml;ne des Proteins zu unterziehen. VPR Verarbeitung trat extracellularly nach engem Kontakt zu Zellen und h&ouml;chstwahrscheinlich beteiligt eine Zelle Oberfl&auml;che-verbundenen PC. PC-Inhibitoren unterdr&uuml;ckt konsequent, Vpr Verarbeitung, w&auml;hrend Ausdruck der extrazellul&auml;ren Matrix-PC5 assoziierte und PACE4 Vpr Dekollet&eacute; verbessert. PC-vermittelte Verarbeitung von extrazellul&auml;ren Vpr f&uuml;hrten zu der Produktion eine abgeschnittene Vpr-Produkt, das f&uuml;r die Induktion der Zellzyklus Verhaftung und Apoptosis in den menschlichen Zellen ausgedr&uuml;ckt defekt war. Diesen Ergebnissen zufolge gemeinsam Zelle Oberfl&auml;chenbearbeitung der extrazellul&auml;ren Vpr von PCs die Ebenen der aktiven l&ouml;slichen Vpr regulieren k&ouml;nnte.

Zelloberfl&auml;che Verarbeitung von extrazellul&auml;ren Human Immunodeficiency Virus geben 1 Vpr proprotein Convertases.

Retroviral DNA integration leaves behind a single-strand DNA discontinuity at each virus:host DNA junction. It has long been proposed that cellular proteins detect and repair the integrated DNA and that failure to do so might lead to apoptotic cell death, but their identity remains unknown. PIKK family members ATM, DNA-PKcs and ATR have all been proposed to be important for HIV-1 replication, but these findings turned out to be very controversial. In order to clarify their role in retroviral replication, we analyzed the effect of pharmacological inhibitors and of a dominant-negative version of ATR on the replication of retroviruses in cell lines relevant to HIV-1 infection. Our data show that ATR and probably other PIKKs as well are involved in retroviral replication in some but not all cell lines and that ATR increases the frequency of retroviral transduction by a mechanism other than the enhancement of infected cell survival.

Cell context-dependent involvement of ATR in early stages of retroviral replication.

HIV up-regulates cell-surface expression of specific ligands for the activating NKG2D receptor, including ULBP-1, -2, and -3, but not MICA or MICB, in infected cells both in vitro and in vivo. However, the viral factor(s) involved in NKG2D ligand expression still remains undefined. HIV-1 Vpr activates the DNA damage/stress-sensing ATR kinase and promotes G(2) cell-cycle arrest, conditions known to up-regulate NKG2D ligands. We report here that HIV-1 selectively induces cell-surface expression of ULBP-2 in primary CD4(+) T lymphocytes by a process that is Vpr dependent. Importantly, Vpr enhanced the susceptibility of HIV-1-infected cells to NK cell-mediated killing. Strikingly, Vpr alone was sufficient to up-regulate expression of all NKG2D ligands and thus promoted efficient NKG2D-dependent NK cell-mediated killing. Delivery of virion-associated Vpr via defective HIV-1 particles induced analogous biologic effects in noninfected target cells, suggesting that Vpr may act similarly beyond infected cells. All these activities relied on Vpr ability to activate the ATR-mediated DNA damage/stress checkpoint. Overall, these results indicate that Vpr is a key determinant responsible for HIV-1-induced up-regulation of NKG2D ligands and further suggest an immunomodulatory role for Vpr that may not only contribute to HIV-1-induced CD4(+) T-lymphocyte depletion but may also take part in HIV-1-induced NK-cell dysfunction.

HIV-1 Vpr up-regulates expression of ligands for the activating NKG2D receptor and promotes NK cell-mediated killing.

HIV-1 viral protein R (Vpr) induces cell cycle arrest at the G(2)/M phase by a mechanism involving the activation of the DNA damage sensor ATR. We and others recently showed that Vpr performs this function by subverting the activity of the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase. Vpr could thus act as a connector between the E3 ligase and an unknown cellular factor whose ubiquitination would induce G(2) arrest. While attractive, this model is based solely on the indirect observation that some mutants of Vpr retain their interaction with the E3 ligase but fail to induce G(2) arrest. Using a tandem affinity purification approach, we observed that Vpr interacts with ubiquitinated cellular proteins and that this association requires the recruitment of an active E3 ligase given that the depletion of VPRBP by RNA interference or the overexpression of a dominant negative mutant of CUL4A decreased this association. Importantly, G(2)-arrest-defective mutants of Vpr in the C-terminal putative substrate-interacting domain displayed a decreased association with ubiquitinated proteins. We also found that the inhibition of proteasomal activity increased this association and that the ubiquitin chains were at least in part constituted of classical K48 linkages. Interestingly, the inhibition of K48 polyubiquitination specifically impaired the Vpr-induced phosphorylation of H2AX, an early target of ATR, but did not affect UV-induced H2AX phosphorylation. Overall, our results provide direct evidence that the association of Vpr with the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase induces the K48-linked polyubiquitination of as-yet-unknown cellular proteins, resulting in their proteasomal degradation and ultimately leading to the activation of ATR and G(2) arrest.

HIV-1 Vpr induces the K48-linked polyubiquitination and proteasomal degradation of target cellular proteins to activate ATR and promote G2 arrest.

Natural killer (NK)-cell killing of virus-infected cells is regulated in part by the engagement of activation and coactivation receptors. In this issue of Cell Host & Microbe, Shah et al. (2010) demonstrate that HIV-1 protects infected cells from NK-cell-mediated killing by hindering NK-cell degranulation through downmodulation of NTB-A coactivation receptor ligands by the Vpu accessory protein.

HIV-1 Vpu disarms natural killer cells.

HIV-1 viral protein R (Vpr) from laboratory-adapted virus strains activates the DNA damage/stress sensor ATR kinase and induces cell cycle arrest at the G(2)/M phase through a process that requires Vpr to engage the DDB1-CUL4A (VprBP/DCAF-1) E3 ligase complex. Activation of this DNA damage/stress checkpoint in G(2) by Vpr was shown to modulate NKG2D-dependent NK cell effector functions via enhancing expression of NKG2D ligands, notably ULBP2. However, it is unknown whether Vpr from HIV-1 primary isolates (groups M, N, O, and P) could modulate NKG2D-mediated cytotoxic functions of NK cells. Here, we report that Vpr from most HIV-1 primary isolates can upregulate ULBP2 expression and induce NKG2D-dependent NK cell killing. Importantly, these activities were always accompanied by an active G(2) cell cycle arrest function. Interestingly, Vpr variants from group P and a clade D isolate of group M were defective at enhancing NKG2D-mediated NK cell lysis owing to their inability to augment ULBP2 expression. However, distinct mechanisms were responsible for their failure to do so. While Vpr from group P was deficient in its ability to engage the DDB1-CUL4A (VprBP/DCAF-1) E3 ligase complex, the Vpr variant from group D was unable to properly localize to the nucleus, underlining the importance of these biological properties in Vpr function. In conclusion, the ability of Vpr from HIV-1 primary isolates to regulate NK cell effector function underscores the importance of this HIV-1 accessory protein in the modulation of the host's innate immune responses.

Modulation of NKG2D-mediated cytotoxic functions of natural killer cells by viral protein R from HIV-1 primary isolates.

HIV-1 Vpr triggers NK cell-mediated lysis of infected cells by upregulating ULBP2, a ligand of the NKG2D receptor, through activation of the ATR-mediated DNA damage response. Herein, we demonstrate that Vpr augments ULBP2 expression on both infected and uninfected bystander cells during HIV-1 infection of primary CD4+ T lymphocytes. Indeed, the frequency of uninfected bystander cells expressing high levels of ULBP2 was elevated in a Vpr-dependent manner. Nevertheless, the same does not hold true for a Vpr mutant that is not packaged into virions, suggesting the involvement of virion-associated Vpr in this process. Additionally, we show that soluble Vpr has the ability to induce a DNA damage response and to augment cell-surface ULBP2 upon transducing target cells, including T cells, conditions known to promote NK cell-mediated killing. Overall, these findings suggest that Vpr could contribute to CD4+ T cell loss by rendering uninfected bystander cells susceptible to NK cell-mediated killing.

Viral protein R upregulates expression of ULBP2 on uninfected bystander cells during HIV-1 infection of primary CD4+ T lymphocytes.

We apply adaptive sensing techniques to the problem of locating sparse metallic scatterers using high-resolution, frequency modulated continuous wave W-band RADAR. Using a single detector, a frequency stepped source, and a lateral translation stage, inverse synthetic aperture RADAR reconstruction techniques are used to search for one or two wire scatterers within a specified range, while an adaptive algorithm determined successive sampling locations. The two-dimensional location of each scatterer is thereby identified with sub-wavelength accuracy in as few as 1/4 the number of lateral steps required for a simple raster scan. The implications of applying this approach to more complex scattering geometries are explored in light of the various assumptions made.

Adaptive millimeter-wave synthetic aperture imaging for compressive sampling of sparse scenes.

Increased attention on the role of Fc-mediated effector functions against HIV-1 has led to renewed interest into the role that antibody-dependent cellular cytotoxicity (ADCC) could play in controlling viral transmission and/or the rate of disease progression. While (51)Chromium release assays have traditionally been used to study ADCC responses against HIV-1, a number of alternative flow-cytometry-based assays were recently developed. In this study, an alternative flow-cytometry-based assay was established to allow non-radioactive measurement of ADCC-mediated elimination of HIV-1 gp120 envelope glycoprotein (Env)-coated target cells. This assay relies on staining target and effector cells with different dyes, which allows precise gating and permits the calculation of the number of surviving target cells by normalization to flow-cytometry particles. By using small concentrations of recombinant gp120 Env, suitable targets cells that recapitulate the ADCC response mediated against HIV-1-infected cells were generated. Finally, this method was applied successfully to screen human sera for ADCC activity directed against HIV-1 gp120 Env.

Flow cytometry-based assay to study HIV-1 gp120 specific antibody-dependent cellular cytotoxicity responses.

Binding to the primary receptor, CD4, triggers conformational changes in the metastable HIV-1 envelope glycoprotein (Env) trimer ((gp120-gp41)3) that are important for virus entry into host cells. These changes include an 'opening' of the trimer, creation of a binding site for the CCR5 co-receptor and formation and/or exposure of a gp41 coiled coil. Here we identify a new compound, 18A (1), that specifically inhibits the entry of a wide range of HIV-1 isolates. 18A does not interfere with CD4 or CCR5 binding, but it inhibits the CD4-induced disruption of quaternary structures at the trimer apex and the exposure of the gp41 HR1 coiled coil. Analysis of HIV-1 variants with increased or reduced sensitivity to 18A suggests that the inhibitor can distinguish distinct conformational states of gp120 in the unliganded Env trimer. The broad-range activity and observed hypersensitivity of resistant mutants to antibody neutralization support further investigation of 18A.

Article	Year
Cell-surface processing of extracellular human immunodeficiency virus type 1 Vpr by proprotein convertases. Virology\| PubMed ID: 18061232	2008
Cell context-dependent involvement of ATR in early stages of retroviral replication. Virology\| PubMed ID: 19913868	2010
HIV-1 Vpr up-regulates expression of ligands for the activating NKG2D receptor and promotes NK cell-mediated killing. Blood\| PubMed ID: 20008788	2010
HIV-1 Vpr induces the K48-linked polyubiquitination and proteasomal degradation of target cellular proteins to activate ATR and promote G2 arrest. Journal of virology\| PubMed ID: 20089662	2010
HIV-1 Vpu disarms natural killer cells. Cell host & microbe\| PubMed ID: 21075348	2010
Modulation of NKG2D-mediated cytotoxic functions of natural killer cells by viral protein R from HIV-1 primary isolates. Journal of virology\| PubMed ID: 21957298	2011
Viral protein R upregulates expression of ULBP2 on uninfected bystander cells during HIV-1 infection of primary CD4+ T lymphocytes. Virology\| PubMed ID: 23726848	2013
Adaptive millimeter-wave synthetic aperture imaging for compressive sampling of sparse scenes. Optics express\| PubMed ID: 24921545	2014
Flow cytometry-based assay to study HIV-1 gp120 specific antibody-dependent cellular cytotoxicity responses. Journal of virological methods\| PubMed ID: 25125129	2014
A broad HIV-1 inhibitor blocks envelope glycoprotein transitions critical for entry. Nature chemical biology\| PubMed ID: 25174000	2014

Jonathan Richard

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