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1.
Virology ; 350(2): 289-301, 2006 Jul 05.
Article in English | MEDLINE | ID: mdl-16513154

ABSTRACT

Glycan heterogeneity of the respiratory syncytial virus (RSV) fusion (F) protein was demonstrated by proteomics. The effect of maturation of the virus glycoproteins-associated glycans on virus infectivity was therefore examined using the alpha-mannosidase inhibitors deoxymannojirimycin (DMJ) and swainsonine (SW). In the presence of SW the N-linked glycans on the F protein appeared in a partially mature form, whereas in the presence of DMJ no maturation of the glycans was observed. Neither inhibitor had a significant effect on G protein processing or on the formation of progeny virus. Although the level of infectious virus and syncytia formation was not significantly affected by SW-treatment, DMJ-treatment correlated with a one hundred-fold reduction in virus infectivity. Our data suggest that glycan maturation of the RSV glycoproteins, in particular those on the F protein, is an important step in virus maturation and is required for virus infectivity.


Subject(s)
Enzyme Inhibitors/pharmacology , Glycoproteins/metabolism , Polysaccharides/metabolism , Respiratory Syncytial Virus, Human/physiology , Viral Proteins/metabolism , alpha-Mannosidase/antagonists & inhibitors , Cell Fusion , Cell Line, Tumor , Electrophoresis, Gel, Two-Dimensional , Glycoside Hydrolases , Humans , Microscopy, Electron, Scanning , Respiratory Syncytial Virus, Human/drug effects , Respiratory Syncytial Virus, Human/pathogenicity , Viral Proteins/genetics , Viral Proteins/isolation & purification
2.
Virology ; 338(1): 69-80, 2005 Jul 20.
Article in English | MEDLINE | ID: mdl-15936795

ABSTRACT

In this report, the interaction between respiratory syncytial virus (RSV) and heat shock protein 70 (HSP70) was examined. Although no significant increase in total HSP70 protein levels was observed during virus infection, analysis of the HSP70 content in lipid-raft membranes from mock- and virus-infected cells revealed an increase in the levels of raft-associated HSP70 during virus infection. Fluorescence microscopy demonstrated that this transport of HSP70 into lipid-raft membranes correlated with the appearance of HSP70 within virus-induced inclusion bodies. Furthermore, co-localisation of HSP70 with the virus N protein and the raft lipid GM1 was observed within these structures. Immunoprecipitation experiments demonstrated the ability of HSP70 to interact with the virus polymerase complex in lipid-rafts in an ATP-dependent manner. Collectively, these data suggest that RSV may induce cellular changes which allow the recruitment of specific host-cell factors, via lipid-raft membranes, to the polymerase complex.


Subject(s)
HSP70 Heat-Shock Proteins/physiology , Membrane Microdomains/physiology , Membrane Microdomains/virology , Respiratory Syncytial Viruses/physiology , Respiratory Syncytial Viruses/pathogenicity , Viral Proteins/physiology , Amino Acid Sequence , Cell Line , HSP70 Heat-Shock Proteins/genetics , Humans , Inclusion Bodies/physiology , Inclusion Bodies/virology , Microscopy, Fluorescence , Molecular Sequence Data , Multiprotein Complexes , Peptide Fragments/genetics , Peptide Fragments/isolation & purification , Respiratory Syncytial Viruses/genetics , Viral Proteins/genetics , Virus Replication
3.
Virology ; 330(1): 147-57, 2004 Dec 05.
Article in English | MEDLINE | ID: mdl-15527841

ABSTRACT

The interaction between the respiratory syncytial virus (RSV) polymerase complex and lipid rafts was examined in HEp2 cells. Lipid-raft membranes were prepared from virus-infected cells and their protein content was analysed by Western blotting and mass spectrometry. This analysis revealed the presence of the N, P, L, M2-1 and M proteins. However, these proteins appeared to differ from one another in their association with these structures, with the M2-1 protein showing a greater partitioning into raft membranes compared to that of the N, P or M proteins. Determination of the polymerase activity profile of the gradient fractions revealed that 95% of the detectable viral enzyme activity was associated with lipid-raft membranes. Furthermore, analysis of virus-infected cells by confocal microscopy suggested an association between these proteins and the raft-lipid, GM1. Together, these results provide evidence that the RSV polymerase complex is able to associate with lipid rafts in virus-infected cells.


Subject(s)
DNA-Directed RNA Polymerases/metabolism , Membrane Microdomains/virology , Respiratory Syncytial Virus, Human/enzymology , Amino Acid Sequence , Cell Line, Tumor , DNA-Directed RNA Polymerases/chemistry , Humans , Mass Spectrometry , Molecular Sequence Data , Peptide Fragments/chemistry
4.
Virology ; 327(2): 175-85, 2004 Oct 01.
Article in English | MEDLINE | ID: mdl-15351205

ABSTRACT

The assembly of respiratory syncytial virus (RSV) in lipid-rafts was examined in Hep2 cells. Confocal and electron microscopy showed that during RSV assembly, the cellular distribution of the complement regulatory proteins, decay accelerating factor (CD55) and CD59, changes and high levels of these cellular proteins are incorporated into mature virus filaments. The detergent-solubility properties of CD55, CD59, and the RSV fusion (F) protein were found to be consistent with each protein being located predominantly within lipid-raft structures. The levels of these proteins in cell-released virus were examined by immunoelectronmicroscopy and found to account for between 5% and 15% of the virus attachment (G) glycoprotein levels. Collectively, our findings suggest that an intimate association exists between RSV and lipid-raft membranes and that significant levels of these host-derived raft proteins, such as those regulating complement activation, are subsequently incorporated into the envelope of mature virus particles.


Subject(s)
CD55 Antigens/metabolism , CD59 Antigens/metabolism , Membrane Microdomains/metabolism , Respiratory Syncytial Virus, Human/pathogenicity , Virus Assembly , Animals , Cell Line, Tumor , Chlorocebus aethiops , Humans , Membrane Microdomains/chemistry , Microscopy, Confocal , Microscopy, Electron , Respiratory Syncytial Virus, Human/metabolism , Vero Cells , Virion/metabolism
5.
J Gen Virol ; 85(Pt 5): 1153-1165, 2004 May.
Article in English | MEDLINE | ID: mdl-15105532

ABSTRACT

The cellular distribution of the small hydrophobic (SH) protein in respiratory syncytial virus (RSV)-infected cells was examined. Although the SH protein was distributed throughout the cytoplasm, it appeared to accumulate in the Golgi complex within membrane structures that were enriched in the raft lipid, GM1. The ability of the SH protein to interact with lipid-raft membranes was further confirmed by examining its detergent-solubility properties in Triton X-100 at 4 degrees C. This analysis showed that a large proportion of the SH protein exhibited detergent-solubility characteristics that were consistent with an association with lipid-raft membranes. Analysis of virus-infected cells by immuno-transmission electron microscopy revealed SH protein clusters on the cell surface, but only very low levels of the protein appeared to be associated with mature virus filaments and inclusion bodies. These data suggest that during virus infection, the compartments in the secretory pathway, such as the endoplasmic reticulum (ER) and Golgi complex, are major sites of accumulation of the SH protein. Furthermore, although a significant amount of this protein interacts with lipid-raft membranes within the Golgi complex, its presence within mature virus filaments is minimal.


Subject(s)
Golgi Apparatus/metabolism , Respiratory Syncytial Virus Infections/metabolism , Respiratory Syncytial Viruses/metabolism , Viral Envelope Proteins/metabolism , Viral Proteins/metabolism , Animals , Chlorocebus aethiops , G(M1) Ganglioside/metabolism , Microscopy, Fluorescence , Vero Cells , Viral Envelope Proteins/analysis , Viral Proteins/analysis
6.
Virology ; 306(2): 254-67, 2003 Feb 15.
Article in English | MEDLINE | ID: mdl-12642099

ABSTRACT

Field emission scanning electron microscopy (FE SEM) was used to visualize the distribution of virus-associated components, the virus-attachment (G) protein, and the host-cell-derived lipid, GM1, in respiratory syncytial virus (RSV) filaments. RSV-infected cells were labeled in situ with a G protein antibody (MAb30) whose presence was detected using a second antibody conjugated to colloidal gold. No bound MAb30 was detected in mock-infected cells, whereas significant quantities bound to viral filaments revealing G protein clusters throughout the filaments. GM1 was detected using cholera toxin B subunit conjugated to colloidal gold. Mock-infected cells revealed numerous GM1 clusters on the cell surface. In RSV-infected cells, these gold clusters were detected on the filaments in low, but significant, amounts, indicating the incorporation of GM1 within the viral envelope. This report describes the first use of FE SEM to map the distribution of specific structural components within the envelope of a Paramyxovirus.


Subject(s)
G(M1) Ganglioside/metabolism , Respiratory Syncytial Viruses/metabolism , Viral Proteins/metabolism , Animals , Antibodies, Monoclonal , Antibodies, Viral , Cell Membrane/metabolism , Cell Membrane/virology , Chlorocebus aethiops , Microscopy, Electron, Scanning/methods , Respiratory Syncytial Viruses/growth & development , Respiratory Syncytial Viruses/ultrastructure , Vero Cells
7.
J Gen Virol ; 83(Pt 8): 1841-1850, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12124448

ABSTRACT

We have previously shown that respiratory syncytial virus (RSV) assembly occurs within regions of the host-cell surface membrane that are enriched in the protein caveolin-1 (cav-1). In this report, we have employed immunofluorescence microscopy to further examine the RSV assembly process. Our results show that RSV matures at regions of the cell surface that, in addition to cav-1, are enriched in the lipid-raft ganglioside GM1. Furthermore, a comparison of mock-infected and RSV-infected cells by confocal microscopy revealed a significant change in the cellular distribution of phosphocaveolin-1 (pcav-1). In mock-infected cells, pcav-1 was located at regions of the cell that interact with the extracellular matrix, termed focal adhesions (FA). In contrast, RSV-infected cells showed both a decrease in the levels of pcav-1 associated with FA and the appearance of pcav-1-containing cytoplasmic vesicles, the latter being absent in mock-infected cells. These cytoplasmic vesicles were clearly visible between 9 and 18 h post-infection and coincided with the formation of RSV filaments, although we did not observe a direct association of pcav-1 with mature virus. In addition, we noted a strong colocalization between pcav-1 and growth hormone receptor binding protein-7 (Grb7), within these cytoplasmic vesicles, which was not observed in mock-infected cells. Collectively, these findings show that the RSV assembly process occurs within specialized lipid-raft structures on the host-cell plasma membrane, induces the cellular redistribution of pcav-1 and results in the formation of cytoplasmic vesicles that contain both pcav-1 and Grb7.


Subject(s)
Caveolins/metabolism , G(M1) Ganglioside/metabolism , Membrane Microdomains/virology , Respiratory Syncytial Virus, Human/growth & development , Respiratory Syncytial Virus, Human/metabolism , Virus Assembly , Animals , Caveolin 1 , Chlorocebus aethiops , Cytoplasmic Vesicles/metabolism , GRB7 Adaptor Protein , Humans , Membrane Microdomains/metabolism , Microscopy, Confocal , Microscopy, Fluorescence , Phosphorylation , Proteins/metabolism , Tyrosine/metabolism , Vero Cells
8.
J Gen Virol ; 83(Pt 3): 611-621, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11842256

ABSTRACT

We have employed immunofluorescence microscopy and transmission electron microscopy to examine the assembly and maturation of respiratory syncytial virus (RSV) in the Vero cell line C1008. RSV matures at the apical cell surface in a filamentous form that extends from the plasma membrane. We observed that inclusion bodies containing viral ribonucleoprotein (RNP) cores predominantly appeared immediately below the plasma membrane, from where RSV filaments form during maturation at the cell surface. A comparison of mock-infected and RSV-infected cells by confocal microscopy revealed a significant change in the pattern of caveolin-1 (cav-1) fluorescence staining. Analysis by immuno-electron microscopy showed that RSV filaments formed in close proximity to cav-1 clusters at the cell surface membrane. In addition, immuno-electron microscopy showed that cav-1 was closely associated with early budding RSV. Further analysis by confocal microscopy showed that cav-1 was subsequently incorporated into the envelope of RSV filaments maturing on the host cell membrane, but was not associated with other virus structures such as the viral RNPs. Although cav-1 was incorporated into the mature virus, it was localized in clusters rather than being uniformly distributed along the length of the viral filaments. Furthermore, when RSV particles in the tissue culture medium from infected cells were examined by immuno-negative staining, the presence of cav-1 on the viral envelope was clearly demonstrated. Collectively, these findings show that cav-1 is incorporated into the envelope of mature RSV particles during egress.


Subject(s)
Caveolins/metabolism , Cell Membrane/metabolism , Cell Membrane/virology , Respiratory Syncytial Virus, Human/growth & development , Respiratory Syncytial Virus, Human/metabolism , Virus Assembly , Animals , Caveolin 1 , Cell Membrane/ultrastructure , Cell Polarity , Chlorocebus aethiops/virology , Fluorescent Antibody Technique , Immunohistochemistry , Inclusion Bodies, Viral/metabolism , Inclusion Bodies, Viral/ultrastructure , Microscopy, Electron , Microscopy, Immunoelectron , Protein Transport , Respiratory Syncytial Virus, Human/ultrastructure , Ribonucleoproteins/metabolism , Vero Cells , Viral Proteins/metabolism
9.
J Gen Virol ; 83(Pt 1): 61-66, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11752701

ABSTRACT

Analysis of the respiratory syncytial virus (RSV) fusion (F) protein in RSV-infected Vero cells showed the presence of a single F1 subunit and at least two different forms of the F2 subunit, designated F2a (21 kDa) and F2b (16 kDa), which were collectively referred to as [F2](a/b). Enzymatic deglycosylation of [F2](a/b) produced a single 10 kDa product suggesting that [F2](a/b) arises from differences in the glycosylation pattern of F2a and F2b. The detection of [F2](a/b) was dependent upon the post-translational cleavage of the F protein by furin, since its appearance was prevented in RSV-infected Vero cells treated with the furin inhibitor dec-RVKR-cmk. Analysis by protein cross-linking revealed that the F1 subunit interacted with [F2](a/b), via disulphide bonding, to produce equivalent F protein trimers, which were expressed on the surface of infected cells. Collectively, these data show that multiple F protein species are expressed in RSV-infected cells.


Subject(s)
Respiratory Syncytial Virus, Human/metabolism , Viral Envelope Proteins/metabolism , Viral Fusion Proteins/metabolism , Viral Proteins/metabolism , Amidohydrolases/metabolism , Animals , Chlorocebus aethiops , Fucose/metabolism , Glucosamine/metabolism , Glycosylation , HeLa Cells , Humans , Mannose/metabolism , Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase/metabolism , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase , Tumor Cells, Cultured , Vero Cells , Viral Envelope Proteins/biosynthesis , Viral Fusion Proteins/biosynthesis
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