Biomedical subjects

N Osterrieder

Publications and source records attributed to N Osterrieder.

At least 19 recordsLinked to original sources

PubMed2004

An enzyme-linked immunosorbent assay (ELISA) for detection of Marek's disease virus-specific antibodies and its application in an experimental vaccine trial.

An enzyme-linked immunosorbent assay (ELISA) for the detection of Marek's disease virus (MDV)-specific antibodies was developed. Chicken embryo cells (CEC) or chicken kidney cells (CKC) were infected with MDV vaccine strain CVI988/Rispens, and infected-cell lysates were prepared at day 5 post-infection by freeze-thawing. Uninfected-cell lysates served as negative controls. Sera were used at a 1 : 100 dilution and were added in parallel to wells containing the infected and uninfected cell lysates. The optical densities at 492 nm (OD(492 nm)) were measured after detection of bound chicken antibodies with anti-chicken IgG peroxidase conjugate and colour reactions using o-phenylenediamine (OPD) as a substrate. The best results concerning the signal-to-noise ratio were obtained by using CKC cells rather than CEC for antigen preparation. The OD(492 nm) of plasma or serum samples with infected CKC was <0.02 when samples of unvaccinated and unchallenged maternal antibody-negative white leghorn chickens were tested. Sera and plasma samples of positive control birds exhibited OD(492 nm) of <0.01 when tested with uninfected CKC. The assay was used to monitor a trial that compared experimental BAC DNA vaccines and a commercial vaccine. Sustained seroconversion and antibody titers that were constantly rising until day 84 after vaccination (71 days after challenge) was observed only when chickens did not develop Marek's disease. In contrast, chickens developing the disease mounted marginal and short-lived antibody titers only. We conclude that the developed ELISA may be a valuable tool for the evaluation of the efficacy of MDV vaccination under experimental but possibly also under field conditions.

Animals↗

PubMed2002

Cloning of the genomes of equine herpesvirus type 1 (EHV-1) strains KyA and racL11 as bacterial artificial chromosomes (BAC).

The genome of equine herpesvirus type 1 (EHV-1) strain RacL11, a highly virulent isolate obtained from an aborted foal, and that of the modified live vaccine strain KyA, were cloned as bacterial artificial chromosomes (BAC) in Eseherichia coli. Mini F plasmid sequences were inserted into the viral genomes by homologous recombination instead of the gene 71 (EUS4) open reading frame after co-transfection of viral DNA and recombinant plasmid pdelta71-pHA2 into RK13 cells. After isolation of recombinant viruses by three rounds of plaque purification, viral DNA was isolated from RK13 cells infected with RacL11 or KyA virus mutants expressing the green fluorescent protein (GFP), and electroporated into Escherichia coli DH10B cells. Several bacterial colonies were shown to contain high-molecular weight BAC DNA with a restriction enzyme fragment pattern indicative of the presence of full-length RacL11 or KyA genomes. Two selected BAC clones were analysed by restriction enzyme analysis and Southern blotting, and were eventually termed pRacLI I and pKyA. respectively. Upon transfection of pRacL11 or pKyA DNA into RK13 cells, GFP-expressing fluorescing virus plaques could be identified from day 1 after transfection. Infectivity after transfection of pRacL11 or pKyA could be readily propagated on RK13 or equine cells, indicating that infectious full-length DNA clones of strains RacL11 and KyA were successfully cloned in Escherichia coli as BACs. The glycoprotein 2-negative progeny reconstituted from pRacL11 and pKyA (L11deltagp2 and KyAdeltagp2) exhibited different growth properties. Whereas both L11deltagp2 and KyAdeltagp2 extracellular titres were reduced by 15- to 32-fold, plaque diameters were only markedly (50%) reduced in the case of KyAdeltagp2.

Animals↗

PubMed2001

Deletion of gene 52 encoding glycoprotein M of equine herpesvirus type 1 strain RacH results in increased immunogenicity.

The immunogenicity of equine herpesvirus type 1 (EHV-1) strain RacH was compared to a RacH virus in which gene 52 encoding glycoprotein M (gM) was interrupted by insertion of LacZ (HDeltagM-Ins) and a RacH with 75% of gene 52 was deleted and replaced by LacZ (HDeltagM-HS). HDeltagM-Ins failed to produce full-length gM, but the carboxy-terminal portion was still expressed. No gM expression was detected in HDeltagM-HS-infected cells. Mice were immunised once with 1x10(3) to 1x10(5) plaque-forming units (PFU) of RacH or mutant viruses and challenged with virulent RacL11 virus 29 days later. A dose-dependence of protection was observed in RacH-immunised mice, and following immunisation with 1x10(4) or 1x10(3) PFU body weight losses and increased virus titres in lungs were observed after challenge infection. HDeltagM-HS-immunised mice were completely protected even after immunisation with 1x10(3) PFU. Mice immunised with 1x10(3) PFU of HDeltagM-Ins but not the higher doses showed signs of disease after challenge infection.

Animals↗

PubMed2001

Glycoproteins E and I of Marek's disease virus serotype 1 are essential for virus growth in cultured cells.

The role of glycoprotein E (gE) and gI of Marek's disease virus serotype 1 (MDV-1) for growth in cultured cells was investigated. MDV-1 mutants lacking either gE (20DeltagE), gI (20DeltagI), or both gE and gI (20DeltagEI) were constructed by recE/T-mediated mutagenesis of a recently established infectious bacterial artificial chromosome (BAC) clone of MDV-1 (D. Schumacher, B. K. Tischer, W. Fuchs, and N. Osterrieder, J. Virol. 74:11088-11098, 2000). Deletion of either gE or gI, which form a complex in MDV-1-infected cells, resulted in the production of virus progeny that were unable to spread from cell to cell in either chicken embryo fibroblasts or quail muscle cells. This was reflected by the absence of virus plaques and the detection of only single infected cells after transfection, even after coseeding of transfected cells with uninfected cells. In contrast, growth of rescuant viruses, in which the deleted glycoprotein genes were reinserted by homologous recombination, was indistinguishable from that of parental BAC20 virus. In addition, the 20DeltagE mutant virus was able to spread from cell to cell when cotransfected into chicken embryo fibroblasts with an expression plasmid encoding MDV-1 gE, and the 20DeltagI mutant virus exhibited cell-to-cell spread capability after cotransfection with a gI expression plasmid. The 20DeltagEI mutant virus, however, was not able to spread in the presence of either a gE or gI expression plasmid, and only single infected cells were detected by indirect immunofluorescence. The results reported here demonstrate for the first time that both gE and gI are absolutely essential for cell-to-cell spread of a member of the Alphaherpesvirinae.

Animals↗

PubMed2001

Egress of alphaherpesviruses: comparative ultrastructural study.

Egress of four important alphaherpesviruses, equine herpesvirus 1 (EHV-1), herpes simplex virus type 1 (HSV-1), infectious laryngotracheitis virus (ILTV), and pseudorabies virus (PrV), was investigated by electron microscopy of infected cell lines of different origins. In all virus-cell systems analyzed, similar observations were made concerning the different stages of virion morphogenesis. After intranuclear assembly, nucleocapsids bud at the inner leaflet of the nuclear membrane, resulting in enveloped particles in the perinuclear space that contain a sharply bordered rim of tegument and a smooth envelope surface. Egress from the perinuclear cisterna primarily occurs by fusion of the primary envelope with the outer leaflet of the nuclear membrane, which has been visualized for HSV-1 and EHV-1 for the first time. The resulting intracytoplasmic naked nucleocapsids are enveloped at membranes of the trans-Golgi network (TGN), as shown by immunogold labeling with a TGN-specific antiserum. Virions containing their final envelope differ in morphology from particles within the perinuclear cisterna by visible surface projections and a diffuse tegument. Particularly striking was the addition of a large amount of tegument material to ILTV capsids in the cytoplasm. Extracellular virions were morphologically identical to virions within Golgi-derived vesicles, but distinct from virions in the perinuclear space. Studies with gB- and gH-deleted PrV mutants indicated that these two glycoproteins, which are essential for virus entry and direct cell-to-cell spread, are dispensable for egress. Taken together, our studies indicate that the deenvelopment-reenvelopment process of herpesvirus maturation also occurs in EHV-1, HSV-1, and ILTV and that membrane fusion processes occurring during egress are substantially different from those during entry and direct viral cell-to-cell spread.

Alphaherpesvirinae↗

PubMed2000

Equine herpesvirus 1 (EHV-1) glycoprotein M: effect of deletions of transmembrane domains.

Equine herpesvirus 1 (EHV-1) recombinants that carry either a deletion of glycoprotein M (gM) or express mutant forms of gM were constructed. The recombinants were derived from strain Kentucky A (KyA), which also lacks genes encoding gE and gI. Plaques on RK13 cells induced by the gM-negative KyA were reduced in size by 80%, but plaque sizes were restored to wild-type levels on gM-expressing cells. Electron microscopic studies revealed a massive defect in virus release after the deletion of gM in the gE- and gI-negative KyA, which was caused by a block in secondary envelopment of virions at Golgi vesicles. Recombinant KyA expressing mutant gM with deletions of predicted transmembrane domains was generated and characterized. It was shown that mutant gM was expressed and formed dimeric and oligomeric structures. However, subcellular localization of mutant gM proteins differed from that of wild-type gM. Mutant glycoproteins were not transported to the Golgi network and consequently were not incorporated into the envelope of extracellular virions. Also, a small plaque phenotype of mutant viruses that was indistinguishable from that of the gM-negative KyA was observed. Plaque sizes of mutant viruses were restored to wild-type levels by plating onto RK13 cells constitutively expressing full-length EHV-1 gM, indicating that mutant proteins did not exert a transdominant negative effect on wild-type gM.

Amino Acid Sequence↗

PubMed2000

Reconstitution of Marek's disease virus serotype 1 (MDV-1) from DNA cloned as a bacterial artificial chromosome and characterization of a glycoprotein B-negative MDV-1 mutant.

The complete genome of Marek's disease virus serotype 1 (MDV-1) strain 584Ap80C was cloned in Escherichia coli as a bacterial artificial chromosome (BAC). BAC vector sequences were introduced into the U(S)2 locus of the MDV-1 genome by homologous recombination. Viral DNA containing the BAC vector was used to transform Escherichia coli strain DH10B, and several colonies harboring the complete MDV-1 genome as an F plasmid (MDV-1 BACs) were identified. DNA from various MDV-1 BACs was transfected into chicken embryo fibroblasts, and from 3 days after transfection, infectious MDV-1 was obtained. Growth of MDV-1 recovered from BACs was indistinguishable from that of the parental virus, as assessed by plaque formation and determination of growth curves. In one of the MDV-1 BAC clones, sequences encoding glycoprotein B (gB) were deleted by one-step mutagenesis using a linear DNA fragment amplified by PCR. Mutant MDV-1 recovered after transfection of BAC DNA that harbored a 2.0-kbp deletion of the 2.6-kbp gB gene were able to grow and induce MDV-1-specific plaques only on cells providing MDV-1 gB in trans. The gB-negative virus reported here represents the first MDV-1 mutant with a deletion of an essential gene and demonstrates the power and usefulness of BACs to analyze genes and gene products in slowly growing and strictly cell-associated herpesviruses.

Animals↗

PubMed2000

Detection of chelonid herpesvirus DNA by nonradioactive in situ hybridization in tissues from tortoises suffering from stomatitis-rhinitis complex in Europe and North America.

Chelonid herpesvirus (ChHV) infection in tortoises associated with stomatitis-rhinitis complex is a severe, mostly epizootic disease characterized by proliferative and diphtheroid-necrotizing glossitis, pharyngitis, rhinitis, and tracheitis, often occurring with pneumonia and encephalitis. The UL5 gene from a German ChHV isolate was used to generate a digoxigenin-labeled 307-base-pair DNA probe by polymerase chain reaction (PCR). ChHV DNA was detected in paraffin-embedded tissues of five naturally infected tortoises (two Afghan tortoises [Testudo horsfieldii], USA; two Hermann's tortoises [Testudo hermanni], Switzerland; one T. hermanni, Germany) by means of in situ hybridization (ISH) and PCR. Distribution of ChHV DNA exhibits many characteristics of alphaherpesvirus but also some characteristics of betaherpesvirus infections. The amino acid sequence of a portion of the ChHV UL5 homolog exhibited more than 50% similarity to alphaherpesvirus UL5 proteins. Nuclear hybridization signals were detected in epithelial cells of the lingual mucosa and glands. Furthermore, ChHV DNA was observed in tracheal epithelium, pneumocytes, hepatocytes, the renal tubular epithelium, cerebral glia cells and neurons, and intramural intestinal ganglia. ChHV DNA in endothelial cells of many organs underlines the systemic character of the disease. Importantly, ChHV DNA was detected by ISH in multiple tissues of tortoises originating from different geographic provenances. This indicates a high degree of conservation of the UL5 gene fragment among viruses prevalent in tortoises on different continents. With the described ISH, a molecular biological tool is available for rapid and specific diagnosis of ChHV infections and, more importantly, comparative pathogenetic studies of ChHV isolates from geographically unrelated regions.

Animals↗

PubMed1999

Sequence and initial characterization of the U(L)10 (glycoprotein M) and U(L)11 homologous genes of serotype 1 Marek's Disease Virus.

The nucleotide sequence of the U(L)10 (glycoprotein M) and the U(L)11 homologs of Marek's Disease Virus 1 strain GA was determined. The U(L)10 open reading frame encodes a type III membrane protein of 424 amino acids that contains eight hydrophobic domains and two consensus N-linked glycosylation sites. The U(L)11 homologous gene encodes an 84 amino acid polypeptide, and contains a highly conserved myristylation site at its aminoterminus. By analysis of infected-cell RNA with strand-specific RNA probes, transcription of both U(L)10 and U(L)11 in infected cells was demonstrated. Coupled in vitro transcription-translation confirmed that the U(L)10 product is a 47 kD N-glycosylated viral protein that aggregated upon boiling, whereas the U(L)11 protein exhibited a size of 12 kD after in vitro translation.

Amino Acid Sequence↗

PubMed1999

Construction and characterization of an equine herpesvirus 1 glycoprotein C negative mutant.

An equine herpesvirus 1 (EHV-1) strain RacL 11 mutant was constructed that carries the Escherichia coli LacZ gene instead of the open reading frame encoding glycoprotein C (gC). The engineered virus mutant (L11(delta)gC) lacked codons 46-440 of the 1404 bp gene. On rabbit kidney cell line Rk13 and equine dermal cell line Edmin337, the L11(delta)gC virus grew to titers which were reduced by approximately 5- to 10-fold compared with wild-type RacL11 virus or a repaired virus (R-L11(delta)gC). However, when L11(delta)gC growth properties were analyzed on primary equine cells a decrease of viral titers was observed such that extracellular L11(delta)gC titers were reduced by 48- to 210-fold compared with those of wild-type or repaired virus. Heparin sensitive and heparin resistant attachment was assessed by binding studies using radiolabeled virion preparations. These studies revealed that EHV-1 gC is important for heparin sensitive attachment to the target cell. Similar results were obtained when cellular glycosaminoglycan (GAG) synthesis was inhibited by chlorate treatment or when cells defective in GAG synthesis were used. L11(delta)gC also exhibited significantly delayed penetration kinetics on Rk13 and primary equine cells. Infection of mice with L11(delta)gC did not cause EHV-1-related disease, whereas mice infected with either RacL11 or R-L11(delta)gC exhibited massive bodyweight losses, high virus titers in the lungs, and viremia. Taken together, EHV-1 gC was shown to play important roles in the early steps of infection and in release of virions, especially in primary equine cells, and contributes to EHV-1 virulence.

Animals↗

PubMed1999

The equine herpesvirus 1 Us2 homolog encodes a nonessential membrane-associated virion component.

Experiments were conducted to analyze the equine herpesvirus 1 (EHV-1) gene 68 product which is encoded by the EHV-1 Us2 homolog. An antiserum directed against the amino-terminal 206 amino acids of the EHV-1 Us2 protein specifically detected a protein with an Mr of 34,000 in cells infected with EHV-1 strain RacL11. EHV-1 strain Ab4 encodes a 44,000-Mr Us2 protein, whereas vaccine strain RacH, a high-passage derivative of RacL11, encodes a 31,000-Mr Us2 polypeptide. Irrespective of its size, the Us2 protein was incorporated into virions. The EHV-1 Us2 protein localized to membrane and nuclear fractions of RacL11-infected cells and to the envelope fraction of purified virions. To monitor intracellular trafficking of the protein, the green fluorescent protein (GFP) was fused to the carboxy terminus of the EHV-1 Us2 protein or to a truncated Us2 protein lacking a stretch of 16 hydrophobic amino acids at the extreme amino terminus. Both fusion proteins were detected at the plasma membrane and accumulated in the vicinity of nuclei of transfected cells. However, trafficking of either GFP fusion protein through the secretory pathway could not be demonstrated, and the EHV-1 Us2 protein lacked detectable N- and O-linked carbohydrates. Consistent with the presence of the Us2 protein in the viral envelope and plasma membrane of infected cells, a Us2-negative RacL11 mutant (L11DeltaUs2) exhibited delayed penetration kinetics and produced smaller plaques compared with either wild-type RacL11 or a Us2-repaired virus. After infection of BALB/c mice with L11DeltaUs2, reduced pathogenicity compared with the parental RacL11 virus and the repaired virus was observed. It is concluded that the EHV-1 Us2 protein modulates virus entry and cell-to-cell spread and appears to support sustained EHV-1 replication in vivo.

Animals↗

PubMed1999

[Mutations in the US2 and glycoprotein B genes of the equine herpesvirus 1 vaccine strain RacH have no effects on its attenuation].

The equine herpesvirus 1 (EHV-1) modified live vaccine strain RacH is apathogenic for both laboratory animals and the natural host. The apathogenicity of RacH was caused by serial passages of the virus in heterologous cells. When compared to the virulent parental strain RacL11 several changes in the RacH genome occurred. Previous results have shown that the loss of the IR6 gene correlated with the loss of virulence. Additional important mutations were observed within the US2 gene which is directly adjacent to the IR6 gene and within the glycoprotein B (gB) gene. To answer the question whether these mutations contribute to the attenuation of RacH several recombinant EHV-1 were constructed: The mutated genes in RacH were replaced by the wild-type US2 gene or the wild-type gB gene, respectively. In addition, a RacL11 recombinant expressing the mutated (RacH) gB instead of the wild-type gene was generated. All recombinant viruses were tested for virulence using the EHV-1 mouse model. The results were as follows: i) The insertion of the RacL11 US2 gene into the RacH virus did not restore virulence and none of the infected mice showed typical signs of EHV-1-caused disease (symptoms and body weight loss). ii) Exchanging gB genes between RacL11 and RacH did not alter their virulence phenotypes remarkably either. Therefore, it is concluded that attenuation of the EHV-1 vaccine strain RacH is caused solely by the absence of the IR6 gene and protein.

Animals↗

PubMed1998

Protective immunity against equine herpesvirus type-1 (EHV-1) infection in mice induced by recombinant EHV-1 gD.

The ability of recombinant preparations of equine herpesvirus type 1 (EHV-1) glycoprotein D (gD) to elicit specific antibody and T lymphocyte responses in the BALB/c mouse model of respiratory infection was investigated. Recombinant gD (rgD) expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli elicited both high titer neutralizing antibody (nAb) and CD4 T cell proliferative responses following subcutaneous or intranasal immunization, but elicited only a weak antibody response after intraperitoneal immunization. Protection against respiratory tract infection with pathogenic EHV-1 RacL11 was observed in mice immunized subcutaneously with GST-gD. Furthermore, the degree of protection correlated to the titer of nAb and the T cell response observed. Finally, GST-gD was more effective in protecting against respiratory RacL11 infection if delivered intranasally. These results confirm that gD plays an important role in eliciting the protective immune response against EHV-1 infection, and indicate that subunit vaccines containing preparations of gD may be very effective if delivered directly to the upper respiratory tract.

Animals↗

PubMed1998

The equine herpesvirus 1 IR6 protein that colocalizes with nuclear lamins is involved in nucleocapsid egress and migrates from cell to cell independently of virus infection.

The equine herpesvirus 1 (EHV-1) IR6 protein forms typical rod-like structures in infected cells, influences virus growth at elevated temperatures, and determines the virulence of EHV-1 Rac strains (Osterrieder et al., Virology 226:243-251, 1996). Experiments to further elucidate the functions and properties of the IR6 protein were conducted. It was shown that the IR6 protein of wild-type RacL11 virus colocalizes with nuclear lamins very late in infection as demonstrated by confocal laser scan microscopy and coimmunoprecipitation experiments. In contrast, the mutated IR6 protein encoded by the RacM24 strain did not colocalize with the lamin proteins at any time postinfection (p.i.). Electron microscopical examinations of ultrathin sections were performed on cells infected at 37 and 40 degreesC, the latter being a temperature at which the IR6-negative RacH virus and the RacM24 virus are greatly impaired in virus replication. These analyses revealed that nucleocapsid formation is efficient at 40 degreesC irrespective of the virus strain. However, whereas cytoplasmic virus particles were readily observed at 16 h p.i. in cells infected with the wild-type EHV-1 RacL11 or an IR6-recombinant RacH virus (HIR6-1) at 40 degreesC, virtually no capsid translocation to the cytoplasm was obvious in RacH- or RacM24-infected cells at the elevated temperature, demonstrating that the IR6 protein is involved in nucleocapsid egress. Transient transfection assays using RacL11 or RacM24 IR6 plasmid DNA and COS7 or Rk13 cells, infection studies using a gB-negative RacL11 mutant (L11DeltagB) which is deficient in direct cell-to-cell spread, and studies using lysates of IR6-transfected cells demonstrated that the wild-type IR6 protein is transported from cell to cell in the absence of virus infection and can enter cells by a yet unknown mechanism.

Animals↗

PubMed1998

Lessons from gene knockouts.

The authors describe the technique for the application of homologous recombination in embryonic stem cells, which is now widely used to engineer mice which carry specific knockouts of genes. A summary is given of some of the knowledge of the pathogenesis of and resistance to infections with parasites, bacteria, or viruses which has accumulated during recent years, based on the investigation of knockout mice. Special emphasis is placed on knockout animals which lack components of the cytokine network, lack genes which are critical for the correct presentation of antigens or are deficient in different immune cell subsets. In addition, a brief explanation is offered of the possibilities for inducing targeted deletions or mutations in genes of livestock species (e.g., by nuclear transfer or by mutagenesis using the alkylating agent N-ethyl-N-nitrosourea) which could lead to the breeding of animals which are resistant to infectious diseases in the future.

Animals↗

PubMed1997

Equine herpesvirus 1 mutants devoid of glycoprotein B or M are apathogenic for mice but induce protection against challenge infection.

Equine herpesvirus 1 (EHV-1) mutants devoid of the open reading frames (ORFs) of either glycoprotein (g) B or M were constructed and tested for their immunogenic potential in a murine model of EHV-1 infection. The mutant viruses were engineered using the virulent EHV-1 strain RacL11 or the modified live vaccine strain RacH by inserting the Escherichia coli LacZ gene into the viral ORFs. RacL11-infected mice showed signs typical of an EHV-1 infection, whereas mice infected with the EHV-1 gB- or gM-negative mutants or with RacH did not develop disease. No difference in the pathogenic potential of RacL11 gB- and gM-negative viruses was observed after application of either phenotypically completed or negative viruses. However, revertant RacL11 viruses in which the gB or gM gene had been restored caused EHV-1-related symptoms that were indistinguishable from those induced by RacL11. Mice that had been immunized with phenotypically negative gB- and gM-deficient EHV-1 were challenged with the RacL11 virus 25 days after immunization. Mock-immunized mice developed EHV-1 disease and high virus loads in their lungs were observed. In contrast, mice developed not exhibit EHV-1-caused disease. It was concluded (i) that deletion of either gB or gM abolished the virulence of strain RacL11 and (ii) that immunization with gB- or gM-negative EHV-1 elicited a protective immunity that was reflected by both virus-neutralizing antibodies and EHV-1-specific T-cells in spleens of immunized mice.

Animals↗

PubMed1997

Increased incorporation of chimeric human immunodeficiency virus type 1 gp120 proteins into Pr55gag virus-like particles by an Epstein-Barr virus gp220/350-derived transmembrane domain.

Noninfectious Pr55gag virus-like particles containing high quantities of oligomeric human immunodeficiency virus type 1 (HIV-1) envelope (Env) proteins represent potential candidate immunogens for a vaccine against HIV-1 infection. Thus, chimeric env genes were constructed encoding the HIV-1 exterior glycoprotein gp120 which was covalently linked at different C-terminal positions to a transmembrane domain (TM) from the Epstein-Barr virus (EBV) major Env glycoprotein gp220/ 350. All chimeric Env-TM polypeptides as well as the wild-type HIV Env proteins were equally produced and incorporated at the outer surface of insect cells using the baculovirus expression system. In the presence of coexpressed HIV Pr55gag polyproteins significantly decreased amounts of wild-type Env proteins were presented at the cell surface, whereas the membrane incorporation of the Env-TM chimeras was not affected. Biochemical and immunoelectron microscopical analysis of particles that were efficiently released from these cells displayed the incorporation of both wild-type Env and chimeric Env-TM proteins on the surface of VLPs. However, the quantities of particle-associated chimeric Env-TM proteins exceeded those of incorporated wild-type Env proteins by a factor of 5-10. Chemical cross-linking and subsequent polyacrylamide gel electrophoresis of VLP-entrapped Env proteins revealed that the chimeric Env-TM proteins form homodimers and a higher-order oligomer, similar to that observed for wild-type Env proteins. Thus, the results of this study clearly demonstrate that the replacement of the gp41 transmembrane protein of gp160 by a heterologous, EBV gp220/350-derived membrane anchor provides an effective strategy to incorporate high quantities of oligomeric HIV gp120 proteins on the surface of Pr55gag virus-like particles.

Amino Acid Sequence↗

PubMed1997

Synthesis and processing of the equine herpesvirus 1 glycoprotein M.

In a previous report, the function of the equine herpesvirus 1 (EHV-1) glycoprotein M (gM) homolog was investigated. It was shown that EHV-1 gM is involved in both virus entry and direct cell-to-cell spread of infection (N. Osterrieder et al., J. Virol. 70, 4110-4115, 1996). In this study, experiments were conducted to analyze the synthesis, posttranslational processing, and the putative ion channel function of EHV-1 gM. It was demonstrated that EHV-1 gM is synthesized as an Mr 44,000 polypeptide, which is cotranslationally N-glycosylated to an Mr 46,000-48,000 glycoprotein. The Mr 46,000-48,000 gM moiety is processed to an Mr 50,000-55,000 glycoprotein, which is resistant to treatment with endoglycosidase H, indicating that processing occurs in the Golgi network. EHV-1 gM forms a dimer in infected cells and the virion, as was demonstrated by the presence of an Mr 105,000-110,000 gM-containing band in electrophoretically separated lysates of infected cells and purified extracellular virions. The Mr 105,000-110,000 protein band containing gM was also observed in lysates of cells that had been transfected with EHV-1 gM DNA. The translation of EHV-1 gM is initiated at the first in-frame methionine of the gM open reading frame as shown by transient transfection experiments of full-length gM and a truncated gM lacking the aminoterminal 83 amino acids. Functional expression of EHV-1 gM in Xenopus laevis oocytes together with voltage-clamp analyses demonstrated that gM per se does not exhibit ion channel activity as had been speculated from the predicted structure of the polypeptide.

Animals↗