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L W Enquist

Publications and source records attributed to L W Enquist.

At least 91 records · Page 5Linked to original sources
PubMed1990

The putative cytoplasmic domain of the pseudorabies virus envelope protein gIII, the herpes simplex virus type 1 glycoprotein C homolog, is not required for normal export and localization.

Glycoprotein gIII of pseudorabies virus is a member of a conserved gene family found in at least seven diverse herpesviruses. We report here that the putative cytoplasmic domain of gIII is not required for transport to the cell surface and, unlike the prototype domain from herpes simplex virus type 1 glycoprotein C, is not required for stable membrane anchoring. Furthermore, this domain does not appear to be essential for incorporation of the glycoprotein into virions.

Amino Acid Sequence↗
PubMed1990

Neurotropic properties of pseudorabies virus: uptake and transneuronal passage in the rat central nervous system.

Uptake, replication, and transneuronal passage of a swine neurotropic herpesvirus (pseudorabies virus, PRV) was evaluated in the rat CNS. PRV was localized in neural circuits innervating the tongue, stomach, esophagus and eye with light microscopic immunohistochemistry. In each instance, the distribution of PRV-immunoreactive neurons was entirely consistent with that observed following injection of cholera toxin-horseradish peroxidase conjugate (CT-HRP). Injections of the tongue resulted in retrograde transport of PRV and CT-HRP to hypoglossal motor neurons, while preganglionic neurons in the dorsal motor vagal nucleus or somatic motor neurons in the nucleus ambiguus were labeled following injections of the stomach or esophagus, respectively. At longer times after infection, viral antigens were found in astrocytes adjacent to infected neurons and their efferent axons and second-order neuron labeling became apparent. The distribution of second-order neurons was also entirely dependent upon the site of PRV injection. Following tongue injection, second-order neurons were observed in the trigeminal complex, the brain-stem tegmentum and in monoaminergic cell groups. Injection of the stomach or esophagus led to second-order neuron labeling confined to distinct subdivisions of the neucleus of the solitary tract and monoaminergic cell groups. Comparative quantitative analysis of the number of PRV immunoreactive neurons present in the diencephalon and brain stem following injection of virus into both the eye and stomach musculature of the same animal demonstrated that retrograde transport of PRV from the viscera was more efficient and occurred at a much faster rate than anterograde transport of virus. These data demonstrate projection-specific transport of PRV in the nervous system and provide further insight into the means through which this neurotropic virus infects the nervous system.

Animals↗
PubMed1989

The gene encoding the gIII envelope protein of pseudorabies virus vaccine strain Bartha contains a mutation affecting protein localization.

Pseudorabies virus (PRV) vaccine strain Bartha has a diminished capacity to cause disease and harbors a variety of mutations affecting virulence. It has been reported that PRV Bartha produces virions with reduced amounts of the major envelope glycoprotein gIII. One hypothesis was that this phenotype was due to reduced expression of the gIII gene. In this report, we demonstrate that the reduced amount of gIII in virions was not mediated at the level of transcription, but rather reflected a defect in protein localization. We describe experiments with gene replacement technology to prove that the expression defect was closely linked to the gIII gene itself. Using pulse-chase experiments, we found a defect similar to that observed for certain signal sequence mutations of PRV Becker gIII. The Bartha gIII protein was translated, but was inefficiently introduced into the membrane protein export pathway. Consequently, only a fraction of the primary Bartha gIII translation product was glycosylated and matured. The remaining fraction stayed presumably in the cytoplasm, where it never became glycosylated or inserted into cell or virus membranes. The result was that Bartha-infected cells produced virions with reduced amounts of gIII in their envelopes. Comparison of the DNA sequence of the promoter and amino-terminal coding regions of Becker and Bartha gIII genes revealed a single base pair difference in Bartha, changing codon 14 of the signal sequence from a leucine (CTC) to a proline (CCC) codon. We suggest that the signal sequence mutation is responsible for the apparent reduced expression phenotype of this attenuated strain. This mutation represents, to our knowledge, the first reported natural signal sequence mutation in a herpesvirus glycoprotein.

Animals↗
PubMed1989

Replacement of the pseudorabies virus glycoprotein gIII gene with its postulated homolog, the glycoprotein gC gene of herpes simplex virus type 1.

gIII, the major envelope glycoprotein of pseudorabies virus (PRV), shares approximately 20% amino acid similarity with glycoprotein gC of herpes simplex virus type 1 (HSV-1) and HSV-2. We describe here our first experiments on the potential conservation of function between these two genes and gene products. We constructed PRV recombinants in which the gIII gene and regulatory sequences have been replaced with the entire HSV-1 gC gene and its regulatory sequences. The gC promoter functions in the PRV genome, and authentic HSV-1 gC protein is produced, albeit at a low level, in infected cells. The gC protein is present at the cell surface but cannot be detected in the PRV envelope.

Animals↗
PubMed1988

An amino-terminal deletion mutation of pseudorabies virus glycoprotein gIII affects protein localization and RNA accumulation.

We have constructed a pseudorabies virus mutant that contains virtually a complete deletion of the predicted signal sequence coding region for a nonessential envelope glycoprotein, gIII. No signal sequence mutants have been reported previously for a herpesvirus glycoprotein. Through endoglycosidase treatments and pulse-chase analysis, we have determined that the mutant gIII protein is not posttranslationally modified like the wild-type polypeptide, but rather is present as a single, stable species within the infected cell. The mutant polypeptide cannot be detected in the virus envelope, nor is it aberrantly localized to the tissue culture medium. Immunofluorescence studies have indicated that the mutant protein also is not localized to the surfaces of infected cells. In addition, Northern (RNA) and slot blot analyses, as well as in vitro translation experiments using infected-cell cytoplasmic RNA, have indicated that the mutant gIII allele is expressed at lower levels than the wild-type gene is. This is despite the fact that no alterations have been made upstream of the gIII coding sequence. From these results, it appears that the first 22 amino acids of the wild-type gIII protein define a necessary signal peptide that is responsible for at least the correct initiation of translocation and subsequent glycosylation of the gIII envelope glycoprotein within infected cells.

Amino Acid Sequence↗
PubMed1988

A herpesvirus vector for expression of glycosylated membrane antigens: fusion proteins of pseudorabies virus gIII and human immunodeficiency virus type 1 envelope glycoproteins.

We describe experiments using the swine herpesvirus, pseudorabies virus (PRV), as a vector for expression of hybrid membrane protein genes. In particular, we present the construction and analysis of three infectious PRV mutants expressing chimeric viral membrane proteins composed of portions of the PRV envelope glycoprotein gIII and of the human retrovirus, human immunodeficiency virus type 1 (HIV-1), envelope glycoproteins gp120 and gp41. All of the chimeric genes contain the transcription control sequences and the first 157 codons of PRV gIII (known to contain signals sufficient for efficient export of the encoded peptide out of the cell) fused to different regions of the HIV-1 envelope. The mutant viruses express novel glycosylated fusion proteins that are immunoprecipitated by polyvalent sera specific for gIII, as well as acquired immunodeficiency syndrome patient sera. The levels of expression are lower than expected due primarily to instability or altered processing of the hybrid mRNA. We could not detect cleavage of chimeric proteins carrying the gp120-gp41 protease processing site. The use of localization signals contained within herpesvirus membrane proteins to direct chimeric proteins to desired cellular locations is discussed.

Acetylglucosaminidase↗
PubMed1988

Pseudorabies virus glycoprotein gIII is required for efficient virus growth in tissue culture.

Glycoprotein gIII of pseudorabies virus is a major antigen found in the envelopes of virus particles as well as in and on the surfaces of infected cells. It is not an essential gene product for virus growth in tissue culture. In this report, we provide evidence that, although it is not essential, the gIII protein is required for efficient virus growth and that gIII mutants are quickly outgrown by wild-type virus in mixed infections.

Animals↗
PubMed1987

Mutational analysis of the lambda int gene: DNA sequence of dominant mutations.

We have combined techniques of genetic and physical mapping with rapid DNA sequence analysis to identify the nucleotide change in lambda int mutations. These mutations define two dominant phenotypic classes: (i) recombination that is partially independent of accessory factors, and (ii) inhibition of wild-type Int by missense or nonsense proteins, i.e., negative complementation.

Bacteriophage lambda↗
PubMed1987

Analysis of pseudorabies virus glycoprotein gIII localization and modification by using novel infectious viral mutants carrying unique EcoRI sites.

We have constructed two pseudorabies virus (PRV) mutants, each with a unique EcoRI restriction site in the nonessential gIII envelope glycoprotein gene. Since no natural PRV isolate has been reported to contain EcoRI sites, the isolation and single-step growth curve analysis of these mutants established that PRV can carry such a site with little ill effect in tissue culture. Virus carrying these defined mutations produced novel gIII proteins that enabled us to begin functional assignment of protein localization information within the gIII gene. Specifically, one viral mutant contained an in-frame synthetic EcoRI linker sequence that was flanked on one side by the first one-third of the gIII gene and on the other side by the last one-third of the gene. The resulting protein lacked the middle one-third of the parental species, including five of eight putative N-linked glycosylation signals, but was still glycosylated and found in enveloped virions; it was not secreted into the medium. A second viral mutant contained an in-frame synthetic EcoRI linker sequence that additionally specified a nonsense codon at position 158, producing a gIII protein that was glycosylated and secreted into the medium; the fragment was not found in enveloped virions. By endoglycosidase and pulse-chase analyses, we established a precursor-product relationship between the various forms of gIII expressed in the parental and mutant strains, and perhaps determined certain features of the gIII protein that are required for its efficient export within the cell.

Alleles↗
PubMed1986

Construction of an infectious pseudorabies virus recombinant expressing a glycoprotein gIII-beta-galactosidase fusion protein.

An infectious herpesvirus mutant has been constructed in which a major structural envelope glycoprotein gene was replaced by a hybrid gene encoding a novel fusion protein consisting of the N-terminus of the viral glycoprotein joined to Escherichia coli beta-galactosidase (beta Gal). Specifically, we fused DNA encoding the first 157 amino acids of the structural glycoprotein gIII from pseudorabies virus strain Becker to the E. coli lacZ gene in a bacterial expression vector. The resulting hybrid gene was then used to replace the wild-type gIII gene in the virus by cotransfection of plasmid and viral DNA. The desired viral recombinants were identified by their inability to react with specific monoclonal antibodies that recognized only wild-type gIII protein. One such mutant virus, PRV-Z1, was chosen for further analysis. PRV-Z1 expressed a glycosylated gIII-beta Gal fusion protein after infection of PK15 cells. The fusion protein has no demonstrable beta Gal activity and, although glycosylated, remains sensitive to the enzyme endo-beta-N-acetylglucosaminidase H, unlike the mature gIII gene product, indicating that the fusion protein was incompletely processed.

Antigens, Viral↗
PubMed1986

Characterization of a pseudorabies virus glycoprotein gene with homology to herpes simplex virus type 1 and type 2 glycoprotein C.

A pseudorabies virus (Becker strain) glycoprotein gene was located in the UL region at map position 0.40. The gene was identified by using open reading frame Escherichia coli plasmid expression vectors and specific antibody reagents. A 1.55-kilobase unspliced transcript from the gene was detected in pseudorabies virus-infected tissue culture cells. The DNA sequence revealed a single open reading frame of 1,437 base pairs encoding 479 amino acids. The predicted primary translation product has a molecular weight of 50,860 and contains features of a typical herpesvirus glycoprotein. An E. coli expression plasmid was constructed that contained essentially all of the open reading frame for this gene. Antibodies raised in rabbits against the protein expressed in bacteria by this plasmid immunoprecipitated pseudorabies virus-specific glycoproteins of 92,000 and 74,000 daltons from infected cell extracts. It is likely that these two forms represent different glycosylation states of the protein.

Cloning, Molecular↗
PubMed1986

Pseudorabies virus gene encoding glycoprotein gIII is not essential for growth in tissue culture.

We have established that in the Becker strain of pseudorabies virus (PRV), the glycoprotein gIII gene is not essential for growth in cell culture. This was accomplished by construction and analysis of viral mutants containing two defined deletion mutations affecting the gIII gene. These mutations were first constructed in vitro and introduced into Escherichia coli expression plasmids to verify structure and protein production. Each mutation was then crossed onto PRV by cotransfection of plasmid DNA and parental viral DNA by using gIII-specific monoclonal antibodies as selective and screening reagents. One resultant virus strain, PRV-2, contained an in-frame deletion of a 402-base-pair (bp) SacI fragment contained within the gIII gene. Another virus strain, PRV-10, contained a deletion of a 1,480-bp XhoI fragment removing 230 bp of the upstream, putative transcriptional control sequences and 87% of the gIII coding sequence. The deletion mutants were compared with parental virus by analysis of virion DNA, gIII specific RNA, and proteins reacting with gIII specific antibodies. Upon infection of PK15 cells, the deletion mutants did not produce any proteins that reacted with two gIII specific monoclonal antibodies. However, two species of truncated glycosylated proteins were observed in PRV-2 infected cells that reacted with antiserum raised against bacterially produced gIII protein. PRV-10 produced no detectable gIII-specific RNA or protein. PRV-10 could be propagated without difficulty in tissue culture. Virus particles lacking gIII were indistinguishable from parental PRV virus particles by analysis of infected-cell thin sections in the electron microscope. We therefore conclude that expression of the gIII gene was not absolutely essential for PRV growth in tissue culture.

Animals↗
PubMed1985

Isolation of novel herpes simplex virus type 1 derivatives with tandem duplications of DNA sequences encoding immediate-early mRNA-5 and an origin of replication.

Two naturally occurring variations of herpes simplex virus type 1 (Patton strain) with novel tandem DNA sequence duplications in the S component were isolated, and the DNA was characterized. These variants were identified among a number of plaque isolates by the appearance of new restriction enzyme fragments that hybridized with radiolabeled DNA from the BamHI Z fragment (map coordinates 0.936 to 0.949) located in the unique S region. One isolate, SP26-3, carried a 3.1-kilobase-pair duplication defined by recombination between a site in the BamHI Z fragment and a site near the origin of replication in the inverted repeat sequence of the S component carried by the EcoRI H fragment. The other isolate, SP22-4, carried a 3.5-kilobase-pair duplication defined by a recombination event between a tandem repeat array in the BamHI Z fragment and a site near the amino terminus of the Vmw175 gene in the S-region inverted repeat sequence contained in the EcoRI K fragment. Both duplicated segments contained the entire immediate early mRNA-5 coding region as well as the origin of replication located in the inverted repeat sequence of the S component. The DNA sequence of each duplication joint was determined.

Base Sequence↗
PubMed1984

An integration-proficient int mutant of bacteriophage lambda.

We have isolated and characterized a novel int mutant of phage lambda. This mutant promotes efficient recombination between the phage and bacterial attachment sites, but, unlike wild type, does not promote efficient recombination of any other pair of attachment sites tested in most conditions. In particular, recombination between two phage or two prophage attachment sites is poor relative to the wild type frequency. We attribute this unusual phenotype to differences in the distribution of int protein binding sites among different attachment sites (Ross and Landy 1982, 1983). We suggest that int protein molecules bound to one of two recombining DNAs interact with empty sites or with bound proteins on the other, and that the mutant protein acts efficiently only if the distribution of protein binding sites within the two attachment sites is that of the attP-attB pair. Similar discrimination among attachment site pairs by wild type int protein may also modulate recombination frequencies.

Bacteriophage lambda↗
PubMed1984

Herpes simplex virus type 1 restriction fragment polymorphism determined using southern hybridization.

Regions of herpes simplex virus type 1 (HSV-1) DNA with variation in the size of restriction endonuclease fragments were identified by comparison of the BamHI, KpnI or SalI restriction endonuclease digestion patterns among 15 HSV-1 isolates after hybridization with specific 32P-labeled cloned HSV-1 DNA fragments. Of the types of restriction fragment polymorphism identified, one was a strain with a distinctly different restriction fragment than the prototype (loss or gain of restriction sites). Another type, the specific fragment varied only in size among strains. Thirteen distinct variations were identified. Ten were mapped to the unique sequence of the L component; two to the inverted repeat of the L component and one to the inverted repeat of the S component. The presence of a common ancestor from which some isolates of HSV-1 might derive was deduced from an analysis of the distribution of the thirteen variations among the 15 HSV-1 isolates.

Base Sequence↗
PubMed1984

Tandem repeated DNA in an intergenic region of herpes simplex virus type 1 (Patton).

When the entire US region of HSV-1 (Patton) was cloned as an EcoRI fragment in bacteriophage lambda gtWES, the BamHI B6B5 fragment was observed to vary in size among independent isolates [Umene and Enquist, Gene 13 (1981) 251-268]. This fragment polymorphism also occurred in DNA of HSV-1 single plaque isolates. We report here that this heterogeneity is due to variation in copy number of a 15-bp tandem repeat of sequence 5'-CCACTCCCCACCCAC-3', which apparently lies in an intergenic region of the HSV-1 DNA.

Base Sequence↗