Multiple nonfunctional alleles of CCR5 are frequent in various human populations

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1638-1645 ◽  
Author(s):  
Cédric Blanpain ◽  
Benhur Lee ◽  
Marie Tackoen ◽  
Bridget Puffer ◽  
Alain Boom ◽  
...  
Keyword(s):  
Cell Surface ◽  
Receptor Activation ◽  
Human Populations ◽  
Type I ◽  
Binding Affinities ◽  
Functional Responses ◽  
Functional Consequences ◽  
Relative Inability ◽  
Specific Alteration ◽  
Hiv 1

CCR5 is the major coreceptor for macrophage-tropic strains of the human immunodeficiency virus type I (HIV-1). Homozygotes for a 32-base pair (bp) deletion in the coding sequence of the receptor (CCR5Δ32) were found to be highly resistant to viral infection, and CCR5 became, therefore, one of the paradigms illustrating the influence of genetic variability onto individual susceptibility to infectious and other diseases. We investigated the functional consequences of 16 other natural CCR5 mutations described in various human populations. We found that 10 of these variants are efficiently expressed at the cell surface, bind [125I]-MIP-1β with affinities similar to wtCCR5, respond functionally to chemokines, and act as HIV-1 coreceptors. In addition to Δ32, six mutations were characterized by major alterations in their functional response to chemokines, as a consequence of intracellular trapping and poor expression at the cell surface (C101X, FS299), general or specific alteration of ligand binding affinities (C20S, C178R, A29S), or relative inability to mediate receptor activation (L55Q). A29S displayed an unusual pharmacological profile, binding and responding to MCP-2 similarly to wtCCR5, but exhibiting severely impaired binding and functional responses to MIP-1α, MIP-1β, and RANTES. In addition to Δ32, only C101X was totally unable to mediate entry of HIV-1. The fact that nonfunctional CCR5 alleles are relatively frequent in various human populations reinforces the hypothesis of a selective pressure favoring these alleles.

Multiple nonfunctional alleles of CCR5 are frequent in various human populations

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1638-1645 ◽  
Author(s):  
Cédric Blanpain ◽  
Benhur Lee ◽  
Marie Tackoen ◽  
Bridget Puffer ◽  
Alain Boom ◽  
...  
Keyword(s):  
Cell Surface ◽  
Receptor Activation ◽  
Human Populations ◽  
Type I ◽  
Binding Affinities ◽  
Functional Responses ◽  
Functional Consequences ◽  
Relative Inability ◽  
Specific Alteration ◽  
Hiv 1

Abstract CCR5 is the major coreceptor for macrophage-tropic strains of the human immunodeficiency virus type I (HIV-1). Homozygotes for a 32-base pair (bp) deletion in the coding sequence of the receptor (CCR5Δ32) were found to be highly resistant to viral infection, and CCR5 became, therefore, one of the paradigms illustrating the influence of genetic variability onto individual susceptibility to infectious and other diseases. We investigated the functional consequences of 16 other natural CCR5 mutations described in various human populations. We found that 10 of these variants are efficiently expressed at the cell surface, bind [125I]-MIP-1β with affinities similar to wtCCR5, respond functionally to chemokines, and act as HIV-1 coreceptors. In addition to Δ32, six mutations were characterized by major alterations in their functional response to chemokines, as a consequence of intracellular trapping and poor expression at the cell surface (C101X, FS299), general or specific alteration of ligand binding affinities (C20S, C178R, A29S), or relative inability to mediate receptor activation (L55Q). A29S displayed an unusual pharmacological profile, binding and responding to MCP-2 similarly to wtCCR5, but exhibiting severely impaired binding and functional responses to MIP-1α, MIP-1β, and RANTES. In addition to Δ32, only C101X was totally unable to mediate entry of HIV-1. The fact that nonfunctional CCR5 alleles are relatively frequent in various human populations reinforces the hypothesis of a selective pressure favoring these alleles.

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

2019 ◽  
Vol 93 (24) ◽  
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.

scholarly journals Functional Analysis of the Transmembrane (TM) Domain of the Autographa californica Multicapsid Nucleopolyhedrovirus GP64 Protein: Substitution of Heterologous TM Domains

2008 ◽  
Vol 82 (7) ◽  
pp. 3329-3341 ◽  
Author(s):  
Zhaofei Li ◽  
Gary W. Blissard
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
Membrane Fusion ◽  
Low Ph ◽  
Autographa Californica ◽  
Virus Infectivity ◽  
Type I ◽  
Gpi Anchor ◽  
Tm Domain ◽  
Hiv 1

ABSTRACT GP64, the major envelope glycoprotein of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) budded virion, is important for host cell receptor binding and mediates low-pH-triggered membrane fusion during entry by endocytosis. In the current study, we examined the functional role of the AcMNPV GP64 transmembrane (TM) domain by replacing the 23-amino-acid GP64 TM domain with corresponding TM domain sequences from a range of viral and cellular type I membrane proteins, including Orgyia pseudotsugata MNPV (OpMNPV) GP64 and F, thogotovirus GP75, Lymantria dispar MNPV (LdMNPV) F, human immunodeficiency virus type 1 (HIV-1) GP41, human CD4 and glycophorin A (GpA), and influenza virus hemagglutinin (HA), and with a glycosylphosphatidylinositol (GPI) anchor addition sequence. In transient expression experiments with Sf9 cells, chimeric GP64 proteins containing either a GPI anchor or TM domains from LdMNPV F or HIV-1 GP41 failed to localize to the cell surface and thus appear to be incompatible with either GP64 structure or cell transport. All of the mutant constructs detected at the cell surface mediated hemifusion (outer leaflet merger) upon low-pH treatment, but only those containing TM domains from CD4, GpA, OpMNPV GP64, and thogotovirus GP75 mediated pore formation and complete membrane fusion activity. This supports a model in which partial fusion (hemifusion) proceeds by a mechanism that is independent of the TM domain and the TM domain participates in the enlargement or expansion of fusion pores after hemifusion. GP64 proteins containing heterologous TM domains mediated virion budding with dramatically differing levels of efficiency. In addition, chimeric GP64 proteins containing TM domains from CD4, GpA, HA, and OpMNPV F were incorporated into budded virions but were unable to rescue the infectivity of a gp64 null virus, whereas those with TM domains from OpMNPV GP64 and thogotovirus GP75 rescued infectivity. These results show that in addition to its basic role in membrane anchoring, the GP64 TM domain is critically important for GP64 trafficking, membrane fusion, virion budding, and virus infectivity. These critical functions were replaced only by TM domains from related viral membrane proteins.

scholarly journals Single-molecule imaging reveals distinct effects of ligands on CCR5 dynamics depending on its dimerization status

2021 ◽  
Author(s):  
Fanny Momboisse ◽  
Giacomo Nardi ◽  
Philippe Colin ◽  
Melanie Hery ◽  
Nelia Cordeiro ◽  
...  
Keyword(s):  
Cell Surface ◽  
Single Molecule ◽  
Opposite Effect ◽  
Receptor Activation ◽  
Agonist Stimulation ◽  
Ligand Free ◽  
Human Immunodeficiency Viruses ◽  
Cc Chemokine Receptor 5 ◽  
G Protein Coupled ◽  
Hiv 1

G protein-coupled receptors (GPCR) are present at the cell surface in different conformational and oligomeric states. However, how these states impact GPCRs biological function and therapeutic targeting remains incompletely known. Here, we investigated this issue in living cells for the CC chemokine receptor 5 (CCR5), a major receptor in inflammation and the principal entry co-receptor for Human Immunodeficiency Viruses (HIV-1). We used TIRF microscopy and an original statistical method to track and classify the motion of different receptors subpopulations. We showed a diversity of ligand-free forms of CCR5 at the cell surface constituted of various oligomeric states and exhibiting transient Brownian and restricted motions. These forms were stabilized differently by distinct ligands. In particular, agonist stimulation restricted the mobility of CCR5 and led to its clustering, a feature depending on b-arrestin, while inverse agonist stimulation exhibited the opposite effect. These results suggest a link between receptor activation and immobilization. Applied to HIV-1 envelope glycoproteins gp120, our quantitative analysis revealed agonist-like properties of gp120s. Distinct gp120s influenced CCR5 dynamics differently, suggesting that they stabilize different CCR5 conformations. Then, using a dimerization-compromized mutant, we showed that dimerization (i) impacts CCR5 precoupling to G proteins, (ii) is a pre-requisite for the immobilization and clustering of receptors upon activation, and (iii) regulates receptor endocytosis, thereby impacting the fate of activated receptors. This study demonstrates that tracking the dynamic behavior of a GPCR is an efficient way to link GPCR conformations to their functions, therefore improving the development of drugs targeting specific receptor conformations.

scholarly journals Human plasmacytoid dendritic cells mount a distinct antiviral response to virus-infected cells

2021 ◽  
Vol 6 (58) ◽  
pp. eabc7302
Author(s):  
Tae Jin Yun ◽  
Suzu Igarashi ◽  
Haoquan Zhao ◽  
Oriana A. Perez ◽  
Marcus R. Pereira ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Virus Detection ◽  
Plasmacytoid Dendritic Cells ◽  
Lung Epithelial Cells ◽  
Live Cells ◽  
Type I ◽  
Antiviral Immune Response ◽  
Lung Epithelial ◽  
Functional Consequences ◽  
Infected Cells

Plasmacytoid dendritic cells (pDCs) can rapidly produce interferons and other soluble factors in response to extracellular viruses or virus mimics such as CpG-containing DNA. pDCs can also recognize live cells infected with certain RNA viruses, but the relevance and functional consequences of such recognition remain unclear. We studied the response of primary DCs to the prototypical persistent DNA virus, human cytomegalovirus (CMV). Human pDCs produced high amounts of type I interferon (IFN-I) when incubated with live CMV-infected fibroblasts but not with free CMV; the response involved integrin-mediated adhesion, transfer of DNA-containing virions to pDCs, and the recognition of DNA through TLR9. Compared with transient polyfunctional responses to CpG or free influenza virus, pDC response to CMV-infected cells was long-lasting, dominated by the production of IFN-I and IFN-III, and lacked diversification into functionally distinct populations. Similarly, pDC activation by influenza-infected lung epithelial cells was highly efficient, prolonged, and dominated by interferon production. Prolonged pDC activation by CMV-infected cells facilitated the activation of natural killer cells critical for CMV control. Last, patients with CMV viremia harbored phenotypically activated pDCs and increased circulating IFN-I and IFN-III. Thus, recognition of live infected cells is a mechanism of virus detection by pDCs that elicits a unique antiviral immune response.

scholarly journals STING and cGAS gene expressions were downregulated among HIV-1-infected persons after antiretroviral therapy

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Lorena Leticia Peixoto de Lima ◽  
Allysson Quintino Tenório de Oliveira ◽  
Tuane Carolina Ferreira Moura ◽  
Ednelza da Silva Graça Amoras ◽  
Sandra Souza Lima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Load ◽  
T Cell ◽  
Antiretroviral Therapy ◽  
Cell Count ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Α Level ◽  
The Impact ◽  
Hiv 1

Abstract Background The HIV-1 epidemic is still considered a global public health problem, but great advances have been made in fighting it by antiretroviral therapy (ART). ART has a considerable impact on viral replication and host immunity. The production of type I interferon (IFN) is key to the innate immune response to viral infections. The STING and cGAS proteins have proven roles in the antiviral cascade. The present study aimed to evaluate the impact of ART on innate immunity, which was represented by STING and cGAS gene expression and plasma IFN-α level. Methods This cohort study evaluated a group of 33 individuals who were initially naïve to therapy and who were treated at a reference center and reassessed 12 months after starting ART. Gene expression levels and viral load were evaluated by real-time PCR, CD4+ and CD8+ T lymphocyte counts by flow cytometry, and IFN-α level by enzyme-linked immunosorbent assay. Results From before to after ART, the CD4+ T cell count and the CD4+/CD8+ ratio significantly increased (p < 0.0001), the CD8+ T cell count slightly decreased, and viral load decreased to undetectable levels in most of the group (84.85%). The expression of STING and cGAS significantly decreased (p = 0.0034 and p = 0.0001, respectively) after the use of ART, but IFN-α did not (p = 0.1558). Among the markers evaluated, the only markers that showed a correlation with each other were STING and CD4+ T at the time of the first collection. Conclusions ART provided immune recovery and viral suppression to the studied group and indirectly downregulated the STING and cGAS genes. In contrast, ART did not influence IFN-α. The expression of STING and cGAS was not correlated with the plasma level of IFN-α, which suggests that there is another pathway regulating this cytokine in addition to the STING–cGAS pathway.

scholarly journals A Single-Pass Type I Membrane Protein from the Apicomplexan Parasite Cryptosporidium parvum with Nanomolar Binding Affinity to Host Cell Surface

2021 ◽  
Vol 9 (5) ◽  
pp. 1015
Author(s):  
Tianyu Zhang ◽  
Xin Gao ◽  
Dongqiang Wang ◽  
Jixue Zhao ◽  
Nan Zhang ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Host Cell ◽  
Binding Affinity ◽  
Cryptosporidium Parvum ◽  
Host Cells ◽  
Watery Diarrhea ◽  
Type I ◽  
Single Pass ◽  
Host Cell Surface

Cryptosporidium parvum is a globally recognized zoonotic parasite of medical and veterinary importance. This parasite mainly infects intestinal epithelial cells and causes mild to severe watery diarrhea that could be deadly in patients with weakened or defect immunity. However, its molecular interactions with hosts and pathogenesis, an important part in adaptation of parasitic lifestyle, remain poorly understood. Here we report the identification and characterization of a C. parvum T-cell immunomodulatory protein homolog (CpTIPH). CpTIPH is a 901-aa single-pass type I membrane protein encoded by cgd5_830 gene that also contains a short Vibrio, Colwellia, Bradyrhizobium and Shewanella (VCBS) repeat and relatively long integrin alpha (ITGA) N-terminus domain. Immunofluorescence assay confirmed the location of CpTIPH on the cell surface of C. parvum sporozoites. In congruence with the presence of VCBS repeat and ITGA domain, CpTIPH displayed high, nanomolar binding affinity to host cell surface (i.e., Kd(App) at 16.2 to 44.7 nM on fixed HCT-8 and CHO-K1 cells, respectively). The involvement of CpTIPH in the parasite invasion is partly supported by experiments showing that an anti-CpTIPH antibody could partially block the invasion of C. parvum sporozoites into host cells. These observations provide a strong basis for further investigation of the roles of CpTIPH in parasite-host cell interactions.

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