Differential IgG Subclass Responses to Epitopes in Transmembrane Protein of HIV-1

ABSTRACT The interferon-induced transmembrane proteins (IFITMs) are a family of highly related antiviral factors that affect numerous viruses at two steps: in target cells by sequestering incoming viruses in endosomes and in producing cells by leading to the production of virions that package IFITMs and exhibit decreased infectivity. While most studies have focused on the former, little is known about the regulation of the negative imprinting of virion particle infectivity by IFITMs and about its relationship with target cell protection. Using a panel of IFITM3 mutants against HIV-1, we have explored these issues as well as others related to the biology of IFITM3, in particular virion packaging, stability, the relation to CD63/multivesicular bodies (MVBs), the modulation of cholesterol levels, and the relationship between negative imprinting of virions and target cell protection. The results that we have obtained exclude a role for cholesterol and indicate that CD63 accumulation does not directly relate to an antiviral behavior. We have defined regions that modulate the two antiviral properties of IFITM3 as well as novel domains that modulate protein stability and that, in so doing, influence the extent of its packaging into virions. The results that we have obtained, however, indicate that, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for negative imprinting. Finally, while most mutations concomitantly affect target cell protection and negative imprinting, a region in the C-terminal domain (CTD) exhibits a differential behavior, potentially highlighting the regulatory role that this domain may play in the two antiviral activities of IFITM3. IMPORTANCE IFITM proteins have been associated with the sequestration of incoming virions in endosomes (target cell protection) and with the production of virion particles that incorporate IFITMs and exhibit decreased infectivity (negative imprinting of virion infectivity). How the latter is regulated and whether these two antiviral properties are related remain unknown. By examining the behavior of a large panel of IFITM3 mutants against HIV-1, we determined that IFITM3 mutants are essentially packaged into virions proportionally to their intracellular levels of expression. However, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for the antiviral effects. Most mutations were found to concomitantly affect both antiviral properties of IFITM3, but one CTD mutant exhibited a divergent behavior, possibly highlighting a novel regulatory role for this domain. These findings thus advance our comprehension of how this class of broad antiviral restriction factors acts.

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Characterization of Endogenous SERINC5 Protein as Anti-HIV-1 Factor

Journal of Virology ◽

10.1128/jvi.01221-19 ◽

2019 ◽

Vol 93 (24) ◽

Cited By ~ 4

Author(s):

Vânia Passos ◽

Thomas Zillinger ◽

Nicoletta Casartelli ◽

Amelie S. Wachs ◽

Shuting Xu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Surface ◽

Subcellular Localization ◽

Transmembrane Protein ◽

Type I ◽

Accessory Gene ◽

Protein Levels ◽

Specificity And Sensitivity ◽

Hiv 1

ABSTRACT When expressed in virus-producing cells, the cellular multipass transmembrane protein SERINC5 reduces the infectivity of HIV-1 particles and is counteracted by HIV-1 Nef. Due to the unavailability of an antibody of sufficient specificity and sensitivity, investigation of SERINC5 protein expression and subcellular localization has been limited to heterologously expressed SERINC5. We generated, via CRISPR/Cas9-assisted gene editing, Jurkat T-cell clones expressing endogenous SERINC5 bearing an extracellularly exposed hemagglutinin (HA) epitope [Jurkat SERINC5(iHA knock-in) T cells]. This modification enabled quantification of endogenous SERINC5 protein levels and demonstrated a predominant localization in lipid rafts. Interferon alpha (IFN-α) treatment enhanced cell surface levels of SERINC5 in a ruxolitinib-sensitive manner in the absence of modulation of mRNA and protein quantities. Parental and SERINC5(iHA knock-in) T cells shared the ability to produce infectious wild-type HIV-1 but not an HIV-1 Δnef mutant. SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. An association of endogenous SERINC5 protein with HIV-1 Δnef virions was consistently detectable as a 35-kDa species, as opposed to heterologous SERINC5, which presented as a 51-kDa species. Nef-mediated functional counteraction did not correlate with virion exclusion of SERINC5, arguing for the existence of additional counteractive mechanisms of Nef that act on virus-associated SERINC5. In HIV-1-infected cells, Nef triggered the internalization of SERINC5 in the absence of detectable changes of steady-state protein levels. These findings establish new properties of endogenous SERINC5 expression and subcellular localization, challenge existing concepts of HIV-1 Nef-mediated antagonism of SERINC5, and uncover an unprecedented role of IFN-α in modulating SERINC5 through accumulation at the cell surface. IMPORTANCE SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions.

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An anti-HIV-1 gp120 antibody expressed as an endocytotic transmembrane protein mediates internalization of HIV-1

Virology ◽

10.1016/s0042-6822(03)00514-2 ◽

2003 ◽

Vol 315 (1) ◽

pp. 80-92 ◽

Cited By ~ 6

Author(s):

Yee-Joo Tan ◽

Siew-Pheng Lim ◽

Anthony E Ting ◽

Phuay-Yee Goh ◽

Y.H Tan ◽

...

Keyword(s):

Transmembrane Protein ◽

Anti Hiv ◽

Hiv 1

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Molecular Mechanism of BST2/Tetherin Downregulation by K5/MIR2 of Kaposi's Sarcoma-Associated Herpesvirus

Journal of Virology ◽

10.1128/jvi.00597-09 ◽

2009 ◽

Vol 83 (19) ◽

pp. 9672-9681 ◽

Cited By ~ 175

Author(s):

Mandana Mansouri ◽

Kasinath Viswanathan ◽

Janet L. Douglas ◽

Jennie Hines ◽

Jean Gustin ◽

...

Keyword(s):

Kaposi's Sarcoma ◽

Defense Mechanism ◽

Transmembrane Protein ◽

Kaposi’S Sarcoma ◽

Dependent Manner ◽

Type Ii ◽

Amino Terminal ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Hiv 1

ABSTRACT K3/MIR1 and K5/MIR2 of Kaposi's sarcoma-associated herpesvirus (KSHV) are viral members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family and contribute to viral immune evasion by directing the conjugation of ubiquitin to immunostimulatory transmembrane proteins. In a quantitative proteomic screen for novel host cell proteins downregulated by viral immunomodulators, we previously observed that K5, as well as the human immunodeficiency virus type 1 (HIV-1) immunomodulator VPU, reduced steady-state levels of bone marrow stromal cell antigen 2 (BST2; also called CD317 or tetherin), suggesting that BST2 might be a novel substrate of K5 and VPU. Recent work revealed that in the absence of VPU, HIV-1 virions are tethered to the plasma membrane in BST2-expressing HeLa cells. By targeting BST2, K5 might thus similarly overcome an innate antiviral host defense mechanism. Here we establish that despite its type II transmembrane topology and carboxy-terminal glycosylphosphatidylinositol (GPI) anchor, BST2 represents a bona fide target of K5 that is downregulated during primary infection by and reactivation of KSHV. Upon exit of the protein from the endoplasmic reticulum, lysines in the short amino-terminal domain of BST2 are ubiquitinated by K5, resulting in rapid degradation of BST2. Ubiquitination of BST2 is required for degradation, since BST2 lacking cytosolic lysines was K5 resistant and ubiquitin depletion by proteasome inhibitors restored BST2 surface expression. Thus, BST2 represents the first type II transmembrane protein targeted by K5 and the first example of a protein that is both ubiquitinated and GPI linked. We further demonstrate that KSHV release is decreased in the absence of K5 in a BST2-dependent manner, suggesting that K5 contributes to the evasion of intracellular antiviral defense programs.

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Characterization of IgG and IgG Subclass Antibodies Present in Paired Maternal and Fetal Serum Which Are Directed Against HIV-1 Proteins

AIDS Research and Human Retroviruses ◽

10.1089/aid.1991.7.847 ◽

1991 ◽

Vol 7 (10) ◽

pp. 847-854 ◽

Cited By ~ 8

Author(s):

TOBIAS R. KOLLMANN ◽

ARYE RUBINSTEIN ◽

WILLIAM D. LYMAN ◽

RUY SOEIRO ◽

HARRIS GOLDSTEIN

Keyword(s):

Igg Subclass ◽

Fetal Serum ◽

Hiv 1

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Identification of a conserved domain of the HIV-1 transmembrane protein gp41 which interacts with cholesteryl groups

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/s0005-2736(02)00611-9 ◽

2002 ◽

Vol 1567 ◽

pp. 157-164 ◽

Cited By ~ 84

Author(s):

Nadine Vincent ◽

Christian Genin ◽

Etienne Malvoisin

Keyword(s):

Transmembrane Protein ◽

Conserved Domain ◽

Protein Gp41 ◽

Hiv 1

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Role of MxB in Alpha Interferon-Mediated Inhibition of HIV-1 Infection

Journal of Virology ◽

10.1128/jvi.00422-18 ◽

2018 ◽

Vol 92 (17) ◽

Cited By ~ 10

Author(s):

Bin Xu ◽

Qinghua Pan ◽

Chen Liang

Keyword(s):

G Protein ◽

Transmembrane Protein ◽

Alpha Interferon ◽

Wild Type Virus ◽

Target Cells ◽

Type I ◽

Wide Range ◽

Anti Hiv ◽

Hiv 1

ABSTRACTType I interferon inhibits viruses through inducing the expression of antiviral proteins, including the myxovirus resistance (Mx) proteins. Compared to the human MxA protein, which inhibits a wide range of viruses, the MxB protein has been reported to specifically inhibit primate lentiviruses, including HIV-1, and herpesviruses. Further, the role of endogenous MxB in alpha interferon-mediated inhibition of HIV-1 infection was questioned by a recent study showing that MxB knockout did not increase the level of infection by HIV-1 which carried the G protein of vesicular stomatitis virus (VSV), allowing infection of CD4-negative HT1080 cells. In order to further examine the anti-HIV-1 activity of endogenous MxB, we have used CRISPR/Cas9 to deplete MxB in different cell lines and observed a substantial restoration of HIV-1 infection in the presence of alpha interferon treatment. However, this rescue effect of MxB knockout became much less pronounced when infection was performed with HIV-1 carrying the VSV G protein. Interestingly, a CRISPR/Cas9 knockout screen of alpha interferon-stimulated genes in U87-MG cells revealed that the genes for interferon-induced transmembrane protein 2 (IFITM2) and IFITM3 inhibited VSV G-pseudotyped HIV-1 much more strongly than the rest of the genes tested, including the gene for MxB. Therefore, our results demonstrate the importance of MxB in alpha interferon-mediated inhibition of HIV-1 infection, which, however, can be underestimated if infection is performed with VSV G protein-pseudotyped HIV-1, due to the high sensitivity of VSV G-mediated infection to inhibition by IFITM proteins.IMPORTANCEThe results of this study reconcile the controversial reports regarding the anti-HIV-1 function of alpha interferon-induced MxB protein. In addition to the different cell types that may have contributed to the different observations, our data also suggest that VSV G protein-pseudotyped HIV-1 is much less inhibited by alpha interferon-induced MxB than HIV-1 itself is. Our results clearly demonstrate an important contribution of MxB to alpha interferon-mediated inhibition of HIV-1 in CD4+T cells, which calls for using HIV-1 target cells and wild-type virus to test the relevance of the anti-HIV-1 activity of endogenous MxB and other restriction factors.

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Bending of the BST-2 coiled-coil during viral budding

10.1101/160242 ◽

2017 ◽

Author(s):

Kadir A. Ozcan ◽

Christopher E. Berndsen

Keyword(s):

Cell Membrane ◽

Host Cell ◽

Transmembrane Protein ◽

Coiled Coil ◽

Membrane Anchor ◽

Mouse Homolog ◽

Host Cell Membrane ◽

Structural Mechanism ◽

Viral Budding ◽

Hiv 1

AbstractBST-2/tetherin is a human extracellular transmembrane protein that serves as a host defense factor against HIV-1 and other viruses by inhibiting viral spreading. Structurally, BST-2 is a homodimeric coiled-coil that is connected to the host cell membrane by N and C terminal transmembrane anchors. The C-terminal membrane anchor of BST-2 is inserted into the budding virus while the N-terminal membrane anchor remains in the host cell membrane creating a viral tether. The structural mechanism of viral budding and tethering as mediated by BST-2 is not clear. To more fully describe the mechanism of viral tethering, we created a model of BST-2 embedded in a membrane and used steered molecular dynamics to simulate the transition from the host cell membrane associated BST-2 and the cell-virus membrane bridging form. We observed that BST-2 did not transition as a rigid structure, but instead bent at sites with a reduced interface between the helices of the coiled-coil. The simulations for the human BST-2 were then compared with simulations on the mouse homolog, which has a more stable coiled-coil. We observed that the mouse homolog spread the bending across the ectodomain, rather than breaking at discrete points as observed with the human homolog. These simulations support previous biochemical and cellular work suggesting some flexibility in the coiled-coil is necessary for viral tethering, while also highlighting how subtle changes in protein sequence can influence the dynamics and stability of proteins with overall similar structure.

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HIV-1 Envelope and MPER antibody structures in lipid assemblies

10.1101/838912 ◽

2019 ◽

Author(s):

Kimmo Rantalainen ◽

Zachary T. Berndsen ◽

Aleksandar Antanasijevic ◽

Torben Schiffner ◽

Xi Zhang ◽

...

Keyword(s):

Electron Microscopy ◽

Transmembrane Protein ◽

Full Length ◽

Structural Studies ◽

Wild Type ◽

Functional Studies ◽

Antibody Complex ◽

Modular Platform ◽

Lipid Assemblies ◽

Hiv 1

SummaryStructural and functional studies of HIV Env as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the membrane-embedded hydrophobic regions. Thus, most structural studies have utilized soluble forms where the regions C-terminal to the ectodomain are deleted. Here, we present approaches for incorporating full-length, wild-type HIV-1 Env, as well as C-terminally truncated and stabilized versions, into lipid assemblies, providing a modular platform for Env structural studies by single particle electron microscopy. We reconstituted a full-length Env clone into a nanodisc with MSP1D1 scaffold, complexed it with an MPER targeting antibody 10E8, and structurally defined the full quaternary epitope of 10E8 consisting of lipid, MPER and ectodomain contacts. By aligning this and other Env-MPER antibody complex reconstructions with the lipid bilayer, we observe evidence of Env tilting as part of the neutralization mechanism for MPER-targeting antibodies. We also adapted the platform toward vaccine design purposes by introducing stabilizing mutations that allow purification of unliganded Env with peptidisc scaffold.

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