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

A facile quantitative assay for viral particle genesis reveals cooperativity in virion assembly and saturation of an antiviral protein

Conventional assays of viral particle assembly and release are time consuming and laborious. We have developed an enzymatic virus-like particle (VLP) genesis assay that rapid and quantitative and is also versatile and applicable to diverse viruses including HIV-1 and Ebola virus. Using this assay, which has a dynamic range of several orders of magnitude, we show that the efficiency of VLP assembly and release, i.e., the fraction of the expressed protein that is assembled into extracellular particles, is dependent on the absolute level of expression of either HIV-1 Gag or Ebola virus VP40. We also demonstrate that the activity of the antiviral factor tetherin is dependent on the level of HIV-1 Gag expression and the numbers of VLPs generated, and appears to become saturated as these parameters are increased

Parental perceptions of the impact of neonatal unit visitation policies during COVID-19 pandemic

Objectives To ascertain parental perceptions of the impact of restricted visiting policies to neonatal intensive care units during the current COVID-19 pandemic. Design Cross-sectional survey of parents impacted by visitation policies. Setting Six tertiary level neonatal units, four from the UK and two from the USA, participated in the study. Participants Parents and families of infants hospitalised in the participating centres between 1 May 2020 and 21 August 2020. Methods Online-based and/or paper-based survey, querying the visitation policies and their impact on parents' ability to visit, care for and bond with their infants. Results A total of 231 responses were received. Visitation limited to a single visitor with no restrictions on duration was the most frequently reported policy; 140/217 (63%). Visitation policies were perceived as being restrictive by 62% (138/219) of the respondents with 37% (80/216) reporting being able to visit less often than desired, 41% (78/191) reporting being unable to bond enough and 27% (51/191) reporting not being able to participate in their baby's daily care. Mild to severe impact on breast feeding was reported by 36% (75/209) of respondents. Stricter policies had a higher impact on families and were significantly associated with a lack of bonding time, inability to participate in care and an adverse impact on breast feeding. Conclusions Visitation policies during the COVID-19 pandemic varied between centres and over time with stricter restrictions implemented earlier on in the pandemic. Parents reported significant impacts on their ability to visit, care for and bond with their infants with perceived severity of impact worse with stricter restrictions

Unknowable bodies, unthinkable sexualities: lesbian and transgender legal invisibility in the Toronto women's bathhouse raid

Although litigation involving sexual orientation and gender identity discrimination claims has generated considerable public attention in recent years, lesbian and transgender bodies and sexualities still remain largely invisible in Anglo-American courts. While such invisibility is generally attributed to social norms that fail to recognize lesbian and transgender experiences, the capacity to 'not see' or 'not know' queer bodies and sexualities also involves wilful acts of ignorance. Drawing from R. v Hornick (2002) a Canadian case involving the police raid of a women's bathhouse, this article explores how lesbian and transgender bodies and sexualities are actively rendered invisible via legal knowledge practices, norms and rationalities. It argues that limited knowledge and limited thinking not only regulate the borders of visibility and belonging, but play an active part in shaping identities, governing conduct and producing subjectivity

Getting Genetic Ancestry Right for Science and Society

There is a scientific and ethical imperative to embrace a multidimensional, continuous view of ancestry and move away from continental ancestry categorie

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