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Biomedical subjects

R Longnecker

Publications and source records attributed to R Longnecker.

61 records · Page 4Linked to original sources
PubMed1988

In vivo behavior of genetically engineered herpes simplex viruses R7017 and R7020: construction and evaluation in rodents.

The herpes simplex virus (HSV) recombinant R7017 was constructed from HSV-1 (strain F) by deleting a portion of the thymidine kinase (tk) gene and by replacing the sequences representing the internal inverted repeats and adjacent genes in the L component with a fragment of the HSV-2 genome encoding the glycoproteins G, D, I, and a portion of E. In addition, the R7020 recombinant contains an HSV-1 DNA fragment encoding the tk gene fused to the alpha 4 gene promoter. The results of studies in mice, guinea pigs, and rabbits were as follows: Both recombinants remained unchanged after nine serial, intracerebral passages in mice; the recombinants could not be differentiated with respect to attenuation in mice injected intracerebrally, in vaginally infected guinea pigs, and in rabbits inoculated on the scarified cornea. Given intradermally or intramuscularly, the recombinants prevented severe infections by virulent challenge viruses, and R7020 established latent infections (at a low frequency) in all species tested, whereas latent R7017 virus was detected in rabbits only.

Animals↗
PubMed1987

Clustering of genes dispensable for growth in culture in the S component of the HSV-1 genome.

The herpes simplex virus 1 genome consists of one long and one short stretch of unique sequences flanked by inverted repeat sequences. The nucleotide sequence and RNA map predict 12 open reading frames designated as US1 through US12 within the short stretch of unique sequences. This paper reports the construction of virus mutants from which US2, US3, or US4 had been deleted that are capable of growth in cell culture. One of the three deleted genes, US4, specifies the viral envelope glycoprotein G. Mutants with deletions in US1, US8, US9, US10, US11, and US12 have been previously reported. The nine genes deleted from this region form two clusters, US1 through US4 and US8 through US12, and encode at least two and possibly more structural proteins. The presence of so many genes dispensable for growth in cell culture suggests several hypotheses regarding their function and evolution.

Base Sequence↗
PubMed1987

Identification of a herpes simplex virus 1 glycoprotein gene within a gene cluster dispensable for growth in cell culture.

The genome of herpes simplex virus 1 consists of two components, L and S, each containing unique sequences flanked by inverted repeats. Current and earlier studies have shown that 11 of the 12 open reading frames contained in the unique sequences of the S component can be deleted and are dispensable for growth in cell culture. Analyses of one recombinant virus containing a deletion in the open reading frame US7 permitted the identification of a monoclonal antibody specific for the product of this gene. The protein encoded by this gene has a predicted translated molecular weight of 41,366 and an apparent molecular weight of approximately 65,000 in denaturing polyacrylamide gels. The electrophoretic mobility of the protein synthesized by cells in the presence of inhibitory concentrations of tunicamycin is faster than that of the protein accumulating in lysates of untreated infected cells. We conclude that the product of US7 is glycoprotein subject to N-linked glycosylation, and we have designated it glycoprotein I. These studies indicate that the unique sequences of the S component encode four glycoproteins (G, D, I, and E) of which at least three (G, I, and E) are dispensable for growth in continuous lines of primate cells.

Animals↗
PubMed1986

Identification, properties, and gene location of a novel glycoprotein specified by herpes simplex virus 1.

We report the identification of a novel herpes simplex virus 1 (HSV-1) glycoprotein reactive with type specific monoclonal antibody H1379. The monoclonal antibody reacted with two broad bands with apparent mol wt of 60K to 68K and 44K to 48K formed by infected cell lysates subjected to electrophoresis in denaturing polyacrylamide gels and electrically transferred to a nitrocellulose sheet. Early in infection the H1379 reactive protein was found in the faster migrating band. The rate of accumulation was highest late in infection and only the slower migrating form incorporates significant amounts of glucosamine. The epitopic site recognized by H1379 was not uniformly distributed among strains. Analyses of HSV-1 X HSV-2 recombinants with monoclonal antibodies to HSV-1 and HSV-2 glycoproteins mapping in the S component of the HSV genomes and marker transfer experiments indicated that the gene specifying the H1379 reactive protein maps within BamHI fragment J to the left of gD most probably within the open reading frame designated as US4 (D. J. McGeoch, A Dolan, S. Donald, and F. J. Rixon, 1985, J. Mol. Biol. 181, 1-13). The gene specifying a recently discovered HSV-2 glycoprotein designated as gG-2 (B. Roizman, B. Norrild, C. Chan, and L. Pereira, 1984, Virology 133, 242-247) maps in the corresponding domain of the HSV-2 genome and marker transfer experiments suggest that the H1379 reactive protein and gG-2 are collinear. We have therefore designated the novel HSV-1 glycoprotein as gG-1.

Antibodies, Monoclonal↗
PubMed1986

Generation of an inverting herpes simplex virus 1 mutant lacking the L-S junction a sequences, an origin of DNA synthesis, and several genes including those specifying glycoprotein E and the alpha 47 gene.

The herpes simplex virus genome consists of two components, L and S, that invert relative to each other to yield four isomeric arrangements, prototype (P), inversion of the S component (Is), inversion of the L component (Il), and inversion of both components (Isl). Previous studies have shown that the 500-base-pair a sequences flanking the two components contain a cis-acting site for inversion. In an attempt to insert a third copy of the alpha 4 gene, the major regulatory gene mapping in the repeats flanking the S component, a fragment containing the alpha 4 gene and an origin of DNA synthesis, was recombined into the thymidine kinase gene mapping in the unique sequences of the L component. The resulting recombinants showed massive rearrangements and deletions mapping in the S component and in the junction between the L and S components. One recombinant (R7023) yielded two isomeric DNA arrangements, a major component consisting of Is and a minor component consisting of Isl. In these arrangements, the genome lacked the gene specifying glycoprotein E and all contiguous genes located between it and the alpha 0 gene in the inverted repeats of the L component. Among the deleted sequences were those encoding an origin of viral DNA synthesis, the alpha 47 gene, and the a sequences located at the junction between the L and S-components. The recombinant grew well in rabbit skin, 143TK-, and Vero cell lines. We conclude that the four unique genes deleted in R7023 are not essential for the growth of herpes simplex virus, at least in the cell lines tested, and that the b sequence of the inverted repeats of the L component also contains cis-acting sites for the inversion of herpes simplex virus DNA sequences.

Animals↗
PubMedDate not supplied

Epstein-Barr virus protein LMP2A regulates reactivation from latency by negatively regulating tyrosine kinases involved in sIg-mediated signal transduction.

Like other herpesviruses, Epstein-Barr Virus (EBV) persists in its host through an ability to establish latent infection with episodic reactivations. In latent infection EBV expresses an integral membrane protein LMP2A that regulates reactivation from latency. LMP2A is constitutively tyrosine phosphorylated and is associated with lyn and syk tyrosine kinases. The activity of lyn is substantially reduced. In EBV-infected cells in which LMP2A is expressed, crosslinking of sIg fails to trigger the protein tyrosine kinase signal cascade, tyrosine phosphorylation of cell proteins does not change, second messengers are not generated, and lytic EBV infection is not induced. In contrast, crosslinking of sIg on cells infected with EBV recombinants with null mutations in LMP2A results in transient tyrosine phosphorylation of lyn, syk, phospholipase C gamma 2 and phosphatidylinositol-3' kinase, transiently increased intracellular free calcium, and reactivation of lytic EBV infection. These studies describe a novel molecular regulator of herpesvirus latency and focus attention on the importance of transmembrane signal transduction in herpes virus reactivation from latency. They support the working hypothesis that the identification of ligand-receptor interactions that can result in the induction of reactivation will provide an important inroad toward the delineation of the molecular mechanism, which govern herpesvirus reactivation from latency.

Amino Acid Sequence↗