Roles Played by Capsid-Dependent Induction of Membrane Curvature and Gag-ESCRT Interactions in Tetherin Recruitment to HIV-1 Assembly Sites

Abstract

Tetherin/BST-2 (here called tetherin) is an antiviral protein that restricts release of diverse enveloped viruses from infected cells through physically tethering virus envelope and host plasma membrane. For HIV-1, specific recruitment of tetherin to assembly sites has been observed as its colocalization with the viral structural protein Gag or its accumulation in virus particles. Because of its broad range of targets, we hypothesized that tetherin is recruited through conserved features shared among various enveloped viruses, such as lipid raft association, membrane curvature, or ESCRT dependence. We observed that reduction of cellular cholesterol does not block tetherin anti-HIV-1 function, excluding an essential role for lipid rafts. In contrast, mutations in the capsid domain of Gag, which inhibit induction of membrane curvature, prevented tetherin-Gag colocalization detectable by confocal microscopy. Disruption of Gag-ESCRT interactions also inhibited tetherin-Gag colocalization when disruption was accomplished via amino acid substitutions in late domain motifs, expression of a dominant-negative Tsg101 derivative, or small interfering RNA (siRNA)-mediated depletion of Tsg101 or Alix. However, further analyses of these conditions by quantitative superresolution localization microscopy revealed that Gag-tetherin coclustering is significantly reduced but persists at intermediate levels. Notably, this residual tetherin recruitment was still sufficient for the full restriction of HIV-1 release. Unlike the late domain mutants, the capsid mutants defective in inducing membrane curvature showed little or no coclustering with tetherin in superresolution analyses. These results support a model in which both Gag-induced membrane curvature and Gag-ESCRT interactions promote tetherin recruitment, but the recruitment level achieved by the former is sufficient for full restriction.

Basic Motifs Target PSGL-1, CD43, and CD44 to Plasma Membrane Sites Where HIV-1 Assembles

“…Sample preparation, conditions for image acquisition, reconstruction of superresolution images, and data analysis methods were described previously (56). Briefly, single fluorophores of Alexa Fluor 647-and mEos3.2-labeled proteins were imaged simultaneously under total internal reflection (TIRF) using an Olympus IX81-XDC inverted microscope with a cellTIRF module, a ϫ100 UAPO TIRF objective (numerical aperture ϭ 1.49), and active Z-drift correction (ZDC) (Olympus America).…”

“…pNL4-3/Gag-mEos3.2 was described previously (56). pNL4-3/ Fyn(10)/Gag-mEos3.2, pNL4-3/Fyn(10)/6A2T/Gag-mEos3.2, pNL4-3/ Fyn(10)/HBR/RKswitch/Gag-mEos3.2, and pNL4-3/Fyn(10)/⌬MA/GagmEos3.2 were constructed using MA-containing sequences from pNL4-3/Gag-Venus versions of these constructs, which were described previously (38,39).…”

“…The VLP release assay used for the Gag-mEos3.2 constructs was described previously (56). For analysis of virion incorporation of CD43, CD43/6A, ICAM-1, or ICAM-1/PCT, HeLa cells cotransfected with pNL4-3 and an expression plasmid carrying one of the membrane proteins were cultured for 16 h or 2 days, and cell and virus lysates were prepared as previously described (61).…”

“…High-resolution microscopy methods not only allow assessment of whether the protein of interest is incorporated into assembling virions but also provide information regarding the degree of enrichment of the protein at assembly sites relative to that in surrounding areas, which is essential in determining whether the protein is actively recruited to assembly sites. Indeed, in our previous study (56), superresolution localization microscopy methods, but not confocal microscopy, allowed us to detect differences in the degree of BST-2/tetherin-Gag coclustering among multimerization-competent Gag mutants. In this study, we employed the same quantitative two-color superresolution localization microscopy techniques to elucidate the molecular mechanism by which a subset of cellular transmembrane proteins is specifically recruited to sites where viral assembly occurs and incorporated into HIV-1 particles.…”

“…A great deal has been learned about mechanisms for the specific incorporation of viral glycoproteins into assembling virus particles using scanning electron microscopy or superresolution fluorescence microscopy (52)(53)(54)(55), which allow analyses at a resolution higher than that possible by conventional fluorescence microscopy. However, except for the molecular determinants for BST-2/tetherin incorporation (56,57), the molecular determinants for the incorporation of host transmembrane proteins have not been examined at a high resolution. High-resolution microscopy methods not only allow assessment of whether the protein of interest is incorporated into assembling virions but also provide information regarding the degree of enrichment of the protein at assembly sites relative to that in surrounding areas, which is essential in determining whether the protein is actively recruited to assembly sites.…”