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1.
Mol Biol Cell ; 12(11): 3451-64, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11694580

ABSTRACT

Melanosomes are tissue-specific organelles within which melanin is synthesized and stored. The melanocyte-specific glycoprotein Pmel17 is enriched in the lumen of premelanosomes, where it associates with characteristic striations of unknown composition upon which melanin is deposited. However, Pmel17 is synthesized as an integral membrane protein. To clarify its physical linkage to premelanosomes, we analyzed the posttranslational processing of human Pmel17 in pigmented and transfected nonpigmented cells. We show that Pmel17 is cleaved in a post-Golgi compartment into two disulfide-linked subunits: a large lumenal subunit, M alpha, and an integral membrane subunit, M beta. The two subunits remain associated intracellularly, indicating that detectable M alpha remains membrane bound. We have previously shown that Pmel17 accumulates on intralumenal membrane vesicles and striations of premelanosomes in pigmented cells. In transfected nonpigmented cells Pmel17 associates with the intralumenal membrane vesicles of multivesicular bodies; cells overexpressing Pmel17 also display structures resembling premelanosomal striations within these compartments. These results suggest that Pmel17 is sufficient to drive the formation of striations from within multivesicular bodies and is thus directly involved in the biogenesis of premelanosomes.


Subject(s)
Melanosomes/physiology , Neoplasm Proteins/physiology , Proteins/physiology , 3T3 Cells , Animals , Cytoplasmic Vesicles/metabolism , Cytoplasmic Vesicles/physiology , Disulfides , Gene Expression , HeLa Cells , Humans , Intracellular Membranes/metabolism , Kinetics , Melanosomes/metabolism , Membrane Glycoproteins , Mice , Morphogenesis , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Protein Processing, Post-Translational , Proteins/genetics , Proteins/metabolism , Tumor Cells, Cultured , gp100 Melanoma Antigen
2.
J Cell Biol ; 152(4): 809-24, 2001 Feb 19.
Article in English | MEDLINE | ID: mdl-11266471

ABSTRACT

Melanosomes and premelanosomes are lysosome-related organelles with a unique structure and cohort of resident proteins. We have positioned these organelles relative to endosomes and lysosomes in pigmented melanoma cells and melanocytes. Melanosome resident proteins Pmel17 and TRP1 localized to separate vesicular structures that were distinct from those enriched in lysosomal proteins. In immunogold-labeled ultrathin cryosections, Pmel17 was most enriched along the intralumenal striations of premelanosomes. Increased pigmentation was accompanied by a decrease in Pmel17 and by an increase in TRP1 in the limiting membrane. Both proteins were largely excluded from lysosomal compartments enriched in LAMP1 and cathepsin D. By kinetic analysis of fluid phase uptake and immunogold labeling, premelanosomal proteins segregated from endocytic markers within an unusual endosomal compartment. This compartment contained Pmel17, was accessed by BSA-gold after 15 min, was acidic, and displayed a cytoplasmic planar coat that contained clathrin. Our results indicate that premelanosomes and melanosomes represent a distinct lineage of organelles, separable from conventional endosomes and lysosomes within pigmented cells. Furthermore, they implicate an unusual clathrin-coated endosomal compartment as a site from which proteins destined for premelanosomes and lysosomes are sorted.


Subject(s)
Lysosomes/metabolism , Melanocytes/metabolism , Melanosomes/metabolism , Membrane Glycoproteins/metabolism , Neoplasm Proteins/metabolism , Clathrin-Coated Vesicles , Endocytosis , Endosomes , Lysosomal Storage Diseases/etiology , Models, Biological , Organelles/classification , Protein Sorting Signals , Protein Transport , Proteins , gp100 Melanoma Antigen
3.
J Biol Chem ; 275(16): 12281-9, 2000 Apr 21.
Article in English | MEDLINE | ID: mdl-10766867

ABSTRACT

Oculocutaneous albinism type 1TS is caused by mutations that render the melanocyte-specific enzyme tyrosinase temperature-sensitive (ts); the enzyme is inactive in cells grown at 37 degrees C but displays full activity in cells grown at 31 degrees C. To distinguish whether the ts phenotype of the common R402Q variant of human tyrosinase is due to altered enzymatic activity or to misfolding and a defect in intracellular trafficking, we analyzed its localization and processing in transiently transfected HeLa cells. R402Q tyrosinase accumulates in the endoplasmic reticulum (ER) at 37 degrees C but exits the ER and accumulates in endosomal structures in cells grown at 31 degrees C. The inability of the R402Q variant to exit the ER is confirmed by the failure to acquire endoglycosidase H resistance at 37 degrees C and cannot be accounted for solely by enhanced proteasome-mediated degradation. ER retention at 37 degrees C is mediated by the lumenal domain of R402Q tyrosinase, is not dependent on tethering to the membrane, and is irreversible. Finally, a wild-type allelic form of tyrosinase is partially ts in transiently transfected HeLa cells. The data show that human tyrosinase expressed in non-melanogenic cells folds and exits the ER inefficiently and that R402Q tyrosinase exaggerates this defect, resulting in a failure to exit the ER at physiologic temperatures.


Subject(s)
Alleles , Endoplasmic Reticulum/enzymology , Monophenol Monooxygenase/genetics , Amino Acid Sequence , Cysteine Endopeptidases/metabolism , HeLa Cells , Humans , Molecular Sequence Data , Multienzyme Complexes/metabolism , Phenotype , Proteasome Endopeptidase Complex , Protein Folding , Protein Processing, Post-Translational , Temperature
4.
J Biol Chem ; 274(18): 12780-9, 1999 Apr 30.
Article in English | MEDLINE | ID: mdl-10212263

ABSTRACT

Distinct cytoplasmic sorting signals target integral membrane proteins to late endosomal compartments, but it is not known whether different signals direct targeting by different pathways. The availability of multiple pathways may permit some cell types to divert proteins to specialized compartments, such as the melanosome of pigmented cells. To address this issue, we characterized sorting determinants of tyrosinase, a tissue-specific resident protein of the melanosome. The cytoplasmic domain of tyrosinase was both necessary and sufficient for internalization and steady state localization to late endosomes and lysosomes in HeLa cells. Mutagenesis of two leucine residues within a conventional di-leucine motif ablated late endosomal localization. However, the properties of this di-leucine-based signal were distinguished from that of CD3gamma by overexpression studies; overexpression of the tyrosinase signal, but not the well characterized CD3gamma signal, induced a 4-fold enlargement of late endosomes and lysosomes and interfered with endosomal sorting mediated by both tyrosine- and other di-leucine-based signals. These properties suggest that the tyrosinase and CD3gamma di-leucine signals are distinctly recognized and sorted by distinct pathways to late endosomes in non-pigmented cells. We speculate that melanocytic cells utilize the second pathway to divert proteins to the melanosome.


Subject(s)
Cytoplasm/enzymology , Endosomes/enzymology , Leucine/metabolism , Lysosomes/enzymology , Monophenol Monooxygenase/metabolism , Amino Acid Sequence , Cell Line , Dipeptides/metabolism , Endocytosis , Humans , Molecular Sequence Data , Monophenol Monooxygenase/chemistry , Protein Sorting Signals/metabolism , Tyrosine/metabolism
5.
J Virol ; 73(4): 2752-61, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10074122

ABSTRACT

The interaction of the chemokine stromal cell-derived factor 1 (SDF-1) with its receptor CXCR4 is vital for cell trafficking during development, is capable of inhibiting human immunodeficiency virus type 1 (HIV-1) utilization of CXCR4 as a coreceptor, and has been implicated in delaying disease progression to AIDS in vivo. Because of the importance of this chemokine-chemokine receptor pair to both development and disease, we investigated the molecular basis of the interaction between CXCR4 and its ligands SDF-1 and HIV-1 envelope. Using CXCR4 chimeras and mutants, we determined that SDF-1 requires the CXCR4 amino terminus for binding and activates downstream signaling pathways by interacting with the second extracellular loop of CXCR4. SDF-1-mediated activation of CXCR4 required the Asp-Arg-Tyr motif in the second intracellular loop of CXCR4, was pertussis toxin sensitive, and did not require the distal C-terminal tail of CXCR4. Several CXCR4 mutants that were not capable of binding SDF-1 or signaling still supported HIV-1 infection, indicating that the ability of CXCR4 to function as a coreceptor is independent of its ability to signal. Direct binding studies using the X4 gp120s HXB, BH8, and MN demonstrated the ability of HIV-1 gp120 to bind directly and specifically to the chemokine receptor CXCR4 in a CD4-dependent manner, using a conformationally complex structure on CXCR4. Several CXCR4 variants that did not support binding of soluble gp120 could still function as viral coreceptors, indicating that detectable binding of monomeric gp120 is not always predictive of coreceptor function.


Subject(s)
Chemokines, CXC/metabolism , HIV Envelope Protein gp120/metabolism , Receptors, CXCR4/metabolism , Amino Acid Sequence , Cell Line , Chemokine CXCL12 , Humans , Molecular Sequence Data , Receptors, CXCR4/genetics , Signal Transduction
6.
Semin Immunol ; 10(3): 237-48, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9653050

ABSTRACT

Ten seven-transmembrane-domain G protein-coupled receptors have been identified that are functional HIV-1, HIV-2 and SIV coreceptors. However, the specific structures these receptors have in common that enable them to mediate HIV entry are unknown. Structure-function analyses have revealed that the determinants of coreceptor activity are distinct for each coreceptor, coreceptor activity is dependent on multiple extracellular domains, and various envelope proteins may interact differently with the same coreceptor. G protein coupling and receptor internalization are not required for fusion and infection of established cell lines, or for inhibition of infection by chemokines. The structure-function studies have also helped determine the mechanism by which previously described small molecules inhibit HIV-1 entry. Finally, these studies have led to a hypothesis as to how coreceptor utilization evolves during the course of an infection.


Subject(s)
HIV Infections/immunology , HIV Infections/virology , HIV-1/immunology , HIV-2/immunology , Receptors, CCR5/chemistry , Receptors, CCR5/physiology , Receptors, CXCR4/chemistry , Receptors, CXCR4/physiology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/immunology , Amino Acid Sequence , Animals , Chemokines/physiology , Humans , Molecular Sequence Data , Receptors, Virus/chemistry , Receptors, Virus/physiology , Structure-Activity Relationship
7.
J Biol Chem ; 273(24): 15007-15, 1998 Jun 12.
Article in English | MEDLINE | ID: mdl-9614108

ABSTRACT

The interaction of human immunodeficiency virus type 1 (HIV-1) with CD4 and one of a cadre of chemokine receptors triggers conformational changes in the HIV-1 envelope (Env) glycoprotein that lead to membrane fusion. The coreceptor activity of the second extracellular loop of CXCR4, which is restricted to dual tropic and T-tropic strains, was insensitive to the removal of charged residues either singly or in combinations by alanine scanning mutagenesis or to the conversion of acidic residues to lysine. Conversion of Asp-187 to a neutral residue exclusively unmasked activity with M-tropic Env in fusion and infection experiments. Insertion of the D187V mutation into chimeras containing extracellular loop 2 of CXCR4 in a CXCR2 framework also resulted in the acquisition of M-tropic coreceptor activity. The independence of CXCR4 coreceptor activity from charged residues and the extension of its repertoire by removing Asp-187 suggest that this interaction is not electrostatic and that coreceptors have the potential to be utilized by a spectrum of Env, which may be masked by charged amino acids in extracellular domains. These findings indicate that the primary structural determinants of coreceptors that program reactivity with M-, dual, and T-tropic Env are surprisingly subtle and that relatively insignificant changes in CXCR4 can dramatically alter utilization by Env of varying tropism.


Subject(s)
HIV-1/metabolism , Receptors, CXCR4/chemistry , Tropism/physiology , Flow Cytometry , HIV Envelope Protein gp120/metabolism , Membrane Fusion/genetics , Membrane Proteins/chemistry , Mutagenesis, Site-Directed/genetics , Receptors, CXCR4/genetics , Receptors, Cell Surface/physiology , Receptors, Chemokine/genetics , Receptors, Interleukin/genetics , Receptors, Interleukin-8B , Recombinant Fusion Proteins/chemistry , T-Lymphocytes/virology
8.
J Virol ; 72(3): 2509-15, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9499115

ABSTRACT

The chemokine receptors CCR5 and CXCR4, in combination with CD4, mediate cellular entry of macrophage-tropic (M-tropic) and T-cell-tropic strains of human immunodeficiency virus type 1 (HIV-1), respectively, while dualtropic viruses can use either receptor. We have constructed a panel of chimeric viruses and envelope glycoproteins in which various domains of the dualtropic HIV-1(DH12) gp160 were introduced into the genetic background of an M-tropic HIV-1 isolate, HIV-1(AD8). These constructs were employed in cell fusion and virus infectivity assays using peripheral blood mononuclear cells, MT4 T cells, primary monocyte-derived macrophages, or HOS-CD4 cell lines, expressing various chemokine receptors, to assess the contributions of different gp120 subdomains in coreceptor usage and cellular tropism. As expected, the dualtropic HIV-1(DH12) gp120 utilized either CCR3, CCR5, or CXCR4, whereas HIV-1(AD8) gp120 was able to use only CCR3 or CCR5. We found that either the V1/V2 or the V3 region of HIV-1(DH12) gp120 individually conferred on HIV-1(AD8) the ability to use CXCR4, while the combination of both the V1/V2 and V3 regions increased the efficiency of CXCR4 use. In addition, while the V4 or the V5 region of HIV-1(DH12) gp120 failed to confer the capacity to utilize CXCR4 on HIV-1(AD8), these regions were required in conjunction with regions V1 to V3 of HIV-1(DH12) gp120 for efficient utilization of CXCR4. Comparison of virus infectivity analyses with various cell types and cell fusion assays revealed assay-dependent discrepancies and indicated that events occurring at the cell surface during infection are complex and cannot always be predicted by any one assay.


Subject(s)
HIV Envelope Protein gp120/metabolism , HIV Envelope Protein gp160/metabolism , HIV-1/metabolism , Receptors, CXCR4/metabolism , Animals , Binding Sites , CD4 Antigens/metabolism , Cell Line , Cell Line, Transformed , Chemokine CCL4 , Chemokine CCL5/metabolism , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp160/genetics , HIV-1/genetics , HIV-1/isolation & purification , Humans , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , Macrophage Inflammatory Proteins/metabolism , Macrophages/virology , Membrane Fusion , Reassortant Viruses/genetics , Reassortant Viruses/isolation & purification , Reassortant Viruses/metabolism , Receptors, CCR3 , Receptors, CCR5/metabolism , Receptors, Chemokine/metabolism , T-Lymphocytes/virology
9.
J Reprod Immunol ; 41(1-2): 197-211, 1998 Dec.
Article in English | MEDLINE | ID: mdl-10213311

ABSTRACT

HIV entry is mediated by an interaction between CD4 and members of the chemokine receptor family of proteins. It is likely that CD4 induces conformational changes in the viral envelope glycoproteins that facilitate a subsequent interaction with the chemokine receptor. To understand these events, variants of HIV-2 and HIV-1 have been derived that are able to interact directly with CXCR4 in the absence of CD4. One HIV-2 variant. termed HIV-2/vcp, has an expanded host range that includes CXCR4+/CD4- lymphoid and nonlymphoid cell lines. In contrast to T-tropic isolates of HIV-1, HIV-2/vcp was shown to induce > 95% downregulation of CXCR4 on chronically infected cells and was able to superinfect HIV-1-infected cells. A variant of HIV-1/IIIB termed HIV-1/IIIBx was also derived that is both replication competent and fusogenic for a CD4-negative subclone of SupT1 cells, termed BC7. Infection of BC7 cells by HIV-1/IIIBx was resistant to anti-CD4 monoclonal antibodies but inhibited by the anti-CXCR4 mAb, 12G5. HIV-1/IIIBx was highly fusogenic on 3T3 cells expressing CXCR4 in the absence of CD4. In contrast to HIV-2/vcp, the host range of HIV-1/IIIBx was highly restricted and replication in several CD4+/CXCR4+ lymphoid cell lines was reduced compared to HIV-1/IIIB. In addition, HIV-1/IIIBx failed to downregulate CXCR4 on chronically infected cells. These studies indicate that HIV-1 and HIV-2 variants can be derived in vitro that utilize CXCR4 in the absence of CD4. Although the mechanism(s) for these changes remain unclear, possibilities include an increased avidity of the viral envelope glycoprotein for CXCR4 and/or the increased exposure of the chemokine receptor binding site. Further biochemical and molecular analysis of the envelope glycoproteins from these viruses should be helpful in addressing these and other possibilities.


Subject(s)
CD4 Antigens/metabolism , HIV-1/metabolism , HIV-2/metabolism , Receptors, CXCR4/metabolism , Animals , Humans
10.
J Virol ; 71(12): 8999-9007, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9371556

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) requires both CD4 and a coreceptor to infect cells. Macrophage-tropic (M-tropic) HIV-1 strains utilize the chemokine receptor CCR5 in conjunction with CD4 to infect cells, while T-cell-tropic (T-tropic) strains generally utilize CXCR4 as a coreceptor. Some viruses can use both CCR5 and CXCR4 for virus entry (i.e., are dual-tropic), while other chemokine receptors can be used by a subset of virus strains. Due to the genetic diversity of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and the potential for chemokine receptors other than CCR5 or CXCR4 to influence viral pathogenesis, we tested a panel of 28 HIV-1, HIV-2, and SIV envelope (Env) proteins for the ability to utilize chemokine receptors, orphan receptors, and herpesvirus-encoded chemokine receptor homologs by membrane fusion and virus infection assays. While all Env proteins used either CCR5 or CXCR4 or both, several also used CCR3. Use of CCR3 was strongly dependent on its surface expression levels, with a larger number of viral Env proteins being able to utilize this coreceptor at the higher levels of surface expression. ChemR1, an orphan receptor recently shown to bind the CC chemokine I309 (and therefore renamed CCR8), was expressed in monocyte and lymphocyte cell populations and functioned as a coreceptor for diverse HIV-1, HIV-2, and SIV Env proteins. Use of ChemR1/CCR8 by SIV strains was dependent in part on V3 loop sequences. The orphan receptor V28 supported Env-mediated cell-cell fusion by four T- or dual-tropic HIV-1 and HIV-2 strains. Three additional orphan receptors failed to function for any of the 28 Env proteins tested. Likewise, five of six seven-transmembrane-domain receptors encoded by herpesviruses did not support Env-mediated membrane fusion. However, the chemokine receptor US28, encoded by cytomegalovirus, did support inefficient infection by two HIV-1 strains. These findings indicate that additional chemokine receptors can function as HIV and SIV coreceptors and that surface expression levels can strongly influence coreceptor use.


Subject(s)
Chemokines, CC , HIV-1/metabolism , HIV-2/metabolism , Receptors, Chemokine/metabolism , Receptors, Virus/metabolism , Simian Immunodeficiency Virus/metabolism , Animals , CX3C Chemokine Receptor 1 , Chemokine CCL1 , HeLa Cells , Herpesviridae/metabolism , Humans , Membrane Fusion , Receptors, CCR3 , Receptors, CCR8 , Receptors, Cytokine/metabolism , Receptors, HIV/metabolism , Tumor Cells, Cultured
11.
Proc Natl Acad Sci U S A ; 94(12): 6426-31, 1997 Jun 10.
Article in English | MEDLINE | ID: mdl-9177234

ABSTRACT

The chemokine receptor CXCR4 functions as a fusion coreceptor for T cell tropic and dual-tropic HIV-1 strains. To identify regions of CXCR4 that are important for coreceptor function, CXCR4-CXCR2 receptor chimeras were tested for the ability to support HIV-1 envelope (env) protein-mediated membrane fusion. Receptor chimeras containing the first and second extracellular loops of CXCR4 supported fusion by T tropic and dual-tropic HIV-1 and HIV-2 strains and binding of a monoclonal antibody to CXCR4, 12G5, that blocks CXCR4-dependent infection by some virus strains. The second extracellular loop of CXCR4 was sufficient to confer coreceptor function to CXCR2 for most virus strains tested but did not support binding of 12G5. Truncation of the CXCR4 cytoplasmic tail or mutation of a conserved DRY motif in the second intracellular loop did not affect coreceptor function, indicating that phosphorylation of the cytoplasmic tail and the DRY motif are not required for coreceptor function. The results implicate the involvement of multiple CXCR4 domains in HIV-1 coreceptor function, especially the second extracellular loop, though the structural requirements for coreceptor function were somewhat variable for different env proteins. Finally, a hybrid receptor in which the amino terminus of CXCR4 was replaced by that of CCR5 was active as a coreceptor for M tropic, T tropic, and dual-tropic env proteins. We propose that dual tropism may evolve in CCR5-restricted HIV-1 strains through acquisition of the ability to utilize the first and second extracellular loops of CXCR4 while retaining the ability to interact with the CCR5 amino-terminal domain.


Subject(s)
HIV-1/physiology , Membrane Proteins/physiology , Receptors, HIV/physiology , Cell Fusion , Cell Line , Gene Products, env/metabolism , Genes, Reporter , HeLa Cells , Humans , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Models, Structural , Protein Structure, Secondary , Receptors, CXCR4 , Receptors, HIV/biosynthesis , Receptors, HIV/chemistry , Receptors, Interleukin/biosynthesis , Receptors, Interleukin/chemistry , Receptors, Interleukin/physiology , Receptors, Interleukin-8B , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Transfection
12.
Immunol Res ; 16(1): 15-28, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9048206

ABSTRACT

CD4 is the primary cellular receptor for human immunodeficiency virus type 1 (HIV-1), but is not sufficient for entry of HIV-1 into cells. After a decade-long search, the cellular coreceptors that HIV-1 requires in conjunction with CD4 have been identified as members of the chemokine receptor family of seven-transmembrane G-protein coupled receptors. The discovery of distinct chemokine receptors that support entry of T-cell tropic (CXCR-4) and macrophage tropic HIV-1 strains (CCR-5) explains the differences in cell tropism between viral strains, the inability of HIV-1 to infect most nonprimate cells, and the resistance of a small percentage of the population to HIV-1 infection. Further understanding of the role of chemokine receptors in viral entry may also help explain the evolution of more pathogenic forms of the virus, viral transmission, and HIV-induced pathogenesis. These recent discoveries will aid the development of strategies for combating HIV-1 transmission and spread, the understanding of HIV-1 fusion mechanisms, and the possible development of small animal models for HIV-1 drug and vaccine testing.


Subject(s)
Chemokines/physiology , HIV-1/immunology , Membrane Fusion , Receptors, Cytokine/physiology , Receptors, HIV/physiology , CD4 Antigens , Chemokines/immunology , HIV-1/pathogenicity , Humans , Membrane Proteins , Polymorphism, Genetic/immunology , Receptors, CCR5 , Receptors, CXCR4
14.
Proc Natl Acad Sci U S A ; 94(26): 14742-7, 1997 Dec 23.
Article in English | MEDLINE | ID: mdl-9405683

ABSTRACT

Brain capillary endothelial cells (BCECs) are targets of CD4-independent infection by HIV-1 and simian immunodeficiency virus (SIV) strains in vitro and in vivo. Infection of BCECs may provide a portal of entry for the virus into the central nervous system and could disrupt blood-brain barrier function, contributing to the development of AIDS dementia. We found that rhesus macaque BCECs express chemokine receptors involved in HIV and SIV entry including CCR5, CCR3, CXCR4, and STRL33, but not CCR2b, GPR1, or GPR15. Infection of BCECs by the neurovirulent strain SIV/17E-Fr was completely inhibited by aminooxypentane regulation upon activation, normal T cell expression and secretion in the presence or absence of ligands, but not by eotaxin or antibodies to CD4. We found that the envelope (env) proteins from SIV/17E-Fr and several additional SIV strains mediated cell-cell fusion and virus infection with CD4-negative, CCR5-positive cells. In contrast, fusion with cells expressing the coreceptors STRL33, GPR1, and GPR15 was CD4-dependent. These results show that CCR5 can serve as a primary receptor for SIV in BCECs and suggest a possible CD4-independent mechanism for blood-brain barrier disruption and viral entry into the central nervous system.


Subject(s)
Brain/blood supply , CD4 Antigens/immunology , Capillaries/virology , Endothelium, Vascular/virology , Receptors, CCR5/immunology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus , Animals , Blood-Brain Barrier/immunology , Capillaries/immunology , Endothelium, Vascular/immunology , HeLa Cells , Humans , Macaca mulatta
15.
Cell ; 87(3): 437-46, 1996 Nov 01.
Article in English | MEDLINE | ID: mdl-8898197

ABSTRACT

Macrophage-tropic (M-tropic) HIV-1 strains use the beta-chemokine receptor CCR5, but not CCR2b, as a cofactor for membrane fusion and infection, while the dual-tropic strain 89.6 uses both. CCR5/2b chimeras and mutants were used to map regions of CCR5 important for cofactor function and specificity. M-tropic strains required either the amino-terminal domain or the first extracellular loop of CCR5. A CCR2b chimera containing the first 20 N-terminal residues of CCR5 supported M-tropic envelope protein fusion. Amino-terminal truncations of CCR5/CCR2b chimeras indicated that residues 2-5 are important for M-tropic viruses, while 89.6 is dependent on residues 6-9. The identification of multiple functionally important regions in CCR5, coupled with differences in how CCR5 is used by M- and dual-tropic viruses, suggests that interactions between HIV-1 and entry cofactors are conformationally complex.


Subject(s)
HIV-1/physiology , Protein Conformation , Receptors, Chemokine , Receptors, Cytokine/chemistry , Receptors, HIV/chemistry , Amino Acid Sequence , CD4 Antigens/physiology , Cytopathogenic Effect, Viral , Glycosylation , HeLa Cells , Humans , Macromolecular Substances , Macrophages/virology , Membrane Fusion , Molecular Sequence Data , Protein Processing, Post-Translational , Receptors, CCR2 , Receptors, CCR5 , Receptors, Cytokine/physiology , Receptors, HIV/physiology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Structure-Activity Relationship
16.
J Virol ; 70(9): 6288-95, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8709256

ABSTRACT

Entry of human immunodeficiency virus type 1 (HIV-1) into cells requires binding to CD4 and fusion with a cellular membrane. Fusion does not occur in most nonhuman cells even when they express human CD4, indicating that one or more human accessory factors are required for virus infection. Recently, a seven-transmembrane domain protein has been shown to serve as an accessory factor for T-cell-tropic (T-tropic) HIV-1 isolates (Y. Feng, C. C. Broder, P. E. Kennedy, and E. A. Berger, Science 272:872-877, 1996). Here we show that expression of this glycoprotein, termed fusin, in murine, feline, simian, and quail cell lines, in conjunction with human CD4, rendered these cells fully permissive for HIV-1 envelope glycoprotein (Env)-mediated membrane fusion. Expression of CD4 or fusin alone did not permit fusion. In addition, introduction of fusin and CD4 into a human cell line, U87MG, that is resistant to HIV-1 induced syncytium formation and to infection by HIV-1 when expressing CD4 alone made this cell line permissive for Env-mediated cell-cell fusion. Fusion was observed only with T-tropic Env proteins. Macrophage-tropic (M-tropic) Env proteins from the SF162, ADA, and Ba-L HIV-1 strains did not fuse with cells expressing fusin and CD4, suggesting that M-tropic viruses utilize an accessory molecule other than fusin. Finally, coexpression of fusin and CD4 made both a murine and feline cell line susceptible to virus infection by T-tropic, but not M-tropic, HIV-1 strains.


Subject(s)
CD4 Antigens/physiology , HIV-1/physiology , Membrane Proteins/physiology , Receptors, HIV/physiology , T-Lymphocytes/immunology , T-Lymphocytes/virology , Amino Acid Sequence , Animals , Antigens, CD/physiology , Astrocytoma , Base Sequence , Cats , Cell Line , Cell Membrane/physiology , Cell Membrane/ultrastructure , Cloning, Molecular , Coturnix , DNA Primers , Fetus , Gene Library , Gene Products, env/physiology , Giant Cells , HIV-1/immunology , HeLa Cells , Humans , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Mice , Molecular Sequence Data , Polymerase Chain Reaction , Protein Structure, Secondary , Receptors, CXCR4 , Spleen , Tumor Cells, Cultured
17.
J Neurovirol ; 1(2): 165-76, 1995 Jun.
Article in English | MEDLINE | ID: mdl-9222355

ABSTRACT

Herpes simplex virus type 1 (HSV-1) replication is thought to occur via a rolling circle type of mechanism, generating large DNA concatemers from which unit length genomes are subsequently cleaved. In this report, we have employed field inversion gel electrophoresis (FIGE), Southern blot hybridization, and endonuclease digestion, to identify and characterize these DNAs. Two species of HSV-1 DNA: (1) genome-length and (2) DNA that remained at the electrophoresis origin (referred to as well-associated DNA) were detected. To ascertain that the latter was large in size and not virion DNA trapped at the origin with high molecular weight cellular DNA, the infected cell DNA was digested with a restriction enzyme that does not cut the viral DNA. In order to do this HSV-1 strain 1702, lacking any XbaI sites in its genome, was utilized. After digestion of samples with XbaI, and FIGE, cellular DNA was seen to migrate into the gel; however, the viral DNA remained in the sample wells. Pulse labeling experiments showed that this large DNA was processed to 150 kb genome lengths. Endonuclease digestion of the well-associated DNA revealed that it contained a greater ratio of joint to terminal fragments than virion DNA-a characteristic of long concatemers. Quantitation of the terminal fragments revealed mainly L termini. Surprisingly, the ratio of joint to terminal fragments was 2.5 suggesting that the lengths of concatemers were short (in the order of 1-2 genome lengths) and that the well association was due to conformation rather than concatemeric length. Because one of these genome lengths is present as the replication intermediate, the growing tail must be less than genome length. Thus genome lengths must be processed from the replication intermediate soon after they are completed.


Subject(s)
Herpesvirus 1, Human/growth & development , Herpesvirus 1, Human/genetics , Virus Replication , Animals , Cell Line , Chlorocebus aethiops , DNA Probes , DNA, Viral/analysis , DNA, Viral/chemistry , DNA, Viral/isolation & purification , Electrophoresis, Gel, Pulsed-Field , Genome, Viral , Kidney/cytology , Molecular Weight
18.
J Virol ; 68(9): 5890-8, 1994 Sep.
Article in English | MEDLINE | ID: mdl-8057468

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) infects some cell types which lack CD4, demonstrating that one or more alternative viral receptors exist. One such receptor is galactosylceramide (GalCer), a glycosphingolipid distributed widely in the nervous system and in colonic epithelial cells. Using a liposome flotation assay, we found that the HIV-1 surface glycoprotein, gp120, quantitatively bound to liposomes containing GalCer but not to liposomes containing phospholipids and cholesterol alone. Binding was saturable and was inhibited by preincubating liposomes with anti-GalCer antibodies. We observed less efficient binding of gp120 to liposomes containing lactosylceramide, glucosylceramide, and galactosylsulfate, whereas no binding to liposomes containing mixed gangliosides, psychosine, or sphingomyelin was detected. Binding to GalCer was rapid, largely independent of temperature and pH, and stable to conditions which remove most peripheral membrane proteins. By contrast, gp120 bound to lactosylceramide could be removed by 2 M potassium chloride or 3 M potassium thiocyanate, demonstrating a less stable interaction. Removal of N-linked oligosaccharides on gp120 did not affect binding efficiency. However, as previously observed for CD4 binding, heat denaturation of gp120 prevented binding to GalCer. Finally, binding was critically dependent on the concentration of GalCer in the target membrane, suggesting that binding to glycolipid-rich domains occurs and that GalCer conformation may be important for gp120 recognition.


Subject(s)
Galactosylceramides/metabolism , HIV Envelope Protein gp120/metabolism , HIV-1/metabolism , HIV Envelope Protein gp120/chemistry , Hydrogen-Ion Concentration , In Vitro Techniques , Liposomes , Osmolar Concentration , Protein Binding , Structure-Activity Relationship , Temperature , Time Factors
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