The endoglycosidase endo-beta-N-acetylglucominidase H (endo H) was used to examine the nature of the oligosaccharides associated with the herpes simplex virus type 1 glycoproteins gA, gB, and gC. Immunoprecipitates from detergent extracts of infected cells, using monospecific antisera to gAB and gC, were treated with endo H. The low-molecular-weight precursor to gC, pgC(105), was found to be sensitive to endo H. Removal of the endo H-sensitive oligosaccharide chains from pgC(105) resulted in a protein with an apparent molecular weight of 75,000. In contrast, the fully glycosylated gC was not sensitive to endo H treatment. These results suggested that the oligosaccharide chains of pgC(105) were primarily of the simple high-mannose type. Both gA and gB were sensitive to endo H treatment; however, gB appeared to be only partially susceptible, whereas [3H]mannose-labeled gA was not detectable after endo H treatment. These results that gB contained both complex- and simple-type oligosaccharides, and gA contained only simple-type oligosaccharides. An accumulation of the high-mannose glycoproteins pgC(105) and gA was observed in monensin-treated infected cells with a concomitant inhibition of gB and gC. Glycoproteins gA and pgC(105) synthesized in the presence of monensin were also sensitive to endo H treatment.
The presence of O-glycosidic linkages on herpes simplex virus type 1 (HSV-1) glycoproteins was indicated by the synthesis and glycosylation of HSV-1 glycoproteins in the presence of tunicamycin. Monospecific antiserum to HSV-1 gC immunoprecipitated a 92,000-molecular-weight protein synthesized in the presence of tunicamycin and isotopically labeled with glucosamine or galactose. Anti-gAB did not immunoprecipitate a carbohydrate-labeled HSV-1 protein synthesized in the presence of tunicamycin. The purified glucosamine-labeled 92,000-molecular-weight protein synthesized in the presence of tunicamycin and the fully glycosylated forms of gAB and gC were tested for their sensitivity to mild alkaline hydrolysis. Purified gAB was resistant to mild alkaline hydrolysis, whereas gC and the 92,000-molecular-weight protein were both sensitive to mild alkaline hydrolysis. These results suggest that O-glycosidic linkages are associated with the HSV-1 gC glycoprotein.
This chapter discusses the genetic diversity of some mammalian reoviruses and considers the structure of reoviruses and function of reovirus genes encoding outer capsid proteins. It presents the genetic diversity among various isolates and how it is being defined at the nucleic acid level. Viral tropism, the specificity of a virus for a particular host tissue, is determined in part by the interaction of viral surface structures with cell-surface receptors on host cells. viruses that have an outer protein shell surrounding an inner shell (or "core"), almost all of the proteins whose functions have been found to be related to interaction with the environment and the extracellular sites in the host, are proteins found in the outer capsid. A variety of mutants have been identified for the reoviruses. In addition, there has been extensive analysis concerning the nature of reversion of the mutants. The mutants are temperature-sensitive mutants, deletion mutants, and extragenic suppression type mutans.
A reovirus variant, 8B, was isolated from a neonatal mouse which had been inoculated with a mixture of two reovirus strains: type 1 Lang (T1L) and type 3 Dearing (T3D) (E. A. Wenske, S.J. Chanock, L. Krata, and B. N. Fields, J. Virol. 56:613-616, 1985). 8B is a reassortant containing eight gene segments derived from the T1L parent and two gene segments derived from the T3D parent. Upon infection of neonatal mice, 8B produced a generalized infection characteristic of many reoviruses, but it also efficiently induced numerous macroscopic external cardiac lesions, unlike either of its parents. Microscopic examination of hearts from infected mice revealed myocarditis with necrotic myocytes and both polymorphonuclear and mononuclear cellular infiltration. Electron microscopy revealed viral arrays in necrotic myocytes and dystrophic calcification accompanying late lesions. Determination of viral titers in hearts from T1L-, T3D-, or 8B-infected mice indicated that growth was not the primary determinant of myocardial necrosis. Results from inoculations of athymic mice demonstrated that T cells were not a requirement for the 8B-induced myocarditis. Finally, 8B was more cytopathic than either of the parent viruses in cultured mouse L cells. Together, the data suggest that 8B-induced myocardial necrosis is due to a direct effect of reovirus on myocytes. Reovirus thus provides a useful model for the study of viral myocarditis.
Reassortments between type 1 (Lang) and type 3 (Dearing) reoviruses were isolated from suckling mice infected perorally with an inoculum containing both type 1 and type 3 viruses. A total of five distinct reassortants (designated as E1 through E5) were isolated from animals during the course of the experiment. Two reassortants (E1 and E2) represented the majority of the reassortants isolated. The majority of genes of types E1 and E2 were derived from type 1 (Lang). However, E1 had an M2 gene and an S1 gene derived from type 3 (Dearing), while E2 had M2 and S2 genes derived from type 3 (Dearing). Thus, nonrandom reassortment between mammalian reoviruses can be demonstrated in vivo.
Inositol-specific phospholipase Cs(PLCs) are a group of enzymes involved in the signal transduction pathway of many plasma membrane receptor mediated events. We developed a modified solid surface to capture [(3)H] PIP(2) onto the Basic FlashPlate(R) in order to monitor PLC activity. Our results clearly demonstrate the utility of [(3)H] PIP(2)-Coated Phospholipid FlashPlate(R) microtiter plates for assessing PLC activity for HTS of receptor-coupled functional assays. The results show that PLC activity can be measured easily from a variety of sources including purified recombinant enzyme preparations, crude HL60 cell lysates and permeabilized A431 human carcinoma cells. Moreover, this format provides a surface comparable to that used for classical solution based radiolabeled mixed phospholipid micelle studies and illustrates the feasibility of this assay for measuring PLC activation in a variety of different drug screening assays.
Journal Article Human Rotaviruses and Genome RNA Get access Stephen J. Chanock, Stephen J. Chanock From the Department of Microbiology and Molecular Genetics, Harvard Medical School; the Department of Medicine, Children's Hospital Medical Center; and the Division of Infectious Disease, Brigham and Women's Hospital, Boston, Massaschusetts Search for other works by this author on: Oxford Academic PubMed Google Scholar Elizabeth A. Wenske, Elizabeth A. Wenske From the Department of Microbiology and Molecular Genetics, Harvard Medical School; the Department of Medicine, Children's Hospital Medical Center; and the Division of Infectious Disease, Brigham and Women's Hospital, Boston, Massaschusetts Search for other works by this author on: Oxford Academic PubMed Google Scholar Bernard N. Fields Bernard N. Fields From the Department of Microbiology and Molecular Genetics, Harvard Medical School; the Department of Medicine, Children's Hospital Medical Center; and the Division of Infectious Disease, Brigham and Women's Hospital, Boston, Massaschusetts Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 148, Issue 1, July 1983, Pages 49–50, https://doi.org/10.1093/infdis/148.1.49 Published: 01 July 1983
