Localization to detergent-resistant membranes and HIV-1 core entry inhibition correlate with HIV-1 restriction by SERINC5

Schulte, Bianca; Selyutina, Anastasia; Opp, Silvana; Herschhorn, Alon; Sodroski, Joseph; Pizzato, Massimo; Díaz-Griffero, Felipe

doi:10.1016/j.virol.2017.12.005

Cited by 44 publications

(86 citation statements)

References 27 publications

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“…We produced fluorescent NL4-3 and THRO virions in 293T or 293T cells stably expressing CD4 (293T-CD4), with or without transient transfection of SERINC5. To limit possible artifacts due to excessive expression of SERINC5 in producer cells, we used 20-fold less SERINC5 plasmid than proviral DNA (57). We tested the infectivity of viral particles and stained producer cells and ultracentrifuged viral particles as described above.…”

Section: Resultsmentioning

confidence: 99%

“…In particular, it will be interesting to investigate directly how SERINC5 affects the exposure of other Env domains, such as the Gp41/Gp120 interface or MPER. Previous studies suggested that SERINC5 significantly increases the neutralization sensitivity to these anti-Env antibod-ies (41,57,67). Also, including Nef mutants unable to counteract SERINC5 (68) will help dissecting the complex interplay between Nef, Env, and SERINC5 proteins.…”

Section: Discussionmentioning

confidence: 99%

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Flow Cytometry Analysis of HIV-1 Env Conformations at the Surface of Infected Cells and Virions: Role of Nef, CD4, and SERINC5

Staropoli

Dufloo

Ducher

et al. 2020

J Virol

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The HIV-1 Env protein is exposed at the surface of virions and infected cells. Env fluctuates between different closed and open structural states and these conformations influence both viral infectivity and sensitivity to antibody binding and neutralization. We established a flow virometry assay to visualize Env proteins at the surface of human immunodeficiency virus type 1 (HIV-1) virions. The assay is performed on ultracentrifuged fluorescent viral particles that are stained with a panel of broadly neutralizing antibodies (bNAbs) and nonneutralizing antibodies (nnAbs) that probe different epitopes of Env. We used this assay to compare Env at the surface of producer cells and viral particles and to analyze the effect of Nef, CD4, and SERINC5 on Env accessibility to antibodies. We studied the laboratory-adapted strain NL4-3 and two transmitted/founder viruses, THRO and CH058. We confirm that antibody accessibility varies between viral strains and show that Nef, CD4, and SERINC5 additively impact Env conformations. We further demonstrate that the Env accessibility profile on virions is globally similar to that observed on HIV-1-infected cells, with some noticeable differences. For instance, nnAbs bind to virions more efficiently than to producer cells, likely reflecting changes in Env conformational states on mature viral particles. This test complements other techniques and provides a convenient and simple tool for quantifying and probing the structure of Env at the virion surface and to analyze the impact of viral and cellular proteins on these parameters. IMPORTANCE HIV-1 Env conformation is one of the key parameters determining viral infectivity. The flow virometry-based assay developed in this study allows for the characterization of proteins incorporated in HIV-1 particles. We studied the conformation of HIV-1 Env and the impact that the viral protein Nef and the cellular proteins CD4 and SERINC5 have on Env accessibility to antibodies. Our assay permitted us to highlight some noticeable differences in the conformation of Env between producer cells and viral particles. It contributes to a better understanding of the actual composition of HIV-1 particles.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Flow Cytometry Analysis of HIV-1 Env Conformations at the Surface of Infected Cells and Virions: Role of Nef, CD4, and SERINC5

Staropoli

Dufloo

Ducher

et al. 2020

J Virol

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“…Further studies will be required to test the functional significance of phosphatidylserine, sulfatide and/or cholesterol binding in biological functions of SERINC proteins. Affinity for cholesterol and/or exposed phosphatidylserine could help explain the association of SERINC5 with lipid rafts 9,26 and ultimately incorporation of the protein into budding virions.…”

Section: Discussionmentioning

confidence: 99%

“…To identify regions of SERINC5 involved in HIV-1 restriction, we conducted an extensive mutagenesis screen, informed by the structure and amino acid sequence conservation with SERINC2, which lacks the HIV-1 restriction activity 9,10 . The mutations were introduced into constructs designed to express SERINC5 harbouring a FLAG epitope implanted within ECL4.…”

Section: Mapping Serinc5 Regions Critical For the Antiviral Activitymentioning

confidence: 99%

A bipartite structural organization defines the SERINC family of HIV-1 restriction factors

Pye

Rosa

Bertelli

et al. 2020

Nat Struct Mol Biol

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108

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(MP). Data Availability. The cryo-EM maps and the refined atomic model of DmSERINC were deposited in the EMDB and wwPDB, respectively, with accession codes EMD-10277 and EMD-10279 and PDB 6SP2. Source data for Figures 4a, 4b, 4c, 4e and for Extended Data Figures 1a, 1c, 1d, 1e, 2b, 3b, 4g-i, 6e-g, are available with the paper online. Author Contributions V.E.P. expressed, purified and characterised DmSERINC, built the atomic model, developed and conducted thermostability assays; V.E.P., P.C., and A.B.-C. prepared and screened cryo-EM grids; A.N. collected all cryo-EM data; V.E.P. and P.C. refined the DmSERINC structure; A.R. and P.C. generated stable cell line for SERINC5 expression, purified and characterised SERINC5 and determined the structure; A.R. conducted thermostability assays on SERINC5 and purified the Fab; P.C. produced mutant SERINC5 constructs; M.P. and C.B. developed and performed assays to measure surface exposure, restriction activity and virion incorporation of SERINC5 variants; W.

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“…SERINC5 seems to be the most potent restrictor and accounts for the majority of the Nef effect (at least in T cell lines); in contrast, SERINC3 has modest restrictive activity, and SERINC2 is devoid of restrictive activity (16,17). The study of chimeras between the nonrestrictive SERINC2 and the restrictive SERINC5 led to a partial mapping of SERINC5's restrictive activity (20). While the ability of SERINC5 to restrict HIV-1 infectivity is conserved among vertebrates, sensitivity to Nef is not.…”

mentioning

confidence: 99%

An N-Glycosylated Form of SERINC5 Is Specifically Incorporated into HIV-1 Virions

Sharma

Lewinski

2018

J Virol

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SERINC5 is an inhibitor of retroviral infectivity that is counteracted by viral proteins, including HIV-1 Nef. Inhibition of infectivity by SERINC5 is associated with its incorporation into virions. Nef counteracts this inhibition, presumably by removing SERINC5 from sites of virion assembly at the plasma membrane. While evaluating the virion incorporation of SERINC5, we observed that a relatively high molecular weight form was preferentially present in virions. We used various glycosidases to establish that virion-associated SERINC5 is modified by N-linked, complex glycans, whereas the majority of SERINC5 in cells is of relatively low molecular weight and is modified by high-mannose glycans. Sequence alignment of SERINC family proteins led us to identify a conserved N-glycosylation site, N294, in SERINC5. We mutated this site to evaluate its effect on glycosylation, the restrictive activity of SERINC5, and the sensitivity of SERINC5 to antagonism by Nef. Our results demonstrate that N294 is the major site of N-glycosylation in SERINC5. Although N-glycosylation was required neither for restrictive activity nor for sensitivity to Nef , we observed a decrease in the steady-state expression of glycosylation-deficient SERINC5 (the N294A mutant) compared to the wild-type protein. Expression of this mutant was partly restored by treatment of cells with MG132 (a proteasome inhibitor) but not with bafilomycin A1 (a lysosomal inhibitor). We conclude that although not required for restrictive activity or Nef sensitivity, N-linked glycosylation is important for maintaining the steady-state expression of SERINC5 and that nonglycosylated SERINC5 is likely subjected to a quality control mechanism that induces its proteasomal degradation. SERINC5 is a member of a family of multipass transmembrane proteins that inhibit the infectivity of retroviruses, including HIV-1. These proteins are incorporated into virions and inhibit infection of target cells unless counteracted by viral antagonists such as HIV-1 Nef. The only other biological function with which these proteins have been associated is the formation of serine-containing membrane lipids. Here we show that SERINC5 is a glycosylated protein and that N-glycosylation is important for its steady-state expression. In the absence of N-glycosylation, SERINC5 is prone to proteasomal degradation. Nonetheless, N-glycosylation is required neither for the ability of SERINC5 to inhibit HIV-1 infectivity nor for its sensitivity to antagonism by Nef.

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Localization to detergent-resistant membranes and HIV-1 core entry inhibition correlate with HIV-1 restriction by SERINC5

Cited by 44 publications

References 27 publications

Flow Cytometry Analysis of HIV-1 Env Conformations at the Surface of Infected Cells and Virions: Role of Nef, CD4, and SERINC5

Flow Cytometry Analysis of HIV-1 Env Conformations at the Surface of Infected Cells and Virions: Role of Nef, CD4, and SERINC5

A bipartite structural organization defines the SERINC family of HIV-1 restriction factors

An N-Glycosylated Form of SERINC5 Is Specifically Incorporated into HIV-1 Virions

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