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R W Compans

Publications and source records attributed to R W Compans.

At least 145 records · Page 8Linked to original sources
PubMed1989

Expression of the human immunodeficiency virus envelope glycoprotein is restricted to basolateral surfaces of polarized epithelial cells.

Polarized epithelial cells exhibit apical (lumenal) and basolateral (serosal) membrane domains that are separated by circumferential tight junctions. In such cells, enveloped viruses that mature by budding at cell surfaces are released at particular membrane domains. We have used a vaccinia virus recombinant to investigate the site of surface expression of the human immunodeficiency virus type 1 envelope glycoprotein in Madin-Darby canine kidney cells. Cells were infected with the vaccinia virus recombinant, and surface expression of the glycoprotein was analyzed by indirect immunofluorescence, 125I-protein A binding, and immunoelectron microscopy. The glycoprotein appeared exclusively at the basolateral surface as early as 2 h postinfection and reached a maximum level at 8 h postinfection. The gp120 glycoprotein was found to be secreted efficiently into culture medium, and this secretion occurred exclusively at the basolateral surface.

Animals↗
PubMed1989

Characterization of simian virus 40 receptor moieties on the surfaces of Vero C1008 cells.

The nature of the simian virus 40 (SV40) receptor on the surfaces of Vero C1008 cells was investigated by a virus binding assay. The optimum pH for SV40 binding to cell surfaces was found to be at 6.5; however, there was little difference in SV40 binding in the range between pH 4.5 and 7.3. The treatment of cell surfaces with several proteases or with an enzyme specific for O-linked carbohydrates significantly reduced virus binding, suggesting that the receptor for SV40 contains protein and O-linked carbohydrates. Treatment of cell monolayers with octyl glucoside removed virus-binding activity from cell surfaces. Recovery of virus-binding activity by octyl glucoside-treated cells took 2.5 h and was inhibited by cycloheximide or tunicamycin. Four polypeptides with molecular weights of 90,000, 58,000, 54,000, and 30,000 were immunoprecipitated from virus-protein complexes derived from octyl glucoside extract solutions and therefore may be components of the SV40 receptor. Competition experiments between SV40 and polyomavirus revealed that these two viruses do not share the same receptor on Vero C1008 cells.

Animals↗
PubMed1989

Release of simian virus 40 virions from epithelial cells is polarized and occurs without cell lysis.

We have investigated the process of release of simian virus 40 (SV40) virions from several monkey kidney cell lines. High levels of virus release were observed prior to any significantly cytopathic effects in all cell lines examined, indicating that SV40 utilizes a mechanism for escape from the host cell which does not involve cell lysis. We demonstrate that SV40 release was polarized in two epithelial cell types (Vero C1008 and primary African green monkey kidney cells) grown on permeable supports; release of virus occurs almost exclusively at apical surfaces. In contrast, equivalent amounts of SV40 virions were recovered from apical and basal culture fluids of nonpolarized CV-1 cells. SV40 virions were observed in large numbers on apical surfaces of epithelial cells and in cytoplasmic smooth membrane vesicles. The sodium ionophore monensin, an inhibitor of vesicular transport, was found to inhibit SV40 release without altering viral protein synthesis or infectious virus production.

Animals↗
PubMed1989

Polarized entry of canine parvovirus in an epithelial cell line.

The binding and uptake of canine parvovirus (CPV) in polarized epithelial cells were investigated by growing the cells on a permeable support and inoculating with the virus either from the apical or basolateral surface. Binding of radiolabeled CPV occurred preferentially on the basolateral surface. In contrast, when a similar experiment was carried out on nonpolarized A72 cells, virus binding occurred regardless of the direction of virus input. Binding appeared to be specific for CPV and could not be competitively inhibited by either bovine or porcine parvovirus. Analysis of the binding data revealed a high-affinity receptor (10(5) per cell) for CPV on the basolateral surfaces of MDCK cells (Kd, 29 pM). In indirect immunofluorescence studies, virus entered only from the basolateral surfaces of MDCK cells. These results provide evidence for a functional CPV-specific receptor that is expressed only on the basolateral surfaces of polarized epithelial cells, a result that has interesting consequences for viral pathogenesis.

Animals↗
PubMed1988

Preliminary X-ray crystallographic analysis of canine parvovirus crystals.

The first diffraction pattern of a crystalline single-stranded DNA virus has been obtained. Canine parvovirus was crystallized in a monoclinic P21 unit cell with a = 264.4 A, b = 350.3 A, c = 267.8 A and beta = 90.86 degrees (1 A = 0.1 nm). The diffraction pattern extends to at least 2.8 A resolution. Packing of the particles suggests that they have a diameter around 257 A, in excellent agreement with the reported molecular weight of 5.5 x 10(6).

Animals↗
PubMed1988

Intracellular accumulation of Punta Toro virus glycoproteins expressed from cloned cDNA.

The Punta Toro virus (PTV) middle size (M) RNA encodes two glycoproteins, G1 and G2, and possibly a nonstructural protein, NSM. A partial cDNA clone of the M segment which contains G1 and G2 glycoprotein coding sequences but lacks most of the NSM sequences was inserted into the genome of vaccinia virus under the control of an early vaccinia promoter. Cells infected with the recombinant virus were found to synthesize two polypeptides with molecular weights of 65,000 (G1) and 55,000 (G2) that reacted specifically with antibody against PTV. Studies using indirect immunofluorescence microscopy revealed that these proteins accumulated intracellularly in the perinuclear region. The results of endoglycosidase H digestion of these glycoproteins suggested that both G1 and G2 glycoproteins were transported from the RER to the Golgi complex. These proteins were not chased out from the Golgi region during a 6-hr incubation in the presence of cycloheximide. Surface immune precipitation and 125I-protein A binding assays also demonstrated that the majority of the G1 and G2 glycoproteins are retained intracellularly. These results indicate that the PTV glycoproteins contain the necessary information for retention in the Golgi apparatus.

Acetylglucosaminidase↗
PubMed1988

Expression of the spleen focus-forming virus envelope gene in a polarized epithelial cell line.

Friend spleen focus-forming virus (F-SFFV) encodes a glycoprotein designated gp52, which is defective in its intracellular transport and accumulates in the rough endoplasmic reticulum. Only 3-5% of the mature form of gp52 eventually reaches the cell surface. Compared to transport-competent murine leukemia virus (MuLV) glycoproteins, the gp52 molecule exhibits several structural differences which may have resulted in the possible loss of signals required for transport to the cell surface. To determine the effect of these alterations on the specific sites of surface expression of the molecule, the SFFV env gene was expressed from a vaccinia virus recombinant in a polarized epithelial cell line in which retrovirus glycoproteins are expressed exclusively on basolateral surfaces. We also determined the site of expression of a chimeric env protein which contains the external domain of SFFV gp52 the transmembrane, and the cytoplasmic tail residues of Friend MuLV. The wild-type and chimeric env gene products were defective in transport, and remained primarily in an unprocessed form in MDCK cells or CV-1 cells. However, both glycoproteins were detected at low levels on the basolateral surfaces of MDCK cells, a line of polarized epithelial cells. These results indicate that the presence or absence of a cytoplasmic tail as well as a 585-base deletion in the external domain has no affect on the site of polarized expression of a murine retrovirus glycoprotein.

Biological Transport↗
PubMed1988

Intranasal immunization of hamsters with envelope glycoproteins of human parainfluenza virus type 3.

Envelope glycoproteins of human parainfluenza virus type 3 (PIV-3) were selectively solubilized with n-octyl beta-D-glucopyranoside and reconstituted into lipid vesicles by dialysis of the detergent. The efficacy of the glycoprotein preparation as a subunit vaccine when administered to hamsters intranasally or subcutaneously was compared. Animals receiving four intranasal immunizations with 5 micrograms of the glycoprotein preparation were completely resistant to challenge infection. Only partial protection, however, was observed in animals immunized subcutaneously with the same dose of antigen. The local glycoprotein-specific IgA response was significantly higher in intranasally immunized animals and was implicated in resistance to challenge infection.

Administration, Intranasal↗
PubMed1988

Role of individual glycoproteins of human parainfluenza virus type 3 in the induction of a protective immune response.

Affinity-purified hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of human parainfluenza virus type 3 (P13 virus) were used to investigate their role in the induction of a protective immune response following immunization of hamsters. The efficacy of immunization with the glycoprotein antigens was tested by challenge infection. Results of virus recovery from lungs and trachea demonstrated that although immunization with HN or F alone induced an antibody response to the respective glycoproteins, it did not provide a significant level of protection. However, immunization with a mixture of both purified glycoproteins induced higher virus-neutralizing activity in bronchial lavages and afforded complete protection from challenge infection. Similarly, incomplete protection was observed after passive transfer of monospecific rabbit antibody to the purified HN or F in baby hamsters. On the other hand, passive transfer of a mixture of antibodies to HN and F conferred a higher level of protection. Thus, the presence of antibody to both glycoproteins of P13 virus may be essential for protective immunity.

Animals↗
PubMed1988

Entry of simian virus 40 is restricted to apical surfaces of polarized epithelial cells.

The uptake of simian virus 40 (SV40) by polarized epithelial cells was investigated by growth of cells on permeable supports and inoculation on either the apical or the basolateral surface. Binding of radiolabeled SV40 occurred on the apical but not the basolateral surfaces of permissive polarized Vero C1008 cells and nonpermissive polarized MDCK cells. When similar experiments were performed on nonpolarized Vero or CV-1 cells, virus binding occurred regardless of the direction of virus input. Electron micrographs of Vero C1008 cells infected at high multiplicities revealed virions lining the surfaces of apically infected cells, while the surfaces of basolaterally infected cells were devoid of virus particles. Analysis of the binding data revealed a single class of virus receptors (9 x 10(4) per cell) with a high affinity for SV40 (Kd = 3.76 pM) on the apical surfaces of Vero C 1008 cells. Indirect immunofluorescence studies revealed that synthesis of viral capsid proteins in Vero C1008 cells occurred only when input virions had access to the apical surface. Virus yields from apically infected Vero C1008 cells were 10(5) PFU per cell, while yields obtained from basolaterally infected cells were less than one PFU per cell. These results indicate that a specific receptor for SV40 is expressed exclusively on the apical surfaces of polarized Vero C1008 cells.

Animals↗
PubMed1987

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

Two integral membrane proteins, influenza virus hemagglutinin (HA) and vesicular stomatitis virus G protein, are transported to and accumulated on the apical and basolateral surfaces, respectively, of the plasma membrane of polarized epithelial cells. We have used chimeric constructions to identify the domains of HA and G proteins which contain the signals for polarized transport. Previously, we have shown that a chimeric protein containing the cleavable leader and the ectodomain of HA fused to the anchoring and cytoplasmic domains of G is transported to the apical surface of polarized MDCK cells (McQueen, N.L., Nayak, D.P., Stephens, E.B., and Compans, R.W. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9318-9322). In this report we show that a chimera containing the cleavable leader and ectodomain of G fused to the anchoring and cytoplasmic domains of HA is transported to the basolateral surface of polarized cells. Another chimera which contains the leader sequence of G fused to leader minus HA is transported to the apical surface of polarized cells. These results taken together suggest that the signals for the polarized transport of HA and G proteins may reside in their ectodomains.

Amino Acid Sequence↗
PubMed1987

Effects of deletion of the cytoplasmic domain upon surface expression and membrane stability of a viral envelope glycoprotein.

The envelope protein (gp52) of Friend spleen focus-forming virus (F-SFFV) is defective in its intracellular transport and accumulates in the rough endoplasmic reticulum of F-SFFV-infected cells. This defect in transport has been attributed to the lack of a cytoplasmic domain, and possible loss of signals required for transport to the cell surface. The mature form of gp52, designated gp65, is also reported to be secreted from SFFV-infected cells. To determine the specific changes in the envelope protein which may lead to its lack of transport and to its lack of stability in associating with membranes, the 3' end of the F-SFFV envelope gene, which encodes the transmembrane domain, was inserted in place of the normal 3' end of the Friend murine leukemia virus genome. This chimeric envelope gene was expressed using the vaccinia virus expression system. The chimeric gp70/p15E glycoprotein molecule lacks the cytoplasmic tail residues and as a consequence is about 3300 daltons smaller. The chimeric PrEnv molecule was found to be cleaved efficiently as indicated by pulse-chase experiments. Immunofluorescence studies demonstrate that the chimeric molecule is efficiently transported to the surface of cells, unlike the SFFV gp52 glycoprotein. The chimeric molecule was found to be unstable in its membrane association and is released into the culture medium. These results indicate that the changes in the membrane spanning region and the lack of a cytoplasmic tail do not determine the defective transport of gp52, but may determine the stability of its association with membranes.

Amino Acid Sequence↗
PubMed1987

Glycoproteins of human parainfluenza virus type 3: affinity purification, antigenic characterization and reconstitution into lipid vesicles.

Monoclonal antibodies to the envelope glycoproteins, HN and F, of human parainfluenza virus type 3 were coupled to a Sepharose 4B matrix and used for affinity purification of the viral glycoproteins. The purity of the glycoproteins was demonstrated by SDS-PAGE followed by fluorography or silver staining. The antigenicity of the glycoproteins was determined by immunization of rabbits; polyclonal rabbit antisera demonstrated inhibition of functional activities of the virus glycoproteins. The F glycoprotein, when reconstituted into lipid vesicles, showed distinct spike-like projections similar to those of intact virions.

Animals↗
PubMed1987

Intracellular transport and leukemogenicity of spleen focus-forming virus envelope glycoproteins with altered transmembrane domains.

Friend murine spleen focus-forming virus (SFFV) encodes a glycoprotein designated gp52, which is responsible for the leukemogenic properties of the virus. gp52 lacks a cytoplasmic domain and is defective in its transport to the cell surface. We constructed a chimeric envelope gene which codes for a molecule with an external domain derived from the SFFV envelope gene and membrane-spanning and cytoplasmic domains derived from the Friend murine leukemia virus envelope gene. Like gp52, the chimeric protein was defective in its transport to the cell surface, indicating that the absence of a cytoplasmic tail is not responsible for the defective intracellular transport of SFFV gp52. However, unlike wild-type SFFV, the chimeric SFFV genome failed to induce erythroleukemia in adult mice. The results indicate that the altered membrane-spanning domain, lack of a detectable cytoplasmic tail in gp52, or both factors are prerequisites for the erythroleukemia-inducing properties of SFFV but are not responsible for the block in intracellular transport of the glycoprotein.

Amino Acid Sequence↗
PubMed1986

Nonpolarized expression of a secreted murine leukemia virus glycoprotein in polarized epithelial cells.

Vaccinia virus recombinants were generated which express the intact gp70/p15E of Friend mink cell focus inducing virus (F-MCFV) or truncated forms of the glycoprotein that lack the transmembrane and cytoplasmic domains. The transport of the intact and truncated envelope glycoproteins to apical or basolateral surfaces was studied in the polarized epithelial MDCK cell line. Infection of MDCK cells with the recombinant expressing the intact F-MCFV envelope glycoprotein resulted in transport exclusively to the basolateral surfaces, whereas the recombinant expressing the truncated glycoprotein was found to be secreted from both the apical and basolateral surfaces. Thus removal of the transmembrane and cytoplasmic domains of the p15E protein results in a loss of directional transport to the basolateral membrane of polarized epithelial cells.

Animals↗
PubMed1986

Monoclonal antibodies reveal extensive antigenic differences between the hemagglutinin-neuraminidase glycoproteins of human and bovine parainfluenza 3 viruses.

A panel of twenty monoclonal antibodies to the hemagglutinin-neuraminidase (HN) glycoprotein of human parainfluenza 3 (PI3) virus has been obtained and tentatively classified into four different groups based on reactivity in hemagglutination inhibition (HI), neuraminidase inhibition (NI), and plaque neutralization (NT) tests. The antibodies were tested for cross-reactivity with bovine PI3 virus, Sendai virus, and simian virus 5 (SV5). Only two of these antibodies showed similar reactivities with human and bovine PI3 viruses in HI and NT tests; a few other antibodies showed low levels of reactivity with the heterologous viruses in HI tests. A competitive binding assay further suggested that the two cross-reactive antibodies are directed against the same domain of the HN molecule. Therefore, the HN glycoproteins of human and bovine PI3 viruses appear to be antigenically dissimilar, although they share at least one common epitope.

Antibodies, Monoclonal↗
PubMed1986

Surface expression of viral glycoproteins is polarized in epithelial cells infected with recombinant vaccinia viral vectors.

In polarized epithelial cells, maturation sites of enveloped viruses that form by budding at cell surfaces are restricted to particular membrane domains. Recombinant vaccinia viruses were used to investigate the sites of surface expression in the Madin-Darby canine kidney (MDCK) cell line of the hemagglutinin (HA) of influenza virus, the G glycoprotein of vesicular stomatitis virus (VSV), and gp70/p15E of Friend murine leukemia virus (MuLV). These glycoproteins could be demonstrated by immunofluorescence on the surfaces of MDCK cells as early as 4 h post-infection. In intact MDCK monolayers, vaccinia recombinants expressing HA produced a pattern of surface fluorescence typical of an apically expressed glycoprotein. In contrast, cells infected with vaccinia recombinants expressing VSV-G or MuLV gp70/p15E exhibited surface fluorescence only when monolayers were treated with EGTA to disrupt tight junctions, as expected of glycoproteins expressed on basolateral surfaces. Immunoferritin labeling in conjunction with electron microscopy confirmed that MDCK cells infected with the HA recombinant exhibited specific labeling of the apical surfaces whereas the VSV-G and MuLV recombinants exhibited the respective antigens predominantly on the basolateral membranes. Quantitation of surface expression by [125I]protein A binding assays on intact and EGTA-treated monolayers confirmed the apical localization of the vaccinia-expressed HA and demonstrated that 95% of the VSV-G and 97% of the MuLV gp70/p15E glycoproteins were localized on the basolateral surfaces. These results demonstrate that glycoproteins of viruses that normally mature at basolateral surfaces of polarized epithelial cells contain all of the structural information required for their directional transport to basolateral plasma membranes.

Animals↗