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R W Compans

Publications and source records attributed to R W Compans.

At least 91 records · Page 5Linked to original sources
PubMed1993

Cytosolic domain of the human immunodeficiency virus envelope glycoproteins binds to calmodulin and inhibits calmodulin-regulated proteins.

Calmodulin (CaM), the major intracellular receptor for calcium, is involved in regulation of diverse cellular functions. Positively charged amphipathic helical segments have been identified as an important structural motif in the recognition of CaM by different CaM-activated enzymes and peptides. The carboxyl-terminal domain of the envelope glycoproteins of human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV) contain regions that can fold into amphipathic helical segments, which closely resemble the amphipathic segments found in CaM-activated enzymes. We show here that synthetic peptide analogs corresponding to the two putative amphipathic helical regions of HIV-1/WMJ gp160 bind to CaM with high affinity (Kd 31-41 nM) in the presence of calcium. They also bind CaM in the absence of calcium, although with much lower affinity. The peptides inhibit CaM-regulated activation of bovine brain phosphodiesterase in vitro. The peptides also inhibit mitogen-induced lymphocyte activation, a property shared by CaM antagonists. Purified HIV-1 gp160 binds to CaM, while gp120, which lacks the putative amphipathic helical segments, does not bind CaM. In HIV-infected cells, the putative CaM-binding regions of gp160 are located intracellularly and may therefore interact with the cytosolic CaM. We postulate that CaM binding by HIV envelope proteins is likely to exert diverse modulatory effects, and the mechanism for HIV-induced cytotoxicity may involve, in part, inhibition of CaM-regulated cellular functions.

Amino Acid Sequence↗
PubMed1993

Protection against vaginal SIV transmission with microencapsulated vaccine.

Although protection in animal models against intravenous challenges with simian immunodeficiency virus (SIV) has been reported, no previous vaccines have protected against a heterosexual route of infection. In this study, five of six macaques were protected against vaginal challenge when immunized with formalin-treated SIV in biodegradable microspheres by the intramuscular plus oral or plus intratracheal route. Oral immunization alone did not protect. After a second vaginal challenge, three of four intramuscularly primed and mucosally boosted macaques remained protected. The data suggest that protection against human immunodeficiency virus vaginal transmission could be provided by microsphere-based booster vaccines when used to immunize women who are systemically primed.

Administration, Oral↗
PubMed1993

Regulation of the late events in flavivirus protein processing and maturation.

In order to determine the requirements for secretion of flavivirus structural proteins, we analyzed the expression of several West Nile flavivirus gene cassettes of different lengths in vaccinia virus expression systems. Expression of the longest cassette coding for the 5'-nontranslated region, proteins C through NS2B, and the protease domain of NS3, resulted in secretion of prM-E complexes and cleavage of prM. The presence and proper processing of the NS2A-NS2B-NS3 region appeared to be necessary for prM-E secretion. These proteins were released from cells mostly as membranous complexes which may represent empty viral envelopes. Cleavage of the membrane-associated intracellular form of protein C (C(i)) to produce the virion form (Ce) appeared to be critical for release of viral proteins. The presence and proper cleavage of the NS2A-NS2B-NS3 region were also found to be necessary for efficient C-prM cleavage by signalases. The NS2B-NS3 complex was implicated in cleavage of the intracellular form of protein C. Formation of a low level of virus-like particles was detected by electron microscopy. A model for virion formation, suggesting a critical role of the NS2B and NS3 proteins, is discussed.

Amino Acid Sequence↗
PubMed1993

Secretion of a truncated form of the human immunodeficiency virus type 1 envelope glycoprotein.

We have characterized a truncated secreted form of the HIV-1 envelope glycoprotein gene. Expression via a recombinant vaccinia virus resulted in a glycoprotein product of approximately 140 kDa (gp160t) and a minor cleavage product of 120 kDa (gp120). Pulse-chase analysis revealed that the majority of gp160t remained cell-associated and underwent degradation within 10-20 hr of synthesis. A secreted form (gp160t/sec) and gp120 were detected in the media 2-4 hr postsynthesis and were not significantly degraded within a period of 20 hr. Most of the cell-associated gp160t remained sensitive to digestion with endoglycosidase H, whereas gp160t/sec and gp120 were largely resistant. Gp160t, gp160t/sec, and gp120 formed oligomers which were stabilized by intermolecular disulfide bonds and/or noncovalent interactions and were also found to bind to soluble CD4. Both wild type gp160 and wild type gp160t were observed to undergo a post-translational modification 4-5 hr postsynthesis, resulting in glycoproteins with a slightly increased electrophoretic mobility. These differences in electrophoretic mobility remained following treatment with N-glycosidase F, indicating that they are not a consequence of N-linked oligosaccharide processing, but may represent an additional modification of the envelope glycoprotein.

Animals↗
PubMed1993

Cell fusion activity of the simian immunodeficiency virus envelope protein is modulated by the intracytoplasmic domain.

The processing and biological activity of envelope glycoproteins of pathogenic and nonpathogenic simian immunodeficiency viruses (SIVs) was compared using recombinant vaccinia viruses (rVVs). The env glycoprotein of the nonpathogenic SIVmac1A11 virus caused much larger and more numerous syncytia than the glycoprotein of the pathogenic SIVmac239 virus in several CD4+ human cell lines. The env gene of SIVmac239 codes for a full-length transmembrane (TM) protein, while the SIVmac1A11 virus has a TM protein with a markedly truncated cytoplasmic domain. To determine if TM protein truncation alone might affect the biological properties of viral glycoproteins, we constructed a rVV which expresses a SIVmac239 env with a site-specific mutation yielding a truncated TM protein. This truncated env protein induced extensive fusion of rVV-infected HeLa T4 cell monolayers, whereas no fusion was observed for the parental SIVmac239 env recombinant. The truncated glycoprotein also caused larger and more numerous syncytia than the wild-type SIVmac239 glycoprotein in the human cell lines HUT 78 and CEM x 174. The mutation altered env glycoprotein transport, but did not significantly affect cell surface expression levels or the amount of secreted soluble SU protein. In coinfection assays, the full-length SIVmac239 env protein was found to interfere with fusion induced by the truncated envelope protein. The results thus demonstrate that changes in the cytoplasmic domain of the SIVmac envelope protein can markedly affect the ability to induce cell fusion, an activity of the external domains of the TM-SU glycoprotein complex.

Animals↗
PubMed1993

Murine model for evaluation of protective immunity to influenza virus.

We have characterized a murine model as an inexpensive, readily available and sensitive animal model for the evaluation of protective immune responses induced by various routes of administration of influenza A vaccine preparations. Using a non-mouse-adapted human influenza virus to infect unanaesthetized animals intranasally, we established that the optimum dose for infection of Balb/c mice was 10(4) plaque forming units of virus and that the optimum sampling time for measurement of virus yields in the organs of the respiratory tract was 72 h after challenge. We found that the infection was initiated in the nose and progressed by descending into the trachea and lungs over a period of days. Evaluation of protection against infection clearly showed that the tissues of the mouse respiratory tract were completely protected after administration of whole killed virus intranasally and partly protected when virus was administered subcutaneously. The protection correlated with the level of virus-specific IgA antibodies in saliva.

Administration, Intranasal↗
PubMed1993

Oral immunization with influenza virus in biodegradable microspheres.

Polymeric microspheres were evaluated as an oral antigen delivery system for immunization with influenza virus. The immune responses obtained were compared after either oral or systemic immunization of BALB/c mice using purified, formalin-inactivated influenza virus type A/H3N2, either encapsulated in biodegradable and biocompatible microspheres or free in solution. The immunogenicity of formalin-treated influenza vaccine was preserved during the microencapsulation process, and the microencapsulated antigen induced protective immune responses after systemic immunization that were equal to or higher than those induced by conventional vaccine. When administered orally to primed animals, microencapsulated antigen induced levels of anti-influenza antibodies in saliva that were higher than and in serum that were comparable to those obtained by systemic immunization. Furthermore, oral booster immunization provided virtually complete protection of animals challenged with live virus.

Administration, Oral↗
PubMed1993

Microencapsulated human parainfluenza virus induces a protective immune response.

Human parainfluenza type 3 (PI3) virus was incorporated into microspheres composed of a biocompatible and biodegradable DL-lactide and glycolide copolymer. Sera from mice immunized with these microspheres showed an antibody response to the viral glycoproteins and neutralized virus infectivity. The microspheres were also evaluated by intraperitoneal, oral, or intranasal administration to determine their protective efficacy in the hamster. After challenge infection of the intraperitoneally immunized hamsters with live PI3 virus, a significant reduction of virus titers in the respiratory tract was observed, demonstrating the protective efficacy of the microencapsulated viral antigens.

Administration, Intranasal↗
PubMed1993

Bidirectional entry of poliovirus into polarized epithelial cells.

The interactions of viruses with polarized epithelial cells are of some significance to the pathogenesis of disease because these cell types comprise the primary barrier to many virus infections and also serve as the sites for virus release from the host. Poliovirus-epithelial cell interactions are of particular interest since this virus is an important enteric pathogen and the host cell receptor has been identified. In this study, poliovirus was observed to adsorb to both the apical and basolateral surfaces of polarized monkey kidney (Vero C1008) and human intestinal (Caco-2) epithelial cells but exhibited preferential binding to the basolateral surfaces of both cell types. Localization of the poliovirus receptor by a receptor-specific monoclonal antibody (D171) revealed a similar distribution predominantly on basolateral membranes, and treatment of cells with antibody D171 inhibited virus adsorption to both membrane surfaces. Poliovirus was able to initiate infection with similar efficiency following adsorption to either surface, and infection was blocked at both surfaces by D171, indicating that functional receptor molecules are expressed on both surfaces at sufficient density to mediate efficient infection at the apical and basolateral plasma membranes. Poliovirus infection resulted in a decrease in transepithelial resistance which was inhibited by prior treatment with monoclonal antibody D171 and occurred prior to other visible cytopathic effects. These results have interesting implications for viral pathogenesis in the human gut.

Adsorption↗
PubMed1993

Role of the cytoplasmic domains of viral glycoproteins in antibody-induced cell surface mobility.

We have investigated the role of the cytoplasmic domains of the influenza virus hemagglutinin (HA) and the parainfluenza virus type 3 (PI3) fusion (F) glycoproteins as a determinant of their ability to undergo antibody-induced redistribution on plasma membranes. The viral envelope genes were truncated in their cytoplasmic domains by using oligonucleotide-directed mutagenesis and expressed by using recombinant vaccinia viruses. In HeLa cells, the truncated HA (HAt), like the full-length HA, did not cap in response to specific antibody. In CV-1 cells, HAt showed patchy surface immunofluorescence with few caps, whereas full-length HA exhibited capping in many cells in response to bivalent antibody. Quantitation of cap formation indicated a sevenfold decrease in the frequency of capping of HAt in comparison with full-length HA. Similarly, truncated F also exhibited a significant decrease in cap formation in comparison with full-length F. These results indicate that the ability of influenza virus HA and PI3 F to undergo redistribution in response to bivalent antibody has been altered by truncation of the viral glycoproteins and suggest that capping may involve interactions between the cytoplasmic domain of the viral glycoproteins and host cell components.

Animals↗
PubMed1993

Alternate pathways of secretion of simian immunodeficiency virus envelope glycoproteins.

A biotinylation assay was used to detect the envelope glycoprotein of the simian immunodeficiency virus (SIV) envelope glycoprotein expressed by a recombinant vaccinia virus on the surface of HeLa T4 cells. The relationship between the detection of the envelope glycoprotein on the cell surface and its secretion from the cell was examined. It was found that much more gp120 was released into the culture medium than could be accounted for by shedding of the biotinylated SIV envelope protein from the cell surface. Treatment with the ionophore monensin showed that this drug did not block the secretion of gp120 into the culture medium even though the expression of gp120 on the cell surface was strongly downregulated. Similar results were observed for the secretion of gp120 in HUT78 cells infected with SIVmac251 virus. Brefeldin A, on the other hand, inhibited both the detection of gp120 on the cell surface and its secretion into the culture medium. On the basis of these results, we propose that gp120 can be secreted into the culture medium via at least two pathways. One pathway involves the dissociation of gp120 from membrane-associated gp41-gp120 complexes on the cell surface. However, the major pathway involves the secretion of gp120 without its transitory appearance on the cell surface as part of a gp41-gp120 complex.

Brefeldin A↗
PubMed1993

Vectorial release of poliovirus from polarized human intestinal epithelial cells.

Polarized epithelial cells represent the primary barrier to virus infection of the host, which must also be traversed prior to virus dissemination from the infected organism. Although there is considerable information available concerning the release of enveloped viruses from such cells, relatively little is known about the processes involved in the dissemination of nonenveloped viruses. We have used two polarized epithelial cell lines, Vero C1008 (African green monkey kidney epithelial cells) and Caco-2 (human intestinal epithelial cells), infected with poliovirus and investigated the process of virus release. Release of poliovirus was observed to occur almost exclusively from the apical cell surface in Caco-2 cells, whereas infected Vero C1008 cells exhibited nondirectional release. Structures consistent with the vectorial transport of virus contained within vesicles or viral aggregates were observed by electron microscopy. Treatment with monensin or ammonium chloride partially inhibited virus release from Caco-2 cells. No significant cell lysis was observed at the times postinfection when extracellular virus was initially detected, and transepithelial resistance and vital dye uptake measurements showed only a moderate decrease. Brefeldin A was found to significantly and specifically inhibit poliovirus biosynthetic processes by an as yet uncharacterized mechanism. The vectorial release of poliovirus from the apical (or luminal) surface of human intestinal epithelial cells has significant implications for viral pathogenesis in the human gut.

Ammonium Chloride↗
PubMed1992

Membrane interactions of synthetic peptides corresponding to amphipathic helical segments of the human immunodeficiency virus type-1 envelope glycoprotein.

The human and simian immunodeficiency virus envelope glycoproteins, which mediate virus-induced cell fusion, contain two putative amphipathic helical segments with large helical hydrophobic moments near their carboxyl-terminal ends. In an attempt to elucidate the biological role of these amphipathic helical segments, we have synthesized peptides corresponding to residues 768-788 and 826-854 of HIV-1/WMJ-22 gp160. Circular dichroism studies of the peptides showed that the alpha helicity of the peptides increased with the addition of dimyristoyl phosphatidylcholine (DMPC) indicating that the peptides form lipid-associating amphipathic helixes. The peptides solubilized turbid suspensions of DMPC vesicles, and electron microscopy of peptide-DMPC mixtures revealed the formation of discoidal complexes, suggesting that the peptides bind to and perturb lipid bilayers. The peptides were found to lyse lipid vesicles and caused carboxyfluorescein leakage from dye-entrapped egg phosphatidylcholine liposomes. The peptides also lysed human erythrocytes and were found to be toxic to cell cultures. At subtoxic concentrations, the peptides effectively inhibited the fusion of CD4+ cells infected with recombinant vaccinia virus expressing human immunodeficiency virus (HIV)-1 envelope proteins. Based on these results, and reported studies on the mutational analysis of HIV envelope proteins, we suggest that the amphipathic helical segments near the carboxyl terminus of HIV envelope proteins may play a role in lysis of HIV-infected cells and also may modulate the extent of cell fusion observed during HIV infection of CD4+ cells.

Amino Acid Sequence↗
PubMed1992

Human immunodeficiency virus type 2 envelope glycoprotein: differential CD4 interactions of soluble gp120 versus the assembled envelope complex.

Utilizing a recombinant vaccinia expression system, we investigated the biological properties and CD4 receptor interactions of the envelope glycoproteins of a noncytopathic human immunodeficiency virus type 2 strain, termed HIV-2/ST, and a highly cytopathic variant derived from it. The efficiency and host cell range of syncytium formation by the recombinant glycoproteins of both viruses were highly restricted compared to those of prototypic strains of HIV (HIV-2/ROD or HIV-1/IIIB). However, the glycoprotein of cytopathic but not wild-type ST generated numerous large syncytia in the human T-cell line Sup T1 from which it was derived. A single cell line (Molt 4 clone 8) was permissive to fusion by both wild-type and cytopathic ST envelopes, but only the glycoprotein of cytopathic ST could be inhibited with a soluble form of the viral receptor CD4 (sCD4). While these results indicated major differences in the envelope glycoprotein-CD4 receptor interactions of wild-type versus cytopathic ST, direct and competition binding assays utilizing soluble external glycoprotein (SU) and sCD4 surprisingly revealed equivalent low binding affinity for both viruses. From these experiments we conclude that relevant biological properties (e.g., CD4 binding, cytopathic potential, and sCD4 neutralization) of HIV viruses which differ in their pathogenic potential are reflected in the sCD4 interactions of the assembled native envelope complex (as on cell or virion surfaces) but not the soluble SU glycoprotein.

CD4 Antigens↗
PubMed1992

The NS and capsid genes determine the host range of porcine parvovirus.

Porcine parvovirus is an autonomous parvovirus which normally infects pigs and multiplies in porcine cells in vitro. In this report, we describe the properties of a variant designated P2, which has extended its host range to include canine cells. The variant was able to produce cytopathic effects (CPE) in canine cells, unlike the prototype NADL-2 strain. The variant also produced higher viral antigen and infectivity titers in canine cells than the NADL-2 strain, whereas both strains produced CPE and similar titers in porcine cells. Generation of recombinant plasmids between the P2 variant DNA and an infectious clone of NADL-2, and analysis of the properties of the virus stocks produced from these recombinant plasmids, indicated that two changes were necessary for this extension in the host range. One change was located in the nonstructural protein coding region and the other in the capsid coding region.

Amino Acid Sequence↗
PubMed1992

A 585-bp deletion found in the spleen focus-forming virus (SFFV) env gene is responsible for the defective intracellular transport of SFFV gp52.

The Friend spleen focus-forming virus (F-SFFV) codes for a transport defective, leukemogenic envelope glycoprotein designated as gp52. Gp52 closely resembles the envelope glycoproteins (gp70-p15E) encoded by the mink cell focus-forming viruses (MCFV). The major differences between SFFV and MCFV include a 585-bp deletion and a frame-shift mutation near the 3' end of the SFFV env gene. We have constructed a mutant MCFV env gene, which contains a 585-bp deletion like that found in the SFFV env gene, and expressed this gene using recombinant vaccinia vectors or retroviral vectors. The mutant MCFV env gene expressed a truncated, transport defective glycoprotein (gp57). Only a small proportion of gp57 underwent further oligosaccharide processing. Intracellular gp57 remained predominantly monomeric and only a small proportion of gp57 (and its processed forms) formed disulfide-linked dimers and trimers which resembled those formed by SFFV gp52. Processed forms of gp57 were found on the cell surfaces and in culture fluids. The extracellular forms had a faster electrophoretic mobility than the intracellular-processed forms of gp57. These results indicate that the 585-bp deletion found in SFFV env gene is responsible for the folding, transport, and secretion of gp52. Retroviral vectors carrying the mutant MCFV env gene were nonpathogenic (or weakly pathogenic) in adult mice. The results indicate that the 585-bp deletion, although essential, is not the sole determinant of SFFV-induced disease in adult mice.

3T3 Cells↗
PubMed1992

Expression of SV40 receptors on apical surfaces of polarized epithelial cells.

We have investigated the interaction of SV40 virions with polarized monkey kidney epithelial cells. Virions were tagged with biotin to facilitate their detection and were found to retain full infectivity. When polarized Vero C1008 cells were incubated with biotinylated virions followed by a strepavidin-rhodamine conjugate, distinct cell populations were identified which expressed very different levels of SV40 receptors. The parental Vero C1008 cells yielded three types of cell clones which exhibited low, moderate, or predominantly high levels of SV40 binding. Virus-binding assays to each of these clones as well as to parental Vero C1008 cells indicated that the level of SV40 receptor expression is cell-cycle-dependent. The cellular receptors for influenza A virus (WSN strain) were also found to be distributed heterogeneously on polarized epithelial cells. In contrast, in several types of nonpolarized cells, SV40 receptors were found to be uniformly distributed over the monolayer. SV40 binding was not found to correlate with HLA expression on Vero C1008 cells or other cell types. Also in contrast to SV40 receptor expression, which is restricted to the apical domain, HLA was found to be distributed on both apical and basolateral domains of Vero C1008 cells.

Animals↗