Tetherin does not significantly restrict dendritic cell-mediated HIV-1 transmission and its expression is upregulated by newly synthesized HIV-1 Nef

<p>Abstract</p> <p>Background</p> <p>Dendritic cells (DCs) are among the first cells to encounter HIV-1 and play important roles in viral transmission and pathogenesis. Immature DCs allow productive HIV-1 replication and long-term viral dissemination. The pro-inflammatory factor lipopolysaccharide (LPS) induces DC maturation and enhances the efficiency of DC-mediated HIV-1 transmission. Type I interferon (IFN) partially inhibits HIV-1 replication and cell-cell transmission in CD4⁺T cells and macrophages. Tetherin is a type I IFN-inducible restriction factor that blocks HIV-1 release and modulates CD4⁺T cell-mediated cell-to-cell transmission of HIV-1. However, the role of type I IFN and tetherin in HIV-1 infection of DCs and DC-mediated viral transmission remains unknown.</p> <p>Results</p> <p>We demonstrated that IFN-alpha (IFNα)-induced mature DCs restricted HIV-1 replication and <it>trans</it>-infection of CD4⁺T cells. Tetherin expression in monocyte-derived immature DCs was undetectable or very low. High levels of tetherin were transiently expressed in LPS- and IFNα-induced mature DCs, while HIV-1 localized into distinct patches in these DCs. Knockdown of induced tetherin in LPS- or IFNα-matured DCs modestly enhanced HIV-1 transmission to CD4⁺T cells, but had no significant effect on wild-type HIV-1 replication in mature DCs. Intriguingly, we found that HIV-1 replication in immature DCs induced significant tetherin expression in a Nef-dependent manner.</p> <p>Conclusions</p> <p>The restriction of HIV-1 replication and transmission in IFNα-induced mature DCs indicates a potent anti-HIV-1 response; however, high levels of tetherin induced in mature DCs cannot significantly restrict wild-type HIV-1 release and DC-mediated HIV-1 transmission. Nef-dependent tetherin induction in HIV-1-infected immature DCs suggests an innate immune response of DCs to HIV-1 infection.</p

Wind-tunnel Tests at Low Speed of Swept and Yawed Wings Having Various Plan Forms

Results are presented of wind-tunnel tests made at low speed of various small-scale models of sweptback, sweptforward, and yawed wings. The tests covered changes in aspect ratio, taper ratio, and tip shape. Some data were obtained with high-lift devices on sweptback wings and with ailerons on sweptforward wings. The data have been briefly analyzed and some comparisons have been made with the available theory

CAML Does Not Modulate Tetherin-Mediated Restriction of HIV-1 Particle Release

Background: Tetherin/BST-2 is a recently-identified potent restriction factor in human cells that restricts HIV particle release following particle formation and budding at the plasma membrane. Vpu counteracts tetherin’s restriction of particle release in a manner that has not yet been fully defined. We recently identified calcium-modulating cyclophilin ligand (CAML) as a Vpu-interacting protein that also restricts particle release. We hypothesized that CAML may act to enhance tetherinmediated restriction of particle release and thereby explain how two distinct factors could be responsible for Vpuresponsive restriction. Methodology/Principal Findings: Endogenous levels of tetherin in human cells correlated well with their restriction pattern and responsiveness to Vpu, while levels of cellular CAML protein did not. Tetherin but not CAML was inducible by interferon in a wide variety of human cells. Stable depletion of human CAML in restrictive HeLa cells had no effect on cell surface levels of tetherin, and failed to relieve tetherin-mediated restriction. Stable depletion of tetherin from HeLa cells, in contrast, rendered HeLa cells permissive and Vpu-unresponsive. Tetherin but not CAML expression in permissive human cells rendered them restrictive and Vpu responsive. Depletion of CAML had no influence on cell surface levels of tetherin. Conclusions/Significance: We conclude that tetherin restricts particle release and does not require CAML for this effect

Expression of Vaccinia E3L and K3L Genes by a Novel Recombinant Canarypox HIV Vaccine Vector Enhances HIV-1 Pseudovirion Production and Inhibits Apoptosis in Human Cells

AbstractPoxviruses that are attenuated for growth in human cells provide a safe means of HIV antigen expression and are capable of eliciting HIV-specific immune responses, including CD8+ cytotoxic T-lymphocyte (CTL) responses. HIV-1 antigen expression in human cells by attenuated poxvirus vectors may be limited by interferon-mediated host defense mechanisms. To enhance HIV antigen expression in human cells, the vaccinia virus E3L and K3L genes were inserted into a canarypox vector that expresses HIV-1 Gag, Env, and a Nef/Pol polyepitope string. E3L and K3L markedly reduced the activation of the double-stranded RNA-dependent protein kinase, PKR, and led to a significant reduction in apoptosis in HeLa cells. Production and release of HIV-1 antigen in the form of pseudovirions was enhanced in both duration and magnitude by this vector modification. The addition of immunomodulatory genes to attenuated poxviruses represents a novel strategy for enhancing antigen production by live vector HIV vaccine candidates

Gp120 stability on HIV-1 virions and Gag-Env pseudovirions is enhanced by an uncleaved Gag core

AbstractHuman immunodeficiency virus type-1 (HIV-1) particles incorporate a trimeric envelope complex (Env) made of gp120 (SU) and gp41 (TM) heterodimers. It has been previously established that soluble CD4 (sCD4) interaction leads to shedding of gp120 from viral particles, and that gp120 may also be easily lost from virions during incubation or particle purification procedures. In the design of HIV particle or pseudovirion-based HIV vaccines, it may be important to develop strategies to maximize the gp120 content of particles. We analyzed the gp120 retention of HIV-1 laboratory-adapted isolates and primary isolates following incubation with sCD4 and variations in temperature. NL4-3 shed gp120 readily in a temperature- and sCD4-dependent manner. Surprisingly, inactivation of the viral protease led to markedly reduced shedding of gp120. Gp120 shedding was shown to vary markedly between HIV-1 strains, and was not strictly determined by whether the isolate was adapted to growth on immortalized T cell lines or was a primary isolate. Pseudovirions produced by expression of codon-optimized gag and env genes also demonstrated enhanced gp120 retention when an immature core structure was maintained. Pseudovirions of optimal stability were produced through a combination of an immature Gag protein core and a primary isolate Env. These results support the feasibility of utilizing pseudovirion particles as immunogens for the induction of humoral responses directed against native envelope structures

Model-Based Systems Engineering and F’: Proof of Concept Via the Creation of an On-Orbit Textual Command Parsing Component for the ABEX Mission

The Alabama Burst Energetics eXplorer (ABEX) mission is defining spacecraft architecture, behavior, mission phases, operational states, risks, and requirements in a Model-Based Systems Engineering (MBSE) Integrated Systems Model (ISM) using SysML in Cameo Enterprise Architecture (CEA). The satellite structural design can be exported from CEA as Extensible Markup Language (XML) specifications and imported to F’, an open-source Flight Software (FSW) framework from NASA’s Jet Propulsion Laboratory. F’ contains background components intended to be connected to user-defined components in the XML after it is exported from the ISM; in this work, ABEX is representing F’ background components in SysML Internal Block Diagrams from which the XML is generated. As a proof of concept for this MBSE-centric FSW implementation, the ABEX FSW team has created a Command Reader component from MBSE-generated XML and tested command enaction on a Raspberry Pi breadboard system for three test cases representing on-orbit command triggers

Association Between Choice of Radical Prostatectomy, External Beam Radiotherapy, Brachytherapy, or Active Surveillance and Patient-Reported Quality of Life Among Men With Localized Prostate Cancer

Importance Patients diagnosed with localized prostate cancer have to decide among treatment strategies that may differ in their likelihood of adverse effects. Objective To compare quality of life (QOL) after radical prostatectomy, external beam radiotherapy, and brachytherapy vs active surveillance. Design, Setting, and Participants Population-based prospective cohort of 1141 men (57% participation among eligible men) with newly diagnosed prostate cancer were enrolled from January 2011 through June 2013 in collaboration with the North Carolina Central Cancer Registry. Median time from diagnosis to enrollment was 5 weeks, and all men were enrolled with written informed consent prior to treatment. Final follow-up date for current analysis was September 9, 2015. Exposures Treatment with radical prostatectomy, external beam radiotherapy, brachytherapy, or active surveillance. Main Outcomes and Measures Quality of life using the validated instrument Prostate Cancer Symptom Indices was assessed at baseline (pretreatment) and 3, 12, and 24 months after treatment. The instrument contains 4 domains—sexual dysfunction, urinary obstruction and irritation, urinary incontinence, and bowel problems—each scored from 0 (no dysfunction) to 100 (maximum dysfunction). Propensity-weighted mean domain scores were compared between each treatment group vs active surveillance at each time point. Results Of 1141 enrolled men, 314 pursued active surveillance (27.5%), 469 radical prostatectomy (41.1%), 249 external beam radiotherapy (21.8%), and 109 brachytherapy (9.6%). After propensity weighting, median age was 66 to 67 years across groups, and 77% to 80% of participants were white. Across groups, propensity-weighted mean baseline scores were 41.8 to 46.4 for sexual dysfunction, 20.8 to 22.8 for urinary obstruction and irritation, 9.7 to 10.5 for urinary incontinence, and 5.7 to 6.1 for bowel problems. Compared with active surveillance, mean sexual dysfunction scores worsened by 3 months for patients who received radical prostatectomy (36.2 [95% CI, 30.4-42.0]), external beam radiotherapy (13.9 [95% CI, 6.7-21.2]), and brachytherapy (17.1 [95% CI, 7.8-26.6]). Compared with active surveillance at 3 months, worsened urinary incontinence was associated with radical prostatectomy (33.6 [95% CI, 27.8-39.2]); acute worsening of urinary obstruction and irritation with external beam radiotherapy (11.7 [95% CI, 8.7-14.8]) and brachytherapy (20.5 [95% CI, 15.1-25.9]); and worsened bowel symptoms with external beam radiotherapy (4.9 [95% CI, 2.4-7.4]). By 24 months, mean scores between treatment groups vs active surveillance were not significantly different in most domains

Immunoelectron Microscopic Evidence for Tetherin/BST2 as the Physical Bridge between HIV-1 Virions and the Plasma Membrane

Tetherin/BST2 was identified in 2008 as the cellular factor responsible for restricting HIV-1 replication at a very late stage in the lifecycle. Tetherin acts to retain virion particles on the plasma membrane after budding has been completed. Infected cells that express large amounts of tetherin display large strings of HIV virions that remain attached to the plasma membrane. Vpu is an HIV-1 accessory protein that specifically counteracts the restriction to virus release contributed by tetherin. Tetherin is an unusual Type II transmembrane protein that contains a GPI anchor at its C-terminus and is found in lipid rafts. The leading model for the mechanism of action of tetherin is that it functions as a direct physical tether bridging virions and the plasma membrane. However, evidence that tetherin functions as a physical tether has thus far been indirect. Here we demonstrate by biochemical and immunoelectron microscopic methods that endogenous tetherin is present on the viral particle and forms a bridge between virion particles and the plasma membrane. Endogenous tetherin was found on HIV particles that were released by partial proteolytic digestion. Immunoelectron microscopy performed on HIV-infected T cells demonstrated that tetherin forms an apparent physical link between virions and connects patches of virions to the plasma membrane. Linear filamentous strands that were highly enriched in tetherin bridged the space between some virions. We conclude that tetherin is the physical tether linking HIV-1 virions and the plasma membrane. The presence of filaments with which multiple molecules of tetherin interact in connecting virion particles is strongly suggested by the morphologic evidence