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R W Compans

Publications and source records attributed to R W Compans.

At least 109 records · Page 6Linked to original sources
PubMed1992

Assembly of G1 and G2 glycoprotein oligomers in Punta Toro virus-infected cells.

We have studied the oligomerization of the membrane glycoproteins of Punta Toro virus (PTV), a member of the Phlebovirus genus of the family Bunyaviridae, and the effect of glycosylation on protein stability and transport. By using sucrose gradient centrifugation, the G1 and G2 glycoproteins in PTV-infected or recombinant-transfected cells were found to sediment as dimers after DSP cross-linking, suggesting that the G 1 and G2 proteins are associated as dimers by non-covalent interactions. Pulse-chase and two-dimensional gel analysis indicate that dimerization occurs between newly synthesized G1 and G2 proteins, and that a small fraction of the G2 proteins is assembled into G2 homodimers. The amounts of G1 and G2 proteins were substantially decreased, while the amounts of nucleocapsid protein remained nearly unchanged, when PTV-infected cells were treated with the glycosylation inhibitor tunicamycin, indicating that the G1 and G2 proteins are unstable if glycosylation is prevented.

Animals↗
PubMed1992

A carboxy-terminal mutant spleen focus-forming virus (SFFV) envelope glycoprotein is transport-competent, but non-leukemogenic.

The Friend spleen focus-forming virus (F-SFFV) codes for a transport-defective leukemogenic envelope glycoprotein designated as gp52. We have previously shown that the external domain of gp52 carries the determinants responsible for its transport defect. Consistent with this idea, truncated gp52 molecules that lack a hydrophobic membrane anchor were transport-defective, and were not secreted from cells. In this report, we describe the construction of a mutant SFFV envelope gene that codes for altered gp52 molecules in which the carboxyl-terminal hydrophobic residues are replaced with exogenous hydrophilic residues encoded by the vector-derived sequences. The mutant env gene was expressed using the retroviral expression vector, pLXSN, and the mutant envelope protein was found to be transport-competent, and efficiently secreted from the cells. However, M-MuLV pseudotypes of the retroviral vectors expressing the mutant genome were found to be non-leukemogenic in mice.

3T3 Cells↗
PubMed1992

Sulfation of the human immunodeficiency virus envelope glycoprotein.

Sulfation is a posttranslational modification of proteins which occurs on either the tyrosine residues or the carbohydrate moieties of some glycoproteins. In the case of secretory proteins, sulfation has been hypothesized to act as a signal for export from the cell. We have shown that the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor (gp160) as well as the surface (gp120) and transmembrane (gp41) subunits can be specifically labelled with 35SO42-. Sulfated HIV-1 envelope glycoproteins were identified in H9 cells infected with the IIIB isolate of HIV-1 and in the cell lysates and culture media of cells infected with vaccinia virus recombinants expressing a full-length or truncated, secreted form of the HIV-1 gp160 gene. N-glycosidase F digestion of 35SO4(2-)-labelled envelope proteins removed virtually all radiolabel from gp160, gp120, and gp41, indicating that sulfate was linked to the carbohydrate chains of the glycoprotein. The 35SO42-label was at least partially resistant to endoglycosidase H digestion, indicating that some sulfate was linked to complex carbohydrates. Brefeldin A, a compound that inhibits the endoplasmic reticulum to Golgi transport of glycoproteins, was found to inhibit the sulfation of the envelope glycoproteins. Envelope glycoproteins synthesized in cells treated with chlorate failed to incorporate 35SO42-. However, HIV glycoproteins were still secreted from cells in the presence of chlorate, indicating that sulfation is not a requirement for secretion of envelope glycoproteins. Sulfation of HIV-2 and simian immunodeficiency virus envelope glycoproteins has also been demonstrated by using vaccinia virus-based expression systems. Sulfation is a major determinant of negative charge and could play a role in biological functions and antigenic properties of HIV glycoproteins.

Antiviral Agents↗
PubMed1992

Multiple glycoproteins synthesized by the smallest RNA segment (S10) of bluetongue virus.

The genome of bluetongue virus, an orbivirus, consists of 10 double-stranded RNAs, each encoding at least one polypeptide. The smallest RNA segment (S10) encodes two minor nonstructural proteins, NS3 and NS3A, the structures and functions of which are not understood. We have expressed these two proteins in mammalian cells by using the T7 cytoplasmic transient expression system. Using a deletion mutant (lacking the first AUG initiation codon), we have demonstrated that the second initiation codon is used to initiate the synthesis of NS3A protein and that the two initiation codons are responsible for the synthesis not only of NS3 and NS3A but also of high-molecular-weight forms of both proteins. These higher-molecular-weight forms (GNS3 and GNS3A) are glycosylated. We have also demonstrated that the carbohydrate chains of GNS3 and GNS3A could be further modified by heterogeneous extension to polylactosaminoglycan forms. The glycosylated and nonglycosylated forms are found in similar intracellular locations in the Golgi complex. In the presence of cycloheximide, NS3 and NS3A immunofluorescence staining was pronounced in the Golgi complex, confirming that NS3 and NS3A are competent for transport to the Golgi apparatus after synthesis. We conclude that S10 gene products are integral membrane glycoproteins.

Amino Acid Sequence↗
PubMed1992

Functional interactions between the fusion protein and hemagglutinin-neuraminidase of human parainfluenza viruses.

The fusion glycoprotein (F) and hemagglutinin-neuraminidase (HN) genes of human parainfluenza virus type 2 (PI2) were molecularly cloned and expressed in HeLa-T4 cells by using the vaccinia virus-T7 transient expression system. Expression of the F and HN proteins was detected by using immunoprecipitation and surface immunofluorescence staining. Although the F protein was found to be cleaved into F1 and F2 and expressed on cell surfaces, no cell fusion was observed. However, cotransfection of the F-protein gene together with the P12 HN gene resulted in significant levels of cell fusion. Cell fusion was also observed when separate cell cultures were transfected with the HN and F genes and the F-expressing cells were mixed with the HN-expressing cells. Surprisingly, when the PI2 F protein was expressed together with the parainfluenza virus type 3 (PI3) HN protein, no fusion was detectable in the transfected cells. Similarly, no fusion was found upon coexpression of the PI2 HN and PI3 F proteins. However, coexpression of the PI3 F and HN proteins resulted in extensive cell fusion, which resembled the PI2 coexpression result. These results indicate that under the conditions used, the F protein is unable to cause fusion by itself and the HN protein provides a specific function in cell fusion which cannot be provided by another paramyxovirus attachment protein. Further, the results suggest that a type-specific functional interaction between the F and HN proteins is involved in mediating cell fusion.

Animals↗
PubMed1992

Cytoplasmic domain truncation enhances fusion activity by the exterior glycoprotein complex of human immunodeficiency virus type 2 in selected cell types.

To investigate the glycoprotein determinants of viral cytopathology, we constructed chimeric env genes between a noncytopathic strain of human immunodeficiency virus type 2 (HIV-2), designated HIV-2/ST, and a highly fusogenic and cytopathic variant derived from this virus. Expression of the resulting chimeric glycoproteins indicated that efficient syncytium formation in the human T-cell line Sup T1 mapped to the C-terminal region of the transmembrane (TM) glycoprotein subunit. In this region, the wild-type and cytopathic ST glycoproteins differed by only four amino acids and by the presence of a premature termination codon in the cytopathic variant. Subsequent site-directed mutagenesis indicated that the cytoplasmic domain truncation was responsible for the enhanced fusion activity. This modification, however, increased the fusion activity of the glycoprotein only in Sup T1 cells (in which the ST variant arose) but not in Molt 4 clone 8 or peripheral blood mononuclear cells. These observations indicate that the length of the cytoplasmic domain of the HIV-2 glycoprotein modulates the fusion activity of the exterior glycoprotein complex in a cell-specific manner. Such adaptability appears to permit the emergence of fusogenic variants during HIV-2 passage in vitro and may also regulate viral growth or cytopathic effects in selected cell types during natural infection in vivo.

Amino Acid Sequence↗
PubMed1992

Expression of the influenza A virus M2 protein is restricted to apical surfaces of polarized epithelial cells.

The M2 protein of influenza A virus is a small, nonglycosylated transmembrane protein that is expressed on surfaces of virus-infected cells. A monoclonal antibody specific for the M2 protein was used to investigate its expression in polarized epithelial cells infected with influenza virus or a recombinant vaccinia virus that expresses M2. The expression of M2 on the surfaces of influenza virus-infected cells was found to be restricted to the apical surface, closely paralleling that of the influenza virus hemagglutinin (HA). Membrane domain-specific immunoprecipitation indicated that the M2 protein was inserted directly into the apical membrane with transport kinetics similar to those of HA. In polarized cells infected with a recombinant vaccinia virus that expresses M2, we found that 86 to 93% of surface M2 was restricted to the apical domain compared with 88 to 90% of HA in a similar assay. These results indicate that the M2 protein undergoes directional transport in the absence of other influenza virus proteins and that M2 contains the structural features required for apical transport in polarized epithelial cells. The ultrastructural localization of the M2 protein in influenza virus-infected MDCK cells was investigated by immunoelectron microscopy using M2 antibody and a gold conjugate. In cells in which extensive virus budding was occurring, the apical cell membrane was labeled with gold particles evenly distributed between microvilli and the surrounding membrane. In addition, a significant fraction of the M2 label was apparently associated with virions. A monoclonal antibody specific for HA demonstrated a similar labeling pattern. These results indicate that M2 is localized in close proximity to budding and assembled virions.

Animals↗
PubMed1992

Migration of polarized epithelial cells through permeable membrane substrates of defined pore size.

We have observed that cells of various epithelial lines exhibit the ability to migrate through permeable membrane substrates containing 3.0 microns pores. Scanning and transmission electron microscopic observations of Vero C1008 and Caco-2 cell lines grown on polycarbonate membranes containing 3.0 microns pores revealed extensive penetration of the filter and the establishment of virtually complete monolayers on the opposing surface. The migration of MDCK cells was also observed to occur under the same conditions; however, the extent of MDCK cell growth on the opposing surface was significantly less than observed for Vero C1008 and Caco-2 cells. Morphological differences were apparent between cells growing on the upper and lower faces of the filter membrane, although cells growing on both surfaces exhibited a polarized phenotype. The cells which invaded the filter were collected and maintained by serial passage. The passaged cells exhibited morphological differences and an altered rate of differentiation in comparison to the parental cell type, suggesting that the invasive cells represent a variant of the parental cell population. Studies using filters of different pore sizes indicated that cellular migration also occurs through pores of 2.0 microns diameter, but not through 1.0 micron (or smaller) pores. These observations have significant implications for studies involving the growth of epithelial cells on permeable membrane substrates containing large pores.

Animals↗
PubMed1991

Human immunodeficiency virus envelope protein determines the site of virus release in polarized epithelial cells.

In polarized epithelial cells, the release of enveloped viruses by budding at the cell surface is restricted to a specific cell membrane domain, either the apical or basolateral domain. To investigate the role of the envelope glycoprotein and the capsid proteins of human immunodeficiency virus type 1 (HIV-1) in determining the site of virus assembly, we analyzed virus maturation in a polarized monkey kidney cell line. A line of cells harboring the HIV-1 provirus (VERO-pFN) was found to differentiate into polarized epithelial cell monolayers upon reaching confluency. By electron microscopy, virus maturation was observed predominantly at the basolateral membranes of VERO-pFN cells. Analysis of HIV-1 proteins revealed that virtually all of glycoprotein gp120 and capsid protein p24 were found in the basolateral medium, while no HIV-1 proteins were detected apically. A recombinant vaccinia virus (VV) expressing the HIV-1 gag polyprotein (VVgag) was used to determine the site of release of HIV-1 core particles in polarized epithelial cells in the presence or absence of envelope glycoproteins. When cells were infected with VVgag in the absence of envelope proteins, similar amounts of the p24 capsid protein were released into virus particles at the apical or basolateral surface. In contrast, when cells were doubly infected with VVgag and a recombinant VV expressing the HIV-1 envelope glycoprotein (VVenv), 94% of p24 and all of gp120 were found to be associated with particles released into the basolateral medium. These results indicate that the HIV-1 envelope glycoprotein directly influences the site of release of virus particles containing the gag protein, probably via a specific interaction between the envelope protein and the gag protein.

Animals↗
PubMed1991

The three-dimensional structure of canine parvovirus and its functional implications.

The three-dimensional atomic structure of a single-stranded DNA virus has been determined. Infectious virions of canine parvovirus contain 60 protein subunits that are predominantly VP-2. The central structural motif of VP-2 has the same topology (an eight-stranded antiparallel beta barrel) as has been found in many other icosahedral viruses but represents only about one-third of the capsid protein. There is a 22 angstrom (A) long protrusion on the threefold axes, a 15 A deep canyon circulating about each of the five cylindrical structures at the fivefold axes, and a 15 A deep depression at the twofold axes. By analogy with rhinoviruses, the canyon may be the site of receptor attachment. Residues related to the antigenic properties of the virus are found on the threefold protrusions. Some of the amino termini of VP-2 run to the exterior in full but not empty virions, which is consistent with the observation that some VP-2 polypeptides in full particles can be cleaved by trypsin. Eleven nucleotides are seen in each of 60 symmetry-related pockets on the interior surface of the capsid and together account for 13 percent of the genome.

Amino Acid Sequence↗
PubMed1991

Inhibition of virus-induced cell fusion by apolipoprotein A-I and its amphipathic peptide analogs.

Apolipoprotein A-I (apoA-I), the major protein component of serum high-density lipoproteins (HDL), was found to inhibit herpes simplex virus (HSV)-induced cell fusion at physiological (approximately 1 microM) concentrations, whereas HDL did not exert any inhibitory effect. Lipid-associating, synthetic amphipathic peptides corresponding to residues 1-33 (apoA-I[1-33]) or residues 66-120 (apoA-I[66-120]) of apoA-I, also inhibited HSV-induced cell fusion, whereas a peptide corresponding to residues 8-33 of apoA-I (apoA-I[8-33]), which fails to associate with lipids, did not exert any inhibitory effect. These results suggest that lipid binding may be a prerequisite for peptide-mediated fusion inhibition. Consistent with this idea, a series of lipid-binding 22-amino-acid-residue-long synthetic amphipathic peptides that correspond to the amphipathic helical domains of apoA-I (A-I consensus series), or 18-residue-long model amphipathic peptides (18A series), were found to exert variable levels of fusion-inhibitory activity. The extent of fusion-inhibitory activity did not correlate with hydrophobic moment, hydrophobicity of the nonpolar face, helix-forming ability, or lipid affinity of the different peptides. Peptides in which the nonpolar face was not interrupted by a charged residue displayed greater fusion-inhibitory activity. Also, the presence of positively charged residues at the polar-nonpolar interface was found to correlate with higher fusion-inhibitory activity.

Amino Acid Sequence↗
PubMed1991

Golgi complex localization of the Punta Toro virus G2 protein requires its association with the G1 protein.

The glycoproteins of bunyaviruses accumulate in membranes of the Golgi complex, where virus maturation occurs by budding. In this study we have constructed a series of full length or truncated mutants of the G2 glycoprotein of Punta Toro virus (PTV), a member of the Phlebovirus genus of the Bunyaviridae, and investigated their transport properties. The results indicate that the hydrophobic domain preceding the G2 glycoprotein can function as a translocational signal peptide, and that the hydrophobic domain near the C-terminus serves as a membrane anchor. A G2 glycoprotein construct with an extra hydrophobic sequence derived from the N-terminal NSM region was stably retained in the ER, and was unable to be transported to the Golgi complex. The full-length G2 glycoprotein, when expressed on its own, was transported out of the ER and expressed on the cell surface, whereas the G1 and G2 proteins when expressed together are retained in the Golgi complex. A truncated anchor-minus form of the G2 glycoprotein was found to be secreted into the culture medium, but was retained in the Golgi complex when coexpressed with the G1 glycoprotein. These results indicate that the G2 membrane glycoprotein is a class I membrane protein which does not contain a signal sufficient for Golgi retention, and suggest that its Golgi localization is a result of association with the G1 glycoprotein.

Amino Acid Sequence↗
PubMed1991

Molecular domains involved in oligomerization of the Friend murine leukemia virus envelope glycoprotein.

The oligomeric structure of the Friend murine leukemia virus envelope glycoprotein has been investigated using crosslinking reagents and sucrose density gradient centrifugation. The results obtained provide evidence that both the precursor and the processed molecules are oligomeric and probably form tetramers. Pulse-chase analyses indicate that assembly occurs sequentially, within 30 min of protein synthesis and prior to cleavage of the precursor. Studies using chimeric envelope glycoproteins and deletion mutants indicate that the transmembrane and cytoplasmic domains are not essential for the formation of oligomers. Evidence is also presented that the SU subunit remains in an oligomeric form following disassociation from the TM subunit. Oligomeric envelope glycoprotein complexes linked by intermolecular disulfide bonds were also observed under certain conditions. Mink cell focus-forming virus envelope glycoprotein constructs lacking the transmembrane domain or both the transmembrane and the cytoplasmic domains formed intermolecular disulfide bonds more readily than the full-length molecule, suggesting that these regions are likely to make a contribution to the conformation of the glycoprotein. These data indicate that there are several points of interaction between retrovirus envelope glycoprotein monomers which contribute to assembly of the oligomer and that contacts within the ectodomain appear to be of critical importance.

Amino Acid Sequence↗
PubMed1991

The hydrophobic membrane-spanning sequences of the gp52 glycoprotein are required for the pathogenicity of Friend spleen focus-forming virus.

Friend spleen focus-forming virus (SFFV) codes for a transport-defective envelope glycoprotein designated gp52, which is responsible for the leukemogenic properties of the virus. gp52 is a monotopic integral membrane protein anchored in the membrane by a stretch of hydrophobic amino acid residues located near the carboxy terminus of the molecule. We have constructed a mutant SFFV envelope gene in which the sequences that code for the hydrophobic membrane-spanning domain have been deleted, and we expressed this gene by using recombinant vaccinia virus vectors or retroviral vectors. The mutant SFFV envelope gene was found to encode a truncated glycoprotein (gp52t) which was also transport defective; a majority of gp52t remained cell associated, while a small proportion of the molecules underwent oligosaccharide processing. The processed form of gp52t was secreted from the cells. Retroviral vectors carrying the mutant SFFV envelope gene were found to be nonpathogenic in adult mice. These results indicate that the hydrophobic membrane-spanning region of gp52 is required for pathogenicity of SFFV and suggest that these sequences may play a role in signal transduction. The results also indicate that the transport defect of SFFV gp52 is due to structural features of the ectodomain of the molecule.

Amino Acid Sequence↗
PubMed1991

Oligomerization, transport, and Golgi retention of Punta Toro virus glycoproteins.

We have investigated the oligomerization and intracellular transport of the membrane glycoproteins of Punta Toro virus, a member of the Phlebovirus genus of the family Bunyaviridae, which is assembled by budding in the Golgi complex. By using one- or two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chemical cross-linking, and sucrose gradient centrifugation, we found that the majority of the G1 and G2 glycoproteins are assembled into noncovalently linked G1-G2 heterodimers. At the same time, a fraction of the G2 protein, possibly produced independently of the G1 protein, is assembled into G2 homodimers. Kinetic analysis indicates that heterodimerization occurs between newly synthesized G1 and G2 within 3 min after protein synthesis, and that the G1 and G2 glycoproteins are associated as dimeric forms both during transport and after accumulation in the Golgi complex. Analysis of a G1-truncated G2 mutant, which is also targeted to the Golgi complex, showed that these molecules also assemble into dimeric forms, which are linked by disulfide bonds. Both the G1-G2 heterodimer and the G2 homodimer were found to be able to exit from the endoplasmic reticulum. Differences in transport kinetics observed for the G1 and G2 proteins may be due to the differences in the transport efficiency between the G1-G2 heterodimer and the G2 homodimer from the endoplasmic reticulum to the Golgi complex. These and previous results (S.-Y. Chen, Y. Matsuoka, and R.W. Compans, Virology 183:351-365, 1991) suggest that Golgi retention of the G2 homodimer occurs by association with the G1-G2 heterodimer, whereas the Golgi targeting of the G1-G2 heterodimer occurs by a specific retention mechanism.

Animals↗
PubMed1991

Inhibition of human immunodeficiency virus type 1-induced cell fusion by recombinant human interferons.

Pretreatment of HeLa T4 cells with recombinant alpha, beta, or gamma interferon (IFN) was found to significantly inhibit syncytium formation induced by the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein. All three IFNs were found to be potent inhibitors of fusion in a system in which Spodoptera frugiperda cells, infected with a baculovirus recombinant expressing the HIV-1 envelope protein, were cocultivated with HeLa T4 cells. In addition, these IFNs were also found to block HeLa T4 cell fusion induced by the HIV-1 envelope proteins expressed from a vaccinia virus recombinant. Furthermore, the IFNs inhibited cell fusion between HIV-1 envelope glycoprotein-expressing cells and either immortalized or fresh CD4+ lymphocytes pretreated with the IFNs. These results suggest that further testing of human IFNs for therapy of HIV-1 infection will be of interest.

Animals↗