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M J Mulligan

Publications and source records attributed to M J Mulligan.

At least 37 records · Page 2Linked to original sources
PubMed1997

A sorting motif localizes the foamy virus glycoprotein to the endoplasmic reticulum.

We recently identified an endoplasmic reticulum (ER) retrieval signal-the dilysine motif-in the glycoproteins of all five foamy viruses (FVs) for which sequences were available (P. A. Goepfert, G. Wang, and M. J. Mulligan, Cell 82:543-544, 1995). In the present study, expression of recombinant human FV (HFV) glycoprotein and analyses of oligosaccharide modifications and precursor cleavage indicated that the protein was localized to the ER. HFV glycoproteins encoding seven different dilysine motif mutations were then expressed. The results indicated that disruptions of the dilysine motif resulted in higher levels of forward transport of the HFV glycoprotein from the ER through the Golgi apparatus to the plasma membrane. We conclude that the dilysine motif is responsible for ER sorting of the FV glycoprotein. Signal-mediated ER localization has not previously been described for a retroviral glycoprotein.

Animals↗
PubMed1997

Induction of vigorous cytotoxic T-lymphocyte responses by live attenuated simian immunodeficiency virus.

Although live attenuated vaccine strains of simian immunodeficiency virus (SIV) have proven highly effective in protecting macaques against challenge with pathogenic SIV strains, little is known about the mechanisms of protective immunity induced by these vaccines. We examined cytotoxic T-lymphocyte (CTL) responses against SIV in animals infected with SIVmac239delta nef (deficient in nef) or SIVmac239delta 3 (deficient in nef, vpr, and upstream sequences in U3). To enhance detection of SIV-specific CTL activity, we stimulated peripheral blood mononuclear cells with autologous B-lymphoblastoid cell lines which had been infected with recombinant vaccinia viruses expressing SIV proteins and subsequently inactivated with psoralen and UV light. Animals chronically infected with SIV239delta nef or SIV239delta 3 mounted vigorous CTL responses against the SIV Gag and Env proteins. This CTL activity was major histocompatibility class restricted and mediated by CD8+ T lymphocytes. CTL responses persisted at relatively high levels for more than 6 years after infection. Limiting dilution precursor frequency assays demonstrated that the frequency of SIV-specific CTLs was as high as 234 CTL precursors per 100,000 cells. Animals acutely infected with SIV239delta nef developed CTL activity by day 14 after infection, coincident with decreases in viral load. Animals acutely infected with SIV239delta 3 developed CTL responses within 4 weeks of infection. Thus, vaccination of juvenile or adult animals with SIV239delta nef or SIV239delta 3 results in the induction of a vigorous CTL response which arises early in the course of infection and persists for years after a single inoculation of virus.

Animals↗
PubMed1996

Analysis of west African hunters for foamy virus infections.

Foamy viruses are a genus of complex retroviruses that infect a wide variety of mammals. However, a clear association with any disease process has yet to be proven for these viruses. A higher human seroprevalence was reported in African populations, perhaps due to exposure to simian foamy viruses (SFV) endemic in primates. However, the earlier serologic surveys were not confirmed by studies employing nucleic acid amplification. Foamy virus infections of humans clearly do occur as rare zoonoses among primate center or laboratory workers exposed to captive primates or their blood. We sought to detect foamy virus infections in a cohort of humans also presumed to be exposed to SFV, i.e., West African hunters. We constructed recombinant vaccinia viruses that expressed human foamy virus (HFV) Gag or Env polyproteins in mammalian cells. The sera from 17 monkey hunters or several controls were tested in radioimmunoprecipitation assays (RIPAs) against the recombinant HFV proteins. Chimpanzee sera or HFV-positive human sera immunoprecipitated gp130, the HFV Env precursor, as well as p74, the HFV Gag polyprotein. None of the hunters' sera recognized both of these recombinant proteins. We then employed a nested polymerase chain reaction (PCR) analysis of the hunters' DNA but also failed to detect foamy virus infections. Therefore, by utilizing a recombinant RIPA and a nested PCR assay, we have not identified foamy virus infections occurring naturally in hunters exposed to wild monkeys in West Africa.

Africa, Western↗
PubMed1996

Human immunodeficiency virus type 2 glycoprotein enhancement of particle budding: role of the cytoplasmic domain.

Previous studies have shown that the glycoprotein cytoplasmic domains of human immunodeficiency virus type 2 (HIV-2) or simian immunodeficiency virus of macaques modulate biological activities of the viral glycoprotein complex, including syncytium formation, exterior glycoprotein conformation, and glycoprotein incorporation into budding virus particles. We have now utilized a recombinant expression system to study interactions of full-length or truncated HIV-2 glycoproteins with coexpressed HIV-2 Gag proteins which self-assemble and bud as virus-like particles. Interestingly, budding of HIV-2 virus-like particles from cells was enhanced 5- to 24-fold when Gag was coexpressed with the full-length HIV-2 glycoprotein, compared with Gag expressed either alone or with a truncated HIV-2 glycoprotein. The results obtained in this model system indicate that an additional effect of the lengthy cytoplasmic domain of the glycoprotein of HIV-2 is enhancement of particle budding. We speculate that the cytoplasmic domain of the viral glycoprotein of HIV-2 enhances budding by (i) potentiation of Gag structure or function or (ii) membrane modulation.

Base Sequence↗
PubMed1995

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↗
PubMed1994

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↗
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

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↗
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

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

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

Amino Acid Sequence↗
PubMed1991

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

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

Animals↗
PubMed1990

Oligopeptide inhibitors of HIV-induced syncytium formation.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is essential for virus entry and the formation of multinucleated giant cells by cell fusion, one of the major virus-induced cytopathic effects. To study the effects of potential fusion inhibitors, a vaccinia virus recombinant expressing the envelope glycoprotein was generated and used to infect HeLa CD4+ cells. Syncytium induction was observed as early as 4 h postinfection and continued until the entire monolayer was fused. The N-terminus of the gp41 subunit of the HIV envelope protein is very hydrophobic, and appears to be involved in virus-induced membrane fusion. We synthesized several oligopeptide analogs of the N-terminal region of gp41 and determined their ability to inhibit HIV-induced cell fusion in CD4+ HeLa cells. A hexapeptide which was identical in amino acid sequence to the N-terminus of gp41 was found to completely inhibit cell fusion, whereas peptides with altered sequences showed reduced inhibitory activity. These peptides had no effect on protein synthesis, processing, or transport to the cell surface, and showed no signs of toxicity to cells even at very high concentrations. These results indicate that oligopeptides which are homologous to the fusion peptide of HIV inhibit virus-induced cytopathology, and should be evaluated further as potential antiviral agents.

Amino Acid Sequence↗
PubMed1990

The env protein of an infectious noncytopathic HIV-2 is deficient in syncytium formation.

A recent isolate of human immunodeficiency virus type 2 (HIV-2) designated HIV-2ST is deficient in its ability to cause the typical cytopathic effects of HIV infection. The pathogenic potential of HIV-2 in inducing human disease may be less than that of HIV-1, and it is of particular interest to establish the basis for the reduced cytopathogenicity of this isolate in vitro. Utilizing recombinant vaccinia viruses (rVV) carrying the envelope genes (env) of HIV-2ST or those of fully cytopathic HIV-1 or HIV-2 isolates, we have investigated envelope glycoprotein expression, processing, transport, and biological function. Radioimmunoprecipitation and polyacrylamide gel electrophoresis (RIP-PAGE) of rVV-infected cell lysates indicated that the proteins expressed by each recombinant were synthesized, processed, and recognized by specific antisera. Immunofluorescence studies showed that the recombinant env gene products of HIV-2ST and HIV-2ROD reach the cell surface and are retained there in similar amounts. Whereas cells expressing the HIV-1 or HIV-2ROD env gene products were found to undergo fusion with uninfected CD4+ cells, no syncytium formation was observed with three CD4+ cell lines exposed to the cells expressing the envelope glycoproteins of HIV-2ST on their surfaces; one CD4+ lymphoid cell line (SupT1) exhibited few very small syncytia in the presence of recombinant HIV-2ST envelope glycoproteins. The failure of the HIV-2ST envelope glycoprotein to induce cell fusion was not the result of an inhibition by cell-associated CD4, since fusion was also not observed when rVVST-infected CD4- cells were cocultured with CD4+ cells. Thus, the HIV-2ST envelope protein itself is defective in its ability to induce cell fusion. Furthermore, the expression, processing, transport, and surface stability of env products of HIV-2ST are unlikely to be responsible for its attenuation, suggesting that the molecular interactions between its env products and target cell membranes are significantly altered.

CD4 Antigens↗
PubMed1989

Bacteriostatic versus bactericidal activity.

Antibiotics alone or in combination may either inhibit or kill bacteria. Laboratory methods are available to determine the activity of various antimicrobial agents and can aid the physician in selecting appropriate antimicrobial therapy for specific infectious disease disorders. A few infectious processes appear to require bactericidal antimicrobial therapy for cure. Additional multicenter trials of the ability of a standardized SBT to predict therapeutic outcome in specific bacterial diseases are required.

Animals↗
PubMed1988

Neoplastic meningitis with eosinophilic pleocytosis in Hodgkin's disease: a case with cerebellar dysfunction and a review of the literature.

A 31-year-old man had Hodgkin's disease (stage IIA, nodular sclerosis) in apparent remission after radiotherapy. Nine months after the diagnosis of Hodgkin's disease, he developed neoplastic meningitis with eosinophilic pleocytosis and neurologic findings suggestive of peri-fourth ventricle infiltration. Morphologic and surface marker analysis of cerebrospinal fluid cells showed large numbers of T-lymphocytes and Reed-Sternberg variant cells positive for CD15, the Lex hapten expressed on myeloid cells and on a variety of malignant cells. Therapy with intrathecal methotrexate, oral dexamethasone, and cranial irradiation resulted in prompt resolution of his cerebrospinal fluid abnormalities and neurologic deficits. Ten months after the diagnosis of eosinophilic meningitis, systemic relapse of Hodgkin's disease occurred in right iliac and inguinal lymph nodes. The diagnosis, pathogenesis, and therapy of this unusual complication of Hodgkin's disease are reviewed.

Adult↗
PubMed1978

Psychiatric disturbances in drunk driving offenders referred for treatment of alcoholism.

The incidence of psychopathology in persons arrested for driving while intoxicated (DWI) has serious implications in both alcoholism treatment and in general highway safety. This study investigated the types of psychopathology, as measured by the Minnesota Multiphasic Personality Inventory (MMPI), in a sample of 116 males referred for treatment by a court diversion program.

Accidents, Traffic↗