Despite its previous classification as a gammaherpesvirus, primarily due to its lymphotropism, Marek's disease virus (MDV), an oncogenic avian herpesvirus, is phylogenetically more related to the "neurotropic" alphaherpesviruses, characterized by its prototype, herpes simplex virus (HSV) (Buckmaster et al., 1988, J. Gen. Virol. 69, 2033-2042). In this report we present the DNA sequence of an 11,286-bp DNA segment encompassing the entire 11,160-bp-long Us region of the oncogenic avian herpesvirus, Marek's disease virus. Eleven open reading frames (ORFs) likely to code for proteins were identified; of these, 7 represent homologs exclusive to alphaherpesvirus S component genes. These include MDV counterparts of HSV US1 (ICP22), US2, US3 (a serine-threonine protein kinase), US6, US7, and US8 (HSV glycoproteins gD, gI, and gE, respectively), and US10. Three additional ORFs were identified with no apparent relation to any sequences currently present in the SwissProt or GenBank/EMBL databases, while a fourth was found to exhibit significant homology to an uncharacterized fowlpox virus (FPV) ORF. Having precisely identified the IRs-U(s) and U(s)-TRs junctions, we have corrected and clarified their previously reported locations. By characterizing genes encoding three new alphaherpesvirus-related homologs (US1, US8, and US10), completing the sequence for a fourth (US7), and identifying 2 new MDV-specific ORFs (SORF1 and SORF3) and a fowlpox homolog (SORF2), our sequence analysis of the "virulent" GA strain of MDV (vMDV) extends upon that of a 5255-bp segment located in the U(s) region of the "very virulent" RB1B strain of MDV (vvMDV) (Ross et al., 1991, J. Gen. Virol. 72, 939-947; 949-954). These two sequences were found to exhibit 99% identity at both nucleotide and predicted amino acid levels. Combined with the fact that MDV U(s) sequences failed to show statistically significant CpG deficiencies, our analysis is consistent with MDV bearing a closer phylogenetic relation to alphaherpesviruses than to gammaherpesviruses. Because alphaherpesvirus-specific U(s) region genes are primarily nonessential for virus replication, they are thought to be important biological property determinants. Thus, our sequence provides a foundation for further MDV studies aimed at resolving the apparent discrepancy between MDV's genetic and biologic properties.
A method was developed for the large-scale production of Marek's disease herpesvirus A antigen in duck embryo fibroblast roller bottle cultures in quantities sufficient to permit its purification and characterization. Maximum yield was obtained in serum-free culture medium harvested daily. The Marek's disease herpesvirus A antigen was stable at pH 2.0 and was a glycoprotein based on its sensitivity to trypsin, specific immune co-precipitation of radioactive amino acids and glucosamine, and detection of radioactive glucosamine by immunodiffusion and autoradiography. The antigen aggregated and lost titer upon storage but dissociated readily and regained titer in 1 or 2 M urea and 0.05% Brij 35. Fresh unaggregated antigen or antigen dissociated with urea and Brij 35 sedimented at 3.7S on sucrose gradients. The apparent molecular weight of the glycoprotein antigen was estimated to be 44,800 by gel filtration on Sephadex G-200 in the presence of 2 M urea and 0.05% Brij 35.
The virions of adenoviruses assemble and accumulate in huge numbers in nuclei of infected cells;1-4 and the viral capsid proteins, identified by immunofluorescent and ferritin-labeled antibody techniques, also can be detected only in the nuclei.5-7 These unequivocal findings led to the question whether the adenovirus proteins were actually synthesized in the nucleus by a unique mechanism since the host proteins, including the nuclear histones,8 seem to be produced in the cytoplasm. It has been reported, however, that some mammalian cells contain nuclear ribosomes9 and that DNA-dependent protein synthesis can take place in their nuclei.9' 10 The investigation to be described was initiated to determine whether a novel system for synthesizing proteins is established in the nuclei of adenovirus-infected cells or whether, utilizing the established cytoplasmic host mechanism, the viral proteins are made and then rapidly transported into the nucleus where they accumulate.Thomas and Green11 provided indirect evidence that cytoplasmic synthesis of viral proteins occurs in KB cells infected with type 2 adenovirus. They detected virus-specific RNA on the polyribosomes of infected cells and showed that amino acids were incorporated into acid-precipitable material on the cytoplasmic polyribosomes. However, the nature of the polypeptides thus made was not studied.Experiments to be reported in this communication indicate that the proteins on cytoplasmic polyribosomes can be identified as viral structural proteins by immunological techniques, and that the newly synthesized viral structural proteins are transported rapidly from the cytoplasmic polyribosomes into the nucleus.Materials and Methods.-The methods for cell growth, viral propagation and purification, viral plaque assay, and extraction of DNA were described previously.12-15 Radioactive labeling of macromolecules: To deplete the amino acid pool before addition of isotope, the cells were resuspended in valine-free or amino-acid-free media supplemented with normal concentrations of tryptophan and glutamine and incubated for 1 hr at 36CC. C1l valine or C14-labeled amino acid mixture was then added and the short pulses were terminated by pouring the cell suspension onto an equal volume of partly frozen medium.Cell fractionation: Cytoplasmic extracts were prepared from 2-10 X 107 cells suspended in 2 ml of hypotonic buffer (RSB) 16 for 5 min and disrupted with five strokes in a tight-fitting Dounce homogenizer (85-90% of the cells were broken). In control experiments with infected or uninfected cells only 1-3% of H3-labeled DNA was released into the cytoplasmic extract by the homogenization procedure. Nuclei devoid of cytoplasmic contamination were prepared with sodium deoxycholate (DOC) and Tween 40 according to Penman's procedure. 17 The detergent released on additional 1-3% of H'-labeled DNA from the nuclei; a similar percentage of nuclei were disrupted.
The 57,000to 65,000-dalton (Da) Marek's disease herpesvirus A (MDHV-A) antigen glycoprotein (gp57-65) has a 47,000-Da unglycosylated precursor polypeptide (pr47), as determined by immunological detection after cell-free translation of infected-cell mRNA. Cleavage of its signal peptide yielded a 44,000-Da precursor polypeptide molecule (pr44), detected both in vivo after tunicamycin inhibition of glycosylation and in vitro after dog pancreas microsome processing of pr47. High-resolution pulse-chase studies showed that pr44 was quickly glycosylated (within 1 min) to nearly full size, a rapid processing time consistent with a cotranslational mode of glycosylation. This major glycosylation intermediate was further modified 6 to 30 min postsynthesis (including the addition of sialic acid), and mature MDHV-A was secreted 30 to 120 min postsynthesis. Limited apparent secretion of pr44 occurred only in the first minute postsynthesis, in contrast to the later secretion of most of the MDHV-A polypeptide as the fully glycosylated form described above. In addition, in the presence of tunicamycin a small fraction of the newly synthesized MDHV-A protein appeared as a secreted, partially glycosylated, heterogeneously sized precursor larger than pr44. pr44 constituted the major fraction of the new MDHV-A made in the presence of the inhibitor but the precursor was smaller than mature MDHV-A. These data indicate that there is a minor glycosylation pathway not sensitive to tunicamycin and that "normal" glycosylation is not necessary for secretion. Collectively, the data demonstrate that the rapid release of most of the fully glycosylated form of MHDV-A from the cell shortly after synthesis is true secretion in a well-regulated and precisely programmed way and not the result of cell death and disruption.
The Marek's disease herpesvirus (MDHV) B antigen (MDHV-B) was identified and molecularly characterized as a set of three glycoproteins of 100,000, 60,000, and 49,000 apparent molecular weight (gplOO, gp6O, and gp49, respectively) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after immu-noprecipitation from [35S]methionine-labeled infected cells by specific rabbit antiserum directed against MDHV-B (RaB), as previously determined by immunodiffusion. Further identification was accomplished by blocking this immunoprecipitation with highly purified MDHV-B. The same set of three polypeptides was also immunoprecipitated from [35S]methionineand 14C-labeled infected cells with two other sera shown to have anti-B activity, i.e., rabbit anti-MDHV-infected-cell plasma membrane (RaLPM) and immune chicken serum from birds naturally infected with MDHV. The three herpesvirus of turkeys (HVT) B-antigen (HVT-B) glycoproteins immunoprecipitated with all three sera containing anti-B activity were also shown to be identical in size to those of MDHV-B by immunoprecipitation and SDS-PAGE. These data serve to clarify the molecular identification of the polypeptides found in common between MDHV and HVT by linking them to MDHV-B, previously identified only by immunodiffusion, and to a similarly sized set of immunologically related common glycoproteins called gplOO, gp6O, and gp49, detected with monoclonal antibody by other workers. Tunicamycin inhibition of N-linked glycosylation resulted in either nonglycosylated or 0-linked glycosylated putative precursors of MDHV-B and HVT-B with apparent molecular weights of 88,000, called pr88, and 44,000, tentatively called pr44, both immunoprecipitable with all three sera. However, the relationships of these two polypeptides to each other and to the overall precursor-processing relationship of the MDHV-B complex remains to be elucidated. The three fully glycosylated B-antigen polypeptides were not connected by disulfide linkage. Collectively, the data presented here and by others support the conclusion that all three glycoproteins now identified as gplOO, gp6O, and gp49 have MDHV-B determinants. Finally, detection of the same three polypeptides with well-absorbed RaPM, which was directed against purified infected-cell plasma membranes, suggests that at least one component of the B-antigen complex has a plasma membrane location in the infected cell. These preliminary data point to the future membrane biochemistry and membrane immunology experiments needed to understand the MDHV system, and they may explain the high level of immunogenicity of MDHV-B in the infected chicken, as shown by its immunoprecipitation with immune chicken serum.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.