Biomedical subjects

J D Baines

Publications and source records attributed to J D Baines.

25 records · Page 2Linked to original sources

PubMed1993

The UL10 gene of herpes simplex virus 1 encodes a novel viral glycoprotein, gM, which is present in the virion and in the plasma membrane of infected cells.

The herpes simplex virus 1 UL10 gene encodes a hydrophobic membrane protein dispensable for viral replication in cell culture (J.D. Baines and B. Roizman, J. Virol. 65:938-944, 1991). We report the following. (i) A fusion protein consisting of glutathione S-transferase fused to the C-terminal 93 amino acids of the UL10 protein was used to produce a rabbit polyclonal antiserum. The antiserum reacted with infected-cell proteins which formed in denaturing polyacrylamide gels a sharp band (apparent M(r) of 50,000) and a very broad band (M(r) of 53,000 to 63,000). These bands were not formed by lysates of UL10- virus or by lysates of infected cells boiled in the presence of sodium dodecyl sulfate before electrophoresis. (ii) The proteins forming both bands were labeled by [3H]glucosamine, indicating that they were glycosylated. (iii) The UL10 protein in cells treated with tunicamycin formed a single band (apparent M(r) of 47,000) reactive with the anti-UL10 antibody, indicating that the 47,000-M(r) protein was a precursor of N-glycosylated, more slowly migrating forms of UL10. Treatment of the immunoprecipitate with endoglycosidase H increased the electrophoretic mobility of the 50,000-M(r) species to that of the 47,000-M(r) species, indicating that the 50,000-M(r) species contained high-mannose polysaccharide chains, whereas the proteins forming the 53,000- to 63,000-M(r) bands contained mature chains inasmuch as they were resistant to digestion by the enzyme. (iv) The UL10 protein of R7221 carrying a 20-amino-acid epitope formed only one band with an M(r) of 53,000. This band was sensitive to endoglycosidase H, suggesting that the epitope inserted in the R7221 UL10 protein may have interfered with glycosylation. (v) The UL10 protein does not contain a cleavable signal sequence inasmuch as the first UL10 methionine codon was reflected in the 50,000-M(r) protein. (vi) The UL10 protein is present in virions and plasma membranes of unfixed cells that were reacted with the polyclonal rabbit antibody. In accordance with the current nomenclature, the UL10 protein is designated glycoprotein M.

Animals↗

PubMed1992

Monoclonal antibodies to the spike protein of feline infectious peritonitis virus mediate antibody-dependent enhancement of infection of feline macrophages.

Antibody-dependent enhancement of virus infection is a process whereby virus-antibody complexes initiate infection of cells via Fc receptor-mediated endocytosis. We sought to investigate antibody-dependent enhancement of feline infectious peritonitis virus infection of primary feline peritoneal macrophages in vitro. Enhancement of infection was assessed, after indirect immunofluorescent-antibody labelling of infected cells, by determining the ratio between the number of cells infected in the presence and absence of virus-specific antibody. Infection enhancement was initially demonstrated by using heat-inactivated, virus-specific feline antiserum. Functional compatibility between murine immunoglobulin molecules and feline Fc receptors was demonstrated by using murine anti-sheep erythrocyte serum and an antibody-coated sheep erythrocyte phagocytosis assay. Thirty-seven murine monoclonal antibodies specific for the nucleocapsid, membrane, or spike proteins of feline infectious peritonitis virus or transmissible gastroenteritis virus were assayed for their ability to enhance the infectivity of feline infectious peritonitis virus. Infection enhancement was mediated by a subset of spike protein-specific monoclonal antibodies. A distinct correlation was seen between the ability of a monoclonal antibody to cause virus neutralization in a routine cell culture neutralization assay and its ability to mediate infection enhancement of macrophages. Infection enhancement was shown to be Fc receptor mediated by blockade of antibody-Fc receptor interaction using staphylococcal protein A. Our results are consistent with the hypothesis that antibody-dependent enhancement of feline infectious peritonitis virus infectivity is mediated by antibody directed against specific sites on the spike protein.

Animals↗

PubMed1992

The UL11 gene of herpes simplex virus 1 encodes a function that facilitates nucleocapsid envelopment and egress from cells.

The UL11 gene of herpes simplex virus 1 was reported to encode a myristylated protein (C. A. MacLean, B. Clark, and D. J. McGeoch, J. Gen. Virol. 70:3147-3157, 1989). To determine the function of the gene product, a recombinant virus (R7219) lacking 61% of the codons (176 bp of the 288-bp coding domain) was genetically engineered. The deletion mutant replicated in all cell lines tested, albeit to titers 30- to 250-fold lower than those obtained from cells infected with wild-type virus. Electron microscopic analyses indicated that both full and empty capsids accumulated in the nuclei, juxtaposed with the inner lamellae of the nuclear membranes, and that increased numbers of naked particles were present in the cytoplasm of cells infected with the mutant virus. There was a greater than 1,000-fold decrease in the amount of infectious extracellular virus released from Vero cells infected with the deletion mutant compared with that from cells infected with wild-type virus. Furthermore, the onset of release of infectious virus from cells infected with the UL11- mutant was significantly delayed: levels of extracellular UL11- virus increased 15-fold between 20 and 26 h after infection, while levels of wild-type extracellular virus increased 500-fold between 8 and 14 h after infection. A virus in which the UL11 gene was restored produced wild-type levels of total and extracellular virus and was indistinguishable from wild-type virus upon analysis by electron microscopy. Taken together, the data indicate that the absence of the UL11 gene causes a reduced capacity to envelope and transport virions into the extracellular space.

Animals↗

PubMed1992

The cDNA of UL15, a highly conserved herpes simplex virus 1 gene, effectively replaces the two exons of the wild-type virus.

The UL15 gene of herpes simplex virus 1 (HSV-1) is encoded by two or more exons in all herpesvirus genomes sequenced to date. The UL15 coding region is highly conserved, and the intron invariably encodes other genes transcribed antisense to the UL15 coding region. Previously we reported that we deleted the intron domain encoding UL16 but were unable to delete UL15 (J. D. Baines and B. Roizman, J. Virol. 65:938-944, 1991). Here we report that we replaced exon I of UL15 with an unspliced cDNA copy of UL15 in HSV-1 DNA and deleted 58% of the carboxyl-terminal sequences of the natural copy of exon II, including the polyadenylation signal. The yields of infectious virus obtained upon infection with viruses containing the cDNA copy of UL15 were similar to those of an isogenic virus with a wild-type UL15 gene. We therefore conclude that the separation of the two exons of UL15 by an intron encoding two genes is not essential for the replication of HSV, at least in cell culture.

Base Sequence↗

PubMed1991

The UL20 gene of herpes simplex virus 1 encodes a function necessary for viral egress.

A recombinant virus from which the start codon and 53% of the UL20 open reading frame had been deleted was constructed and characterized. We report the following: (i) The UL20- mutant formed small plaques in 143 tk- cells but failed to form plaques in Vero cells. Virus yields were approximately 10- to 100-fold lower than those of wild-type virus in all cell lines tested. (ii) Electron microscopic examination of Vero cells infected with the UL20- mutant revealed that enveloped and unenveloped capsids accumulated in the cytoplasm, possibly in the space between the inner and outer lamellae of the nuclear membrane, and that virtually no virus was present in the extracellular space. (iii) Glycoproteins B, C, D, E, H, and I recovered from lysates of cells infected with the UL20- mutant could not be differentiated from those present in lysates of cells infected with the wild-type parent virus with respect to the electrophoretic mobility of mature and precursor forms. (iv) Repair of the deleted sequences restored the wild-type phenotype. (v) The gene product of the UL20 gene was shown to be associated with cellular membranes and to possess characteristics of integral membrane proteins. We conclude that the UL20 gene encodes an integral membrane protein with a hitherto unrecognized function in that it enables the transit of virions to the extracellular space. The function of the UL20 gene product is complemented by some cell lines but not by Vero cells. The vesicles which serve to transport virions may have an origin different from those associated with transport of normal cellular proteins.

Amino Acid Sequence↗

PubMed1991

The open reading frames UL3, UL4, UL10, and UL16 are dispensable for the replication of herpes simplex virus 1 in cell culture.

By means of insertion and deletion mutagenesis, we have constructed four herpes simplex virus 1 recombinants, each lacking most sequences encoding a different open reading frame. The deleted genes are located in the unique sequences of the long component and include those designated UL3, UL4, UL10, and UL16. The recombinant virus R7211 lacks 579 of the 696 bp of UL3. The recombinant virus R7217 lacks 307 of the 597 bp of the UL4 open reading frame. R7216 contains a 972-bp deletion within the 1,419-bp open reading frame of UL10, whereas R7210 lacks 988 bp of the 1,119-bp UL16 open reading frame. Growth curves indicated that the yields of these viruses in Vero and BHK cell cultures were only slightly reduced from or in some instances equivalent to that of the parent virus. The function of the gene products is not known. It is of interest to note that (i) the UL16 open reading frame maps entirely within the single intron of UL15 and (ii) on the basis of the extent and size of hydrophobic domains, the UL3 and UL10 gene products were predicted to be membrane proteins.

Animals↗

PubMed1981

Effect of carbon source on growth rate and phospholipid composition of Escherichia coli 15T- and an unsaturated fatty acid auxotroph.

Escherichia coli 15T- was grown with glucose, succinic acid, aspartic acid, oleic acid, and oleic plus aspartic acids as carbon sources, and a fatty acid auxotroph derived from 15T- was grown on oleic acid and oleic plus aspartic acids. The doubling time, phospholipid composition, phosphorus content, and the fatty acid composition of the phospholipids of cells in each of the media were determined. In all cases, phosphatidylethanolamine was the major phospholipid present; but with 15T- its concentration was inversely proportional to the doubling time in unsupplemented media. With the auxotroph the phosphatidylethanolamine concentration was essentially unchanged with growth. Total lipid phosphorus was inversely proportional to doubling time, an effect particularly evident with the auxotroph. Without oleic acid supplementation, the major effects of carbon source on fatty acid composition are decreases in the content of palmitoleic acid and increases in the content of cis-9, 10-methylene hexadecanoic acid as growth rate decreases. Oleic acid supplementation elevated 18:1 fatty acid content in both 15T- and the auxotroph.

Aspartic Acid↗