Compartmentalization within the common mucosal immune system.
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Administration, Intranasal↗
Biomedical subjects
R W Compans
Publications and source records attributed to R W Compans.
Virus entry and release in polarized epithelial cells.
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Animals↗
A polarized human endometrial cell line that binds and transports polymeric IgA.
We have demonstrated that a human endometrial cell line, HEC-1, maintains a transepithelial electrical resistance, directionally transports fluids across the cell monolayer, and releases enveloped viruses at distinct plasma membrane domains: influenza virus is released at the apical surfaces and vesicular stomatitis virus (VSV) at the basolateral surfaces. In addition, we have examined the expression of domain-specific endogenous proteins, including the polyimmunoglobulin receptor. Multiple endogenous polypeptides were found to be secreted into the culture medium at basolateral surfaces, whereas no secretion of specific polypeptides was observed from apical cell surfaces. Distinct patterns of endogenous proteins were also observed on apical and basolateral cell surfaces, with a much more complex polypeptide pattern on the basolateral membranes. Using surface biotinylation and immunofluorescence, the polyimmunoglobulin receptor was found to be expressed on the basolateral surface of HEC-1 monolayers. The specific binding of poly-immunoglobulin A (pIgA) was found to occur on the basolateral surface, and was followed by transcytosis to the apical surface and release into the apical medium. The observed characteristics indicate that the endometrium-derived HEC-1 epithelial cell line can be employed as a model for studies of protein transport in polarized epithelial cells of human endometrial tissues, as well as for studies of the interaction of microorganisms with epithelial cells in the genital tract.
Biological Transport↗
Differences in the role of the cytoplasmic domain of human parainfluenza virus fusion proteins.
We have investigated the roles of the cytoplasmic domains of the human parainfluenza virus type 2 (PI2) and type 3 (PI3) fusion (F) proteins in protein transport and cell fusion activity. By using the vaccinia virus-T7 transient expression system, a series of F protein cytoplasmic tail truncation mutants was studied with respect to intracellular and surface expression and the ability to induce cell fusion when coexpressed with the corresponding hemagglutinin-neuraminidase (HN) proteins. All of the cytoplasmic tail truncation mutants of PI2F were expressed at high levels intracellularly or on cell surfaces as measured by immunoprecipitation and cell surface biotinylation assays. In addition, when coexpressed with PI2HN, these truncation mutants of PI2F were all found to be essentially unimpaired in the ability to induce cell fusion as measured by a quantitative cell fusion assay. In contrast, surface expression and cell fusion activity were found to be eliminated by a mutant of PI3F in which the entire cytoplasmic tail was deleted, and the mutant protein appeared to be unable to assemble into a high-molecular-weight oligomeric structure. To further investigate whether there is a specific sequence requirement in the cytoplasmic tail of PI3F, a chimeric protein consisting of the PI3F extracellular and transmembrane domains and the PI2F cytoplasmic tail was constructed. This chimeric protein was detected on the surface, and it was capable of inducing cell fusion when expressed together with PI3HN, although the fusogenic activity was reduced compared with that of wild-type PI3F. These results demonstrate that although PI2 and PI3 viruses belong to the same parainfluenza virus genus, these viruses show marked differences with respect to functional requirements for the cytoplasmic tail of the F glycoprotein.
Amino Acid Sequence↗
Influenza type A virus neuraminidase does not play a role in viral entry, replication, assembly, or budding.
We have used a neuraminidase-deficient influenza virus, NWS-Mvi, which was selected by supplying bacterial neuraminidase in the medium (C. Liu and G. M. Air, Virology 194:403-407, 1993), to define the role of neuraminidase in influenza virus replication. Electron microscopy showed that virions of the NWS-Mvi mutant assembled normally and formed large aggregates associated with cell surfaces. The NWS-Mvi virus grown in the absence of neuraminidase was able to carry out a second round of replication in MDCK cells without added neuraminidase, indicating that the virus particles contained in these aggregates were infectious. Aggregates of virus were also found in cytoplasmic vacuoles. When virus-infected cells were incubated in the presence of ferritin, such aggregates were found to be labeled with ferritin, indicating that they are derived from uptake at the cell surface. When the neuraminidase-deficient virus was administered intranasally to C57BL/6 mice, low titers of virus were recovered from the lungs and major histocompatibility complex class I-restricted cytotoxic T cells were generated: evidence that cells were infected in vivo. In C57BL/6 nu/nu mice, the low level of virus persisted for at least 28 days but never increased. These results suggest that neuraminidase is not required for influenza virus entry, replication, or assembly in cell culture or in mice.
Animals↗
Formation of the flavivirus envelope: role of the viral NS2B-NS3 protease.
One of the late processing events in the flavivirus replication cycle involves cleavage of the intracellular form of the flavivirus capsid protein (Cint) to the mature virion form (Cvir) lacking the carboxy-terminal stretch of hydrophobic amino acids which serves as a signal peptide for the downstream prM protein. This cleavage event was hypothesized to be effected by a viral protease and to be associated with virion formation. We have proposed a model of flavivirus virion formation in which processing of the C-prM precursor at the upstream signalase site is upregulated by interaction of the NS2B part of the protease with the prM signal peptide or with an adjacent carboxy-terminal region of the capsid protein in the precursor, and processing of Cint by the NS2B-NS3 protease follows the signalase cleavage. Recently, an alternative hypothesis was proposed which suggests a reverse order of these two cleavage events, namely, that cleavage of the C-prM precursor by the NS2B-NS3 protease at the Cint-->Cvir dibasic cleavage site is a prerequisite for the subsequent signalase cleavage of the prM signal peptide. To distinguish between these alternative models, we prepared a series of expression cassettes carrying mutations at the Cint-->Cvir dibasic cleavage site and investigated the effects of these mutations on signalase processing of C-prM and on formation and secretion of prM-E heterodimers. For certain mutated C-prM precursors, namely, for those with Lys-->Gly disruption of the dibasic site, efficient formation of prM was observed upon expression from larger cassettes encoding the viral protease, despite the absence of processing at the Cint-->Cvir cleavage site. Surprisingly, formation and secretion of prM-E heterodimers accompanied by late cleavage of prM was also observed for these cassettes, with an efficiency comparable to that of the wild-type expression cassette. These observations contradict the model in which cleavage of the C-prM precursor at the Cint-->Cvir dibasic site is a prerequisite for signalase cleavage.
Base Sequence↗
Respiratory syncytial virus matures at the apical surfaces of polarized epithelial cells.
Respiratory syncytial (RS) virus infects the epithelium of the respiratory tract. We examined the replication and maturation of RS virus in two polarized epithelial cell lines, Vero C1008 and MDCK. Electron microscopy of RS virus-infected Vero C1008 cells revealed the presence of pleomorphic viral particles budding exclusively from the apical surface, often in clusters. The predominant type of particle was filamentous, 80 to 100 nm in diameter, and 4 to 8 microns in length, and evidence from filtration studies indicated that the filamentous particles were infectious. Cytopathology produced by RS virus infection of polarized Vero C1008 cells was minimal, and syncytia were not observed, consistent with the maintenance of tight junctions and the exclusively apical maturation of the virus. Infectivity assays with MDCK cells confirmed that in this cell line, RS virus was released into the apical medium but not into the basolateral medium. In addition, the majority of the RS virus transmembrane fusion glycoprotein on the cell surface was localized to the apical surface of the Vero C1008 cells. Taken together, these results demonstrate that RS virus matures at the apical surface of polarized epithelial cell lines.
Animals↗
Oligomerization of the hydrophobic heptad repeat of gp41.
The transmembrane protein of human immunodeficiency virus type 1 (HIV-1) contains a leucine zipper-like (hydrophobic heptad) repeat which has been predicted to form an amphipathic alpha helix. To evaluate the potential of the hydrophobic heptad repeat to induce protein oligomerization, this region of gp41 has been cloned into the bacterial expression vector pRIT2T. The resulting plasmid, pRIT3, expresses a fusion protein consisting of the Fc binding domain of monomeric protein A, a bacterial protein, and amino acids 538 to 593 of HIV-1 gp41. Gel filtration chromatography demonstrated the presence of oligomeric forms of the fusion protein, and analytical centrifugation studies confirmed that the chimeric protein formed a higher-order multimer that was greater than a dimer. Thus, we have identified a region of HIV-1 gp41 which is capable of directing the oligomerization of a fusion protein containing monomeric protein A. Point mutations, previously shown to inhibit the biological activity of the HIV-1 envelope glycoprotein, have been engineered into the segment of gp41 contained in the fusion protein, and expressed mutant proteins were purified and analyzed via fast protein liquid chromatography. A point mutation in the heptad repeat, which changed the central isoleucine to an alanine, caused a significant (> 60%) decrease in oligomerization, whereas changing the central isoleucine to aspartate or proline resulted in almost a complete loss of oligomerization. Deletions of one, two, or three amino acids following the first isoleucine also resulted in a profound decrease in oligomerization. The inhibitory effects of the mutations on oligomer formation correlated with the effects of the same mutations on envelope glycoprotein-mediated fusion. A possible role of the leucine zipper-like region in the fusion process and in an oligomerization event distinct from assembly of the envelope glycoprotein complex is discussed.
Amino Acid Sequence↗
Generation of long flavivirus expression cassettes by in vivo recombination and transient dominant selection.
Assembly of expression cassettes coding for large segments of viral polyproteins is often complicated or impossible due to the instability of the resulting recombinant (re-) plasmids during propagation in Escherichia coli. Using the transient dominant selection approach described for the construction of vaccinia virus recombinants (re-VV), we have constructed several intermediate vectors and developed a procedure which enables direct assembly of long expression cassettes in the VV genome by in vivo recombination and does not require preliminary assembly of long cassettes in intermediate plasmids, thus eliminating the instability problems. The procedure was used to construct re-VV carrying fragments of the West Nile (WN), Murray Valley encephalitis (MVE), tick-borne encephalitis (TBE) and dengue type-2 (DEN2) viral genomes. Using this procedure, we have assembled a WN expression cassette which represents 86% of the WN genome and codes for 91% of its polyprotein and constitutes the longest flavivirus (FV) expression cassette inserted so far into the VV genome. Analysis of FV protein expression from the obtained recombinants indicates that recombination occurs with a high degree of specificity and the ORF remains intact. The procedure described offers a possible approach for the assembly of infectious cDNA clones.
Base Sequence↗
A signal for Golgi retention in the bunyavirus G1 glycoprotein.
The G1 and G2 glycoproteins of Punta Toro virus, a member of the bunyaviruses, are targeted to the Golgi complex, where viral budding occurs. We found that the G1 protein, when expressed in the absence of G2, is also targeted to the Golgi complex. A series of G1 proteins truncated at the carboxyl-terminal region was constructed, and the localization of the expressed proteins was examined. It was found that the proteins expressed from constructs with partial deletions in the cytoplasmic domain were transported to the Golgi complex at a significantly slower rate than G1. Although a major fraction of these proteins was eventually transported to the Golgi complex, they did not exhibit as clearly defined a pattern of accumulation as G1, but rather appeared to be distributed throughout the endoplasmic reticulum as well as the Golgi complex. The proteins expressed from constructs lacking most of the cytoplasmic domain and, in some cases, part of the transmembrane domain sequences as well were transported to the cell surface. We have also constructed chimeric proteins with the envelope protein of a murine leukemia virus (MCFenv), which is efficiently transported to the plasma membrane. A MCF-G1 chimera that contained the G1 transmembrane and cytoplasmic domains was found to be efficiently retained in the Golgi complex, and a construct that contained only the G1 transmembrane domain was also partially retained in the Golgi complex. Thus, the transmembrane domain as well as a portion of the cytoplasmic domain adjacent to the transmembrane domain are apparently crucial for Golgi retention of the G1 protein.
Animals↗
Effects of cytoplasmic domain length on cell surface expression and syncytium-forming capacity of the simian immunodeficiency virus envelope glycoprotein.
We previously reported that truncation of the terminal 146 amino acids of the macaque simian immunodeficiency virus SIVmac239 envelope glycoprotein enhanced envelope-specific syncytium formation in HeLa T4, CEM X 174, and HUT 78 cell lines and caused a change in the conformation of the transmembrane subunit of the envelope complex on the surface of these cells [Ritter et al. (1993) Virology 197, 255-264; Spies et al. (1994) J. Virol. 68, 585-591]. To investigate the effects of different lengths of the cytoplasmic domain on syncytium formation and cell surface expression, we have compared the expression and cytopathic effects induced by five SIVmac239 envelope constructs which vary in the lengths of their cytoplasmic domains. In contrast to the envelope protein truncated by 146 amino acids, the ability of proteins truncated by 98 or 161 amino acids to form syncytia was substantially reduced in CEM X 174 and HUT 78 cells, while syncytium formation by a protein truncated by 53 amino acids was only slightly reduced compared to the full-length protein. Furthermore, only the glycoprotein which was truncated by 146 amino acids induced syncytium formation in HeLa T4 cells. When examining the expression of the truncated proteins on the surface of HeLa T4 cells, we found that, in contrast to the full-length SIVmac239 protein, each of the truncated transmembrane subunits could be efficiently biotinylated with the membrane-impermeable reagent NHS-SS-biotin. Furthermore, using cell surface iodination, we found stable oligomeric forms of both the transmembrane subunits and the uncleaved precursor proteins of each mutant protein on the surface of HeLa T4 cells. Using pulse-chase analysis, we also found that the precursor of the protein truncated by 98 residues was degraded more rapidly than the wild-type and the other mutant proteins. Finally, we constructed two mutants which expressed a full-length TM protein or a TM protein with a 146 amino acid C-terminal deletion and had most of the coding sequences of their SU subunits deleted. Neither of these two proteins was found to cause syncytium formation in HeLa T4, CEM X 174, or HUT 78 cell lines even though we could detect both proteins on the surfaces of HeLa T4 cells using iodination. These results could explain why the selection of truncated variants of SIV which emerge after prolonged passage in human cell lines is restricted to truncations which remove close to 146 amino acids in the cytoplasmic domain of the TM protein.
Amino Acid Sequence↗
Virus-like particles in MDCK cells persistently infected with Borna disease virus.
A line of Madin Darby canine kidney (MDCK) cells persistently infected with Borna disease virus was examined by electron microscopy. Thin sections revealed the presence of intracytoplasmic virus-like particles ranging from 50-100 nm in diameter. Nuclei of the infected cells exhibited accumulation of electron-dense granular structures 15-18 nm in diameter. The intracytoplasmic particles were roughly spherical with a limiting membrane, suggesting the presence of a lipid-containing envelope. The internal structure consisted of strand-like material which in some cases was condensed underneath the envelope. The possible relationship of these particles to Borna disease virions is discussed.
Animals↗
Viruses as model systems in cell biology.
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Animals↗
Calmodulin antagonists inhibit human immunodeficiency virus-induced cell fusion but not virus replication.
We have reported that amphipathic helical segments in the cytoplasmic domain of the HIV-1 envelope glycoproteins bind to calmodulin (CaM) with high affinity, and inhibit calmodulin-regulated proteins. To investigate the possible role of calmodulin activity in HIV-1 replication, we investigated the anti-HIV activity of various CaM antagonists--trifluoperazine and naphthalenesulfonamide W13 or W7--in HeLa T4 cells, PBMCs, and various T lymphocytic cell lines. The different CaM antagonists were found to inhibit the proliferation of the different cell types to varying extent. Also, the CaM antagonists were found to exert a greater antiproliferative effect on H9/HIV-1IIIB, as compared to uninfected H9 cells, suggesting a deficit of CaM function in HIV-infected cells. The CaM antagonists inhibited virus-induced cell fusion in HeLa T4 cells infected with a recombinant vaccinia virus expressing HIV-1 envelope proteins at threshold concentrations that do not inhibit cell proliferation. The fusion-inhibitory effects of the CaM antagonists were also observed in cocultures of HIV-infected (H9/HIV-1IIIB) and uninfected H9 cells. Under these conditions, the synthesis and surface expression of the viral glycoproteins were not affected, although the kinetics of processing of HIV envelope precursor was delayed. Virus production from both HIV-infected peripheral blood mononuclear cell (PBMC) and MT-2 cell cultures was inhibited by CaM antagonists at concentrations that were inhibitory to cell proliferation. Surprisingly, threshold concentrations of CaM antagonists that do not inhibit cell proliferation were found to enhance virus production from HIV-infected MT-2 cells, but not PBMCs.(ABSTRACT TRUNCATED AT 250 WORDS)
Calmodulin↗
Mucosal immunity and strategies for novel microbial vaccines.
Infectious diseases continue to be the leading cause of morbidity and mortality worldwide. Increased awareness of the fact that mucosal membranes are the most frequent portals of entry of pathogenic microorganisms has prompted studies aimed at the development of vaccination protocols and antigen delivery systems that would lead to an increased protection of mucosae. Although systemic and strictly local immunizations are of limited effectiveness in the induction of mucosal protection, ingestion or inhalation of antigens results in a generalized immune response manifested by the appearance of specific antibodies of the secretory immunoglobulin (Ig) isotype in external secretions due to the dissemination of IgA precursor cells from IgA-inductive lymphoid tissues. Furthermore, additional inductive sites strategically positioned at the opening of the respiratory and digestive tracts may also be suitable targets for induction of immune responses at desired effector sites. To prevent degradation and the increase of ingested antigens absorption, novel strategies including enclosure of antigens into biodegradable microspheres, liposomes or their expression in viral and bacterial vectors and plants are currently being considered. Forthcoming technological advances in antigen preparation and routes of delivery will undoubtedly have a profound impact on immunization practices in the future.
Animals↗
Human immunodeficiency virus type 1 envelope glycoprotein is modified by O-linked oligosaccharides.
The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been shown to be extensively modified by N-linked glycosylation; however, the presence of O-linked carbohydrates on the glycoprotein has not been firmly established. We have found that enzymatic deglycosylation of the HIV-1 envelope glycoprotein with neuraminidase and O-glycosidase results in a decrease in the apparent molecular weight of the envelope glycoprotein. This result was observed in both vaccinia virus recombinant-derived envelope glycoproteins and glycoproteins derived from the IIIB, SG3, and HXB2, strains of HIV-1. The decrease in molecular weight was also observed when the envelope glycoprotein had been deglycosylated with N-glycanase F after treatment with neuraminidase and O-glycosidase, indicating that the decrease in apparent molecular weight was not attributable to the removal of N-linked carbohydrate. Treatment with neuraminidase, O-glycosidase, and N-glycanase F was found to be necessary to remove all radiolabel from [3H]glucosamine-labelled envelope glycoprotein, a result seen for both recombinant and HIV-1-derived envelope glycoprotein. [3H]glucosamine-labelled carbohydrates liberated by O-glycosidase treatment were separated by paper chromatography and were found to be of a size consistent with O-linked oligosaccharides. We, therefore, conclude that the HIV-1 envelope glycoprotein is modified by the addition of O-linked carbohydrates.
Amidohydrolases↗
Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein alters the conformation of the external domain.
We previously reported that truncation of the cytoplasmic domain of the macaque simian immunodeficiency virus SIVmac239 envelope glycoprotein enhanced its ability to induce cell fusion in a variety of cell lines. In the present study, we examined the expression of the full-length and truncated SIVmac239 envelope glycoprotein complex on cell surfaces. Using a membrane-impermeable reagent to biotinylate proteins on cell surfaces followed by immunoprecipitation, we found that under conditions in which the full-length TM protein could not be detected on the surfaces of CD4-positive or CD4-negative cell lines, the truncated TM protein was detected efficiently. In contrast, using a membrane-impermeable iodination reagent to label proteins on cell surfaces, we could detect both the full-length and truncated TM proteins. No difference between the full-length and truncated proteins was observed in the detection of the SU proteins in the biotinylation assay. Additionally, we used an assay in which SIV-specific antibodies are prebound to the native envelope proteins expressed on the cell surface and then the proteins are immunoprecipitated. Using this assay, we could not detect the truncated or full-length TM protein on the cell surface, whereas we could detect the SU subunits of both proteins. We also observed that the truncated TM protein formed more stable sodium dodecyl sulfate-resistant oligomers than the full-length TM protein did. These results indicate that truncation of the cytoplasmic domain of the SIVmac239 envelope glycoprotein affects the conformation of the external domain of the TM protein on the cell surface, even though the two proteins have no differences in the amino acid sequences of their external domains. This altered conformation could play a role in the enhanced fusion activity of the truncated SIV glycoprotein.
Antibodies, Viral↗
Processing of the intracellular form of the west Nile virus capsid protein by the viral NS2B-NS3 protease: an in vitro study.
According to the existing model of flavivirus polyprotein processing, one of the cleavages in the amino-terminal part of the flavivirus polyprotein by host cell signalases results in formation of prM (precursor to one of the structural proteins, M) and the membrane-bound intracellular form of the viral capsid protein (Cint) retaining the prM signal sequence at its carboxy terminus. This hydrophobic anchor is subsequently removed by the viral protease, resulting in formation of the mature viral capsid protein found in virions (Cvir). We have prepared in vitro expression cassettes coding for both forms of the capsid protein, for the prM protein, for the C-prM precursor, and for the viral protease components of West Nile flavivirus and characterized their translation products. Using Cint and Cvir translation products as molecular markers, we have observed processing of the intracellular form of the West Nile capsid protein by the viral protease in vitro both upon cotranslation of the C-prM precursor and the viral protease-encoding cassette and by incubation of C-prM translation products with a detergent-solubilized extract of cells infected with a recombinant vaccinia virus expressing the active viral protease. The cleavage of Cint by the viral protease at the predicted dibasic site was verified by introduction of point mutations into the cleavage site and an adjacent region. These studies provide the first direct demonstration of processing of the intracellular form of the flavivirus capsid protein by the viral protease.
Amino Acid Sequence↗