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R W Compans

Publications and source records attributed to R W Compans.

At least 163 records · Page 9Linked to original sources
PubMed1986

Polarized expression of a chimeric protein in which the transmembrane and cytoplasmic domains of the influenza virus hemagglutinin have been replaced by those of the vesicular stomatitis virus G protein.

In polarized epithelial cells, influenza virus buds exclusively from the apical domain of the plasma membrane, whereas vesicular stomatitis virus (VSV) buds exclusively from the basolateral domain. In virus-infected cells, the envelope proteins, influenza hemagglutinin (HA) and vesicular stomatitis virus G (VSV G), are likewise transported to and localized in the same domain of the plasma membrane from which the viruses bud. Previous studies have shown that influenza HA and VSV G proteins, when expressed from cloned cDNAs, are accumulated preferentially on the proper domains (apical and basolateral, respectively), indicating that the signal(s) for polarized transport resides in the polypeptide backbone of the proteins. To further elucidate the structural features required for apical vs. basolateral transport, we have constructed a gene that encodes a chimeric protein (H1GA) containing the external domain of HA and the transmembrane and cytoplasmic domains of VSV G. When the chimeric protein (H1GA) is expressed in CV1 cells using a simian virus 40 late expression vector, it is transported to the cell surface with kinetics similar to that of the native HA protein. Further, the chimeric protein, when expressed in polarized MDCK cells using a vaccinia virus early expression vector, is transported only to the apical surface, suggesting that the ectodomain of HA contains a signal for apical transport.

Cell Compartmentation↗
PubMed1986

Expression of herpes simplex virus glycoproteins in polarized epithelial cells.

Members of the herpesvirus family mature at inner nuclear membranes, although a fraction of the viral glycoproteins is expressed on the cell surface. In this study, we investigated the localization of herpes simplex virus type 2 (HSV-2) glycoproteins in virus-infected epithelial cells by using a panel of monoclonal antibodies directed against each of the major viral glycoproteins. All of the HSV-2 glycoproteins were localized exclusively on the basolateral membranes of Vero C1008, Madin-Darby bovine kidney, and mouse mammary epithelial cells. Using a monoclonal antibody to HSV-2 gD which cross-reacts with HSV-1 strains, we could also localize HSV-1 gD on the basolateral membranes of Madin-Darby bovine kidney cells. These results indicate that these molecules contain putative sorting signals that direct them to basolateral membrane domains.

Animals↗
PubMed1985

Synthesis of Tacaribe virus polypeptides in an in vitro coupled transcription and translation system.

We have analyzed polypeptides synthesized in a coupled in vitro transcription and translation system in response to detergent-disrupted Tacaribe virus. Analysis of the major Tacaribe virus-specified product by two-dimensional polyacrylamide gel electrophoresis indicated that it had an isoelectric point similar to that of the Tacaribe nucleocapsid polypeptide N; however, the in vitro product had an approximate mol. wt. of 73 000, compared to a mol. wt. of 68 000 for the N protein. The 73 000 dalton product was found to yield proteolytic cleavage products with similar electrophoretic mobilities to those obtained from the virion P and N proteins. These results, as well as pulse-chase experiments in Tacaribe virus-infected cells, suggest that a 73 000 dalton polypeptide may be processed to yield the N polypeptide. The polypeptides synthesized in the coupled system depended on the amount and type of virus added; addition of purified Shark River (SR) virus, a member of the Patois group of bunyaviruses, resulted in synthesis of a polypeptide of mol. wt. 22 000 which corresponds to the SR nucleocapsid protein.

Arenaviridae↗
PubMed1985

Sialic acid is incorporated into influenza hemagglutinin glycoproteins in the absence of viral neuraminidase.

We have analyzed the pronase-derived glycopeptides of the hemagglutinin glycoproteins expressed from SV40 vectors carrying cloned cDNA copies of the HA gene and of HA isolated from influenza virions (A/Jap/305/57). The glycopeptides derived from he HA glycoprotein obtained from cloned genes were heterogeneous, ranging in size from 3800 to 2800 daltons. Upon treatment with neuraminidase, sialic acid was released from the glycopeptides and their size was reduced to 2900-2400 daltons. However, under the same conditions, no sialic acid was detected in the virion HA. The presence of sialic acid was confirmed by monosaccharide analysis of the HA glycoprotein derived from products of cloned genes. These results support the idea that during replication of influenza virus, the viral neuraminidase cleaves sialic acid from the HA glycoprotein in infected cells.

Glycopeptides↗
PubMed1985

Structure and variation of the influenza C glycoprotein.

The amino acid sequence of the influenza C/JHB/1/66 glycoprotein was predicted from sequence analysis of cloned DNA. The glycoprotein exhibited several similarities to the haemagglutinin (HA) glycoproteins of influenza A and B viruses, although its overall homology to these glycoproteins was low. A comparison between the sequences of C/JHB/1/66 and C/Cal/78 strains revealed 96% homology. The nucleotide and amino acid changes appeared to be non-random, with a high proportion occurring between amino acid positions 182-212. A comparison between the JHB/1/66 glycoprotein and the influenza A/X31 HA sequence suggested that this region may be structurally analogous to the 'A' antigenic site of the HA glycoprotein.

Amino Acid Sequence↗
PubMed1985

Glycoproteins of human parainfluenza virus type 3: characterization and evaluation as a subunit vaccine.

The envelope glycoproteins of human parainfluenza type 3 virus were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reactivity with specific monoclonal antibodies. The molecular weight of the hemagglutinin-neuraminidase (HN) glycoprotein was found to be 72,000, and the fusion (F) glycoprotein appeared to consist of 74,000 (F0) or 56,000 (F1) species. Envelope glycoproteins were solubilized with octyl-glucoside and, after removal of the detergent by dialysis, were used for immunization of hamsters. Other animals were immunized with a formalin-inactivated preparation of whole virus. A single subcutaneous immunization with these antigen preparations induced a serum antibody response to the HN and F glycoproteins, as determined by plaque neutralization, hemagglutination inhibition, inhibition of virus-induced cell fusion, and immune precipitation tests. An IgG antibody response to both glycoproteins was also observed in bronchial washings. Animals immunized with the highest dose of envelope glycoproteins showed complete protection from challenge infection, whereas immunization with inactivated virus did not completely protect animals.

Animals↗
PubMed1985

Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells.

We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold- or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and the first 10 amino-terminal residues of the NA polypeptide are not involved in this process.

Animals↗
PubMed1984

Structure of the influenza C glycoprotein gene as determined from cloned DNA.

The complete nucleotide sequence of RNA segment 4 of influenza C/JHB/1/66 virus has been determined, utilizing cloned cDNA derived from the viral RNA segment. The gene is 2073 nucleotides in length, and can code for a polypeptide of 655 amino acids, which corresponds to the viral glycoprotein. The predicted polypeptide has a molecular weight of 72 063, not including oligosaccharides linked to eight predicted glycosylation sites. The influenza C glycoprotein shares structural features with hemagglutinin (HA) glycoproteins of influenza A and B viruses, including three stretches of hydrophobic amino acids believed to function as a signal sequence, a fusion function, and a membrane anchor. However, a substantial part of the protein lacks direct sequence homology to these HA glycoproteins. The results suggest a more distant evolutionary relationship between influenza C virus and influenza A and B viruses, compared to that between influenza A and B themselves.

Amino Acid Sequence↗
PubMed1984

Replication and morphogenesis of avian coronavirus in Vero cells and their inhibition by monensin.

Avian infectious bronchitis virus (IBV) was adapted to Vero cells by serial passage. No significant inhibition of IBV replication was observed when infected Vero cells were treated with alpha-amanitin or actinomycin D. In thin sections of infected cells, assembly of IBV was observed at the rough endoplasmic reticulum (RER), and mature IBV particles were located in dilated cisternae of the RER as well as in smooth cytoplasmic vesicles. In addition to typical IBV particles, enveloped particles containing numerous ribosomes were identified at later times postinfection. Monensin, a sodium ionophore which blocks glycoprotein transport to plasma membranes at the level of the Golgi complex, was found to inhibit the formation of infectious IBV. In thin sections of infected Vero cells treated with the ionophore, IBV particles were located in dilated cytoplasmic vesicles, but fewer particles were found when compared to controls. A similar pattern of virus-specific proteins was detected in control or monensin-treated IBV-infected cells, which included two glycoproteins (170 000 and 24 000 daltons) and a polypeptide of 52 000 daltons. These results suggest that the ionophore inhibits assembly of a virus which matures at intracellular membranes.

Animals↗
PubMed1984

Chimeric influenza virus hemagglutinin containing either the NH2 terminus or the COOH terminus of G protein of vesicular stomatitis virus is defective in transport to the cell surface.

Chimeric cDNA clones of influenza virus hemagglutinin (HA) were constructed in which the DNA encoding either the NH2 terminus or the COOH terminus of HA was replaced with that of a vesicular stomatitis virus G protein. The chimeric cDNAs (GHA or HAG) were expressed in CV1 cells using the simian virus 40 late replacement promoter. Both chimeric proteins are synthesized, glycosylated, and transported to the rough endoplasmic reticulum. These results show that the NH2-terminal sequences of vesicular stomatitis virus G protein can provide a signal function for translocation and the COOH-terminal sequences can provide the anchor function for the influenza virus HA, when substituted for similar sequences. However, the chimeric glycoproteins were not transported to the Golgi complex or the plasma membrane. The implication of these results in translocation, sorting, and transport processes is discussed.

Animals↗
PubMed1983

Membrane association and defective transport of spleen focus-forming virus glycoproteins.

The gp52 glycoprotein of the spleen focus-forming virus found in the Friend and Rauscher complexes of murine leukemia viruses (MuLV) has been previously identified as a recombinant molecule involving substitutions and deletions of the MuLV env gene. Unlike the MuLV structural glycoproteins, gp52 is defective in its transport to the cell surface. We have studied aspects of the intracellular transport and membrane association of gp52 to investigate the possible mechanisms underlying the defective transport process. It was found that a panel of monoclonal antibodies to different epitopes of p 15E, as well as an antiserum to a synthetic peptide corresponding to the carboxy terminus of MuLV envelope precursors, failed to react with gp52. Despite the possible absence of membrane-anchoring regions of MuLV envelope proteins known to reside on p 15E, gp52 was not found to be secreted into the culture fluids. Detergent extraction studies indicated that gp52 is associated with the membranes and not the contents of microsomal vesicles in speen focus-forming virus-infected cells. gp65, the processed form of gp52, could be labeled with [3H]palmitic acid, suggesting a membrane association. To determine whether a spontaneous denaturation occurs leading to aggregation and defective transport of gp52, we studied the surface expression of gp52 in cells grown at different temperatures, as well as the solubility of gp52 in low concentrations of Triton X-100. No evidence of aggregation or of a temperature-dependent difference in transport was obtained. gp52 appears to be a monotopic integral membrane protein, unlike MuLV envelope proteins which are bitopic integral membrane proteins; proteolytic digestion of intact microsomal vesicles did not reveal a detectable cytoplasmic tail under conditions where this could be demonstrated on MuLV envelope precursors. We suggest that a loss of putative signals involved in mediating intracellular transport is a likely cause for the defective transport of the spleen focus-forming virus glycoproteins.

Animals↗
PubMed1983

Analysis of polypeptides in Tacaribe virus-infected cells.

Two virus-induced polypeptides designated p79 (mol wt, 79,000) and p105 (mol wt, 105,000) in BHK21 or Vero cells infected with Tacaribe (Tac) virus have been identified. Both polypeptides were found in immune precipitates with antiserum to Tac virus, suggesting that they are virus specific. Two-dimensional gel analysis of Tac virus-infected Vero cell extracts indicated that p79 and p105 were acidic proteins which did not comigrate with any polypeptides from uninfected cells. Neither of the polypeptides was found to be phosphorylated under conditions in which phosphorylation of the N (nucleocapsid) protein was detected. Comparison of one-dimensional peptide maps of the p79 polypeptide and the nucleoprotein indicated that they are unrelated in primary sequences.

Amino Acid Sequence↗
PubMed1983

Studies on the role of glycosylation in the functions and antigenic properties of influenza virus glycoproteins.

The biological and antigenic roles of glycosylation were investigated in the influenza hemagglutinin (HA) glycoprotein using the glycosylation inhibitor tunicamycin (TM). Under conditions where only the nonglycosylated form of HA was detected by immunoprecipitation and gel electrophoresis, the migration of glycoproteins to the cell surface was observed by immunofluorescence using either monospecific or monoclonal antibody to the HA polypeptide. Analysis of the surface fluorescence in TM-treated infected cells by a fluorescence-activated cell sorter (FACS) showed that all cells exhibited fluorescence in the complete absence of glycosylation. The relative amount of HA antigen on cell surfaces was found to be reduced by only 30-40% in TM-treated cells, and this reflected a similar reduction in intracellular synthesis. Electron microscopic studies using ferritin labeling also demonstrated that the nonglycosylated HA glycoprotein was present in significant amounts on surfaces of infected cells. Virions with nonglycosylated glycoproteins were purified, and were found to have an approximate 30-fold decrease in both hemagglutinin and neuraminidase specific activities. The possible role of oligosaccharides in antigenic variation among various H1N1 strains was investigated. Immunoprecipitation reactions involving five different monoclonal antibodies and five antigenic variants of A/USSR/90/77 revealed no major antigenic differences between the glycosylated and nonglycosylated forms of HA.

Animals↗
PubMed1983

Glycosylation and intracellular transport of spleen focus-forming virus glycoproteins.

We have investigated the pattern of glycosylation of the membrane glycoproteins encoded by a polycythemic strain of spleen focus-forming virus (SFFV). These include a major species designated gp52 and its processed form which is designated gp65. The SFFV glycoproteins were found to be predominantly intracellular, although a portion of gp65 is expressed on the cell surface. gp65 was observed to be highly sialylated and resistant to digestion with endoglycosidase-H (endo-H). In contrast, gp52 was endo-H sensitive and the polyacrylamide gel electrophoresis profile of the endo-H digests suggested the presence of four glycosylation sites. Analysis of tryptic glycopeptides from gp52 by reverse-phase high-performance liquid chromatography also suggested the presence of four glycosylation sites. Glycopeptide analysis of Pronase digests of gp52 revealed two major size classes with molecular weights of 2200 and 1500, which correspond to two of the four oligosaccharide size classes reported previously for MuLV gp70's (M.C. Kemp, N.G. Famulari, P.V. O'Donnell, and R.W. Compans, 1980, J. Virol. 34, 154). Both glycopeptide size classes were sensitive to digestion with endo-H. The glycopeptide profile of gp65 was found to be very heterogeneous and the predominant form was a 2900-dalton size class. In addition a fucosyl glycopeptide of 2500 daltons was observed in gp65, but not in F-MuLV or F-MCF glycoproteins. In the presence of the sodium ionophore monensin, the processing of gp52 to gp65 was inhibited. Instead a smaller protein of about 60,000 daltons was observed, which did not arrive at the cell surface, a situation analogous to the processing and post-translational modification reported for gp52 from anemic isolates of SFFV (S.K. Ruscetti, J.A. Field, and E.M. Scolnick, 1981, Nature (London) 294, 663).

Animals↗
PubMed1983

Effects of hexose starvation and the role of sialic acid in influenza virus release.

We previously reported that growth of influenza virus in the presence of cytochalasin B (CB), a drug that disrupts microfilaments and blocks hexose transport, yields particles with glycoproteins that are heterogeneous and unlabeled by [3H]glucosamine. When the virus was grown in glucose-free medium, we observed reduced virus titers similar to those produced by CB. In contrast, treatment of cells with cytochalasin D (CD) and dihydrocytochalasin B (H2CB), drugs which are known to inhibit microfilament function without affecting hexose transport, did not cause a reduction in virus titers or a change in the electrophoretic mobility of viral glycoproteins. Partial inhibition of glycosylation of viral glycoproteins resulting from either CB-induced inhibition of hexose transport or from glucose starvation resulted in the formation of aggregates of virions on cell surfaces. These aggregates can be dissociated by exogenous neuraminidase. Under these conditions the virions contained a functional hemagglutinin glycoprotein (HA) but an inactive neuraminidase glycoprotein (NA) which was not able to cleave sialic acid, the HA receptor, from viral glycoproteins, or from cellular glycoproteins and glycolipids. Neuraminidase treatment of membrane fractions of CB-treated cells did not cause a shift in the electrophoretic mobility of HA or in the gel elution profile of HA glycopeptides obtained after extensive pronase digestion from HA synthesized in glucose-free medium. These findings suggest that sialic acid is not present on labeled glycoproteins in either of these preparations. We obtained evidence that the sialic acid to which HA binds when NA is inactive is on glycoproteins and glycolipids of cellular origin. Our results support the idea that even when NA is functional, sialylated cellular components impede influenza virus release.

Animals↗
PubMed1983

Influenza virus hemagglutinin expression is polarized in cells infected with recombinant SV40 viruses carrying cloned hemagglutinin DNA.

Primary cell cultures of African Green monkey kidney (AGMK) contain polarized epithelial cells in which influenza virus matures predominantly at the apical surfaces above tight junctions. Influenza virus glycoproteins were found to be localized at the same membrane domain from which the virus budded. When polarized primary AGMK cells were infected with recombinant SV40 viruses containing DNA coding for either an influenza virus H1 or H2 subtype hemagglutinin (HA), the HA proteins were preferentially expressed at the apical surface in a manner identical to that observed in influenza virus-infected cells. Thus, cellular mechanisms for sorting membrane glycoproteins recognize some structural feature of the HA glycoprotein itself, and other viral proteins are not necessary for this process.

Animals↗
PubMed1983

Immune response to human influenza virus hemagglutinin expressed in Escherichia coli.

Cloned DNA fragments coding for parts of strain WSN (H1N1) influenza virus hemagglutinin (HA) were fused to a bacterial leader DNA derived from the Escherichia coli trp operon. Fusion proteins produced consisted of 190 amino acids of trpLE' protein at the amino terminus, and HA amino acids, either 1-308, 1-396, or 1-548 (complete HA), at the carboxyl terminus. These proteins were expressed at high levels (10-20% of total protein) in E. coli starved for tryptophan. A CNBr fragment (HA1-211) was derived from HA-308. Each of the proteins was purified and used for immunizing mice and rabbits. The antibody produced was shown to bind to (i) the HA fusion proteins, (ii) detergent-treated viral HA, (iii) HA, on intact virions, and (iv) the HA on the surface of cells infected with influenza virus. This shows that the HA fusion proteins expressed in bacteria can elicit antibodies that recognize at least some determinants of the native viral HA, and probably could lead to development of an anti-influenza vaccine.

Animals↗