Marke''s disease herpesviruses. III. Purification and characterization of Marek''s disease herpesvirus B antigen.

Sera from chickens naturally infected with Marek's disease herpesvirus (MDHV) form preciptin lines with at least two immunologically distinct soluble antigens designated MDHV-A and MDHV-B. Partial purification and characterization of the glycoprotein MDHV-A antigen was previously reported. MDHV-B was found predominantly in the sonically treated extracts of infected cells, in contrast to the predominantly extracellular MDHV-A. Analysis of these extracts from [14C]glucosamine-labeled cells by immunodiffusion with chicken anti MDHV-B serum negative for MDHV-A followed by autoradiography confirmed that MDHV-B was a common antigen between MDHV and herpesvirus of turkeys and revealed that it was also a glycoprotein. Because of their glycoprotein nature, both MDHV-A and MDHV-B bound to concanavalin A affinity chromatography columns and could then be eluted by alpha-methyl-D-mannoside and recovered for further analysis. Concanavalin A affinity chromatography was an excellent technique for initial purification of MDHV-A and MDHV-B, since approximately 5- and 15- fold purification, respectively, was achieved in a single simple step. MDHV-B was resistant to trypsin under conditions where MDHV-A was sensitive, but was similar to MDHV-A in resistance to pH 2.0 and to 1.0 or 2.0 M urea and 0.05% Brij 35. Partially purified MDHV-B was analyzed by sucrose gradient sedimentation, isoelectric focusing, and gel filtration on Sephadex G-200 in the presence of 1.0 or 2.0 M urea and 0.05% Brij 35 to purify the antigen and to determine its physical and chemical properties in comparison with those already reported for MDHV-A. MDHV-B had a much lower isoelectric point in pH 4,54, a higher sedimentation coefficient of 4.4S, and a greater molecular weight of 58,250. These data indicate that MDHV-B is physically distinct from MDHV-A antigen, although the size difference is not sufficient to allow for effective separation. In contrast, the isoelectric point difference of greater than 2 pH units makes isoelectric focusing an effective means of purifying the antigens free of one another. The four-step purification procedure achieved greater than 200-fold purification of MDHV-B. Immunization of rabbits with this highly purified antigen results in the preparation of antisera that appeared monospecific for MDHV-B in immunodiffusion.     

Identification of the gene encoding Marek''s disease herpesvirus A antigen.

The gene encoding the glycoprotein Marek's disease herpesvirus A antigen (MDHV-A) precursor polypeptide pr47 was delineated by using Northern blot (RNA blot) analysis and hybrid selection of its mRNA with cloned MDHV DNA, cell-free translation of the mRNA, and immunoprecipitation of the polypeptide. The resulting piece of DNA with strongly positive hybrid selection results was a 2.2-kilobase-pair (kbp) PvuII-EcoRI restriction fragment localized to the center of the 18.3-kbp MDHV BamHI B fragment of the total virus genome. The localization was specific since no other small restriction subfragment of the larger BamHI B fragment was able to hybrid select significant MDHV-A mRNA and the gene mapped only in the BamHI B fragment of the total virus genome. Northern blot analysis confirmed the localization of the MDHV-A gene on the 2.2-kbp fragment and detected its mRNA as a 1.8-kilobase species, a size consistent with encoding a 47-kilodalton polypeptide. This is the first report of an MDHV gene being mapped to the MDHV viral genome. This opens the way for the use of recombinant DNA technology to study the nature of the gene encoding a secreted virus-specific glycoprotein that could possibly be involved in immunoprevention, immunosuppression, or immunoevasion, immune phenomena known or speculated to be involved in this oncogenic herpesvirus system.     

Molecular characterization of Marek's disease herpesvirus B antigen.

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 (gp100, gp60, and gp49, respectively) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after immunoprecipitation from [35S]methionine-labeled infected cells by specific rabbit antiserum directed against MDHV-B (R alpha B), 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]methionine- and 14C-labeled infected cells with two other sera shown to have anti-B activity, i.e., rabbit anti-MDHV-infected-cell plasma membrane (R alpha PM) 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 gp100, gp60, and gp49, detected with monoclonal antibody by other workers. Tunicamycin inhibition of N-linked glycosylation resulted in either nonglycosylated or O-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 gp100, gp60, and gp49 have MDHV-B determinants. Finally, detection of the same three polypeptides with well-absorbed R alpha PM, 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.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis, processing, and secretion of the Marek''s disease herpesvirus A antigen glycoprotein.

The 57,000- to 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.     

Synthesis and processing of the Marek''s disease herpesvirus B antigen glycoprotein complex.

The Marek's disease herpesvirus B antigen (MDHV-B) complex was previously immunologically identified and molecularly characterized as a set of three glycoproteins designated gp100, gp60, and gp49 on the basis of apparent molecular weight and immunoprecipitation with both polyclonal and monoclonal antibodies. Immunoprecipitation analysis, previously with polyclonal and more recently with monoclonal antibodies, of infected cell lysates labeled with [35S]methionine in the presence of tunicamycin, an inhibitor of N-linked glycosylation, revealed two putative precursor molecules of 88,000 daltons (pr88) and 44,000 daltons (pr44). High-resolution pulse-chase studies revealed that gp100 was a glycosylated intermediate which was processed to yield gp60 and gp49. This cleavage was inhibited by monensin, an inhibitor of glycoprotein processing. Endo-beta-N-acetylglucosaminidases F and H (endo-F, endo-H) reduced gp100 to pr88, indicating that the latter is an intermediate in the biosynthetic pathway. These same enzymes reduced gp49, and to a lesser extent gp60, to pr44, suggesting that pr44 is their polypeptide backbone. Significant support for this concept is the fact that the same monoclonal antibody recognized all three molecules, gp60, gp49, and pr44. In the presence of monensin, terminal addition of complex sugars was also prevented, since gp60 was replaced by a slightly faster migrating component which was insensitive to both endo-F and endo-H. Cell-free translation of infected-cell mRNA, followed by immunoprecipitation analysis with either polyclonal or monoclonal antibody, resulted in detection of a putative unglycosylated precursor polypeptide of 44,000 daltons. Since pr88 was not the initial precursor polypeptide of the MDHV-B complex, its existence may have resulted from dimerization of pr44. Again, detection of both pr88 and pr44 with the same monoclonal antibody is consistent with this interpretation. These collective data obtained from the cell-free and in vivo studies with polyclonal and monoclonal antibodies reactive with MDHV-B are consistent with the concept that pr44, the initial gene product, dimerizes to form pr88 and demonstrate that pr88 is actually a processing intermediate glycosylated to gp100, another processing intermediate, which is then processed to gp60 and gp49.     

Transformation of T-lymphocyte subsets by Marek''s disease herpesvirus.

Marek's disease herpesvirus (MDV)-transformed lymphoblastoid tumor cell lines were characterized for the presence of the surface markers. Monoclonal antibodies were used for CD3 (T-cell receptor [TCR] complex), TCR1, TCR2, and TCR3, CD4, CD8, and Ia antigen by indirect fluorescence staining followed by microscopic examination or flow cytometry. The lymphoblastoid cell lines were obtained from tumors from chickens infected with MDV (n = 44) or from local lesions induced by inoculation of allogeneic, MDV-infected chick kidney cells (n = 56). Lymphocytes were harvested from these lesions between 4 and 16 days postinoculation and cultured in vitro to establish cell lines. All cell lines expressed Ia antigen and CD3 and/or TCR and thus are activated T cells. Most of the cell lines developed from tumors were CD4+ CD8-; only one cell line was negative for both markers. Sixteen percent of the cell lines were TCR3+, while the remainder were TCR2+. The cell lines developed from local lesions were much more heterogeneous: 45% were CD4- CD8+, 34% were CD4- CD8-, and only 21% were CD4+ CD8-. The number of TCR3+ cell lines was larger than expected for the CD4- CD8+ and CD4- CD8- cell lines, as judged from the presence of these cells in the blood. These results indicate that several subsets of T lymphocytes can be transformed by MDV, depending on the pathogenesis of infection. Activation of T cells as a consequence of the normal pathogenesis or by allogeneic stimulation seem to be a first important step in the process of transformation.     

Augmentation of retrovirus-induced lymphoid leukosis by Marek''s disease herpesviruses in White Leghorn chickens.

Our objective was to determine whether the cell-associated herpesvirus vaccines used in chickens to control Marek's disease tumors can augment development of lymphoid leukosis (LL) induced by exogenous avian leukosis virus (ALV). Various single or mixed Marek's disease vaccines were inoculated at day 1, and ALV was injected at 1 to 10 days, with chickens of several experimental or commercial strains. Development of LL was monitored at 16 to 48 weeks in various experiments. In several strains of chickens we repeatedly found that the widely used serotype 3 turkey herpesvirus vaccine did not augment LL in comparison with unvaccinated controls. However, LL development and incidence were prominently augmented in several chicken strains vaccinated with serotype 2 vaccines, used alone or as mixtures with other serotypes. In one chicken strain, augmentation was demonstrated after natural exposure to ALV or serotype 2 Marek's disease virus viremic shedder chickens. Augmentation of LL by virulent or attenuated Marek's disease viruses of serotype 1 was intermediate in effect. Serotype 2 Marek's disease virus augmentation of LL was prominent in three laboratory lines and one commercial strain of White Leghorns, but it was not observed in an LL-resistant laboratory line or four commercial strains susceptible to ALV infection. Chickens developed similar levels of viremia and neutralizing antibodies to ALV regardless of the presence of augmentation of LL, suggesting that the mechanism of enhanced LL did not result from differences in susceptibility or immune response to ALV. We postulate that the serotype 2 herpesviruses may augment LL through one of several possible influences on bursal cells that are subsequently transformed by exogenous ALV.     

Identification of Marek''s disease virus nuclear antigen in latently infected lymphoblastoid cells.

A new Marek's disease virus (MDV) nuclear antigen (MDNA) was identified in two MDV-transformed T-lymphoblastoid cell lines, MKT-1 and MSB-1, derived from chickens bearing tumors induced by MDV. This MDNA was not detected in MSB-1 cells maintained in iododeoxyuridine, which activates the latent MDV genome. Moreover, it was not found in chicken embryo fibroblasts undergoing productive and cytolytic infection with MDV. Expression of MDNA is not related to strain pathogenicity in chickens, because chicken embryo fibroblasts productively infected with the pathogenic RBIB strain or the nonpathogenic CV-1 strain of MDV did not express this antigen. DNA-protein immunoprecipitation studies revealed that MDNA bound to two sites in the 190,00-base-pair (bp) MDV genome. One of these loci identified by MDNA obtained from MKT-1 and MSB-1 cells corresponded to a 476-bp segment within the short unique region of BamHI-A MDV DNA. A second locus located in a 280-bp segment within the short inverted repeat region of BamHI-A was also identified by MDNA from MSB-1 cells but not by MDNA obtained from MKT-1 cells. Analyses of the nucleotide sequence by DNase digestion showed that MDNA protected a 60-bp segment spanning a 22-bp palindromic sequence of the short unique region and a 103-bp sequence encompassing a 32-bp palindrome in the short inverted repeat region of BamHI-A MDV DNA.     

A repeat sequence, GGGTTA, is shared by DNA of human herpesvirus 6 and Marek''s disease virus.

Some regions of the genomes of human B-lymphotrophic virus (HBLV), also designated as human herpesvirus 6, and Marek's disease virus were found to hybridize to each other under moderate to stringent conditions, scoring from 10 to 30% base-pair mismatch. Nucleotide sequence analysis showed that a 6-base-pair repetitive sequence, GGGTTA (DR2), present in the IRS-IRL junction region of the Marek's disease virus genome, was also reiterated in the HBLV genome. The function(s) of such a sequence is unknown, but this is the first report of homology between HBLV and a nonhuman herpesvirus.     

Sequential autoprocessing of Marek's disease herpesvirus protease differs from that of other herpesviruses

Herpesviruses encode a unique serine protease essential for viral capsid maturation. This protease undergoes autoprocessing at two sites, R and M, at the consensus sequence (V, L, I)(P3)-X(P2)-A(P1)/S(P1') (where X is a polar amino acid). We observed complete autoprocessing at the R and M sites of Marek's disease virus (MDV) protease following production of the polyprotein in Escherichia coli. Site-directed mutagenesis confirmed the predicted sequence of the R and M sites, with the M site sequence being nonconsensual: M(P3)-N(P2)-A(P1)/S(P1'). Mutagenesis and expression kinetics studies suggested that the atypical MDV M site was cleaved exclusively by the processed short protease, a feature making MDV unique among herpesviruses.     

Identification with monoclonal antibodies of glycoproteins of Marek''s disease virus and herpesvirus of turkeys related to virus neutralization.

By use of monoclonal antibodies cross-reactive with Marek's disease virus and herpesvirus of turkeys, three glycoproteins (for Marek's disease virus, gp115/110, gp63, and gp50; for herpesvirus of turkeys, gp115, gp62 and gp52) related to virus neutralization were identified. Immunization of chickens or rabbits with these glycoproteins purified by affinity chromatography resulted in production of neutralizing antibodies.     

Protection against Marek''s disease by a fowlpox virus recombinant expressing the glycoprotein B of Marek''s disease virus.

Fowlpox virus (FPV) recombinants expressing the glycoprotein B and the phosphorylated protein (pp38) of the GA strain of Marek's disease virus (MDV) were assayed for their ability to protect chickens against challenge with virulent MDV. The recombinant FPV expressing the glycoprotein B gene elicited neutralizing antibodies against MDV, significantly reduced the level of cell-associated viremia, and, similar to the conventional herpesvirus of turkeys, protected chickens against challenge with the GA strain and the highly virulent RB1B and Md5 strains of MDV. The recombinant FPV expressing the pp38 gene failed to either elicit neutralizing antibodies against MDV or protect the vaccinated chickens against challenge with MDV.     

Conservation of gene organization in the lymphotropic herpesviruses herpesvirus Saimiri and Epstein-Barr virus.

By analyses of short DNA sequences, we have deduced the overall arrangement of genes in the (A + T)-rich coding sequences of herpesvirus saimiri (HVS) relative to the arrangements of homologous genes in the (G + C)-rich coding sequences of the Epstein-Barr virus (EBV) genome and the (A + T)-rich sequences of the varicella-zoster virus (VZV) genome. Fragments of HVS DNA from 13 separate sites within the 111 kilobase pairs of the light DNA coding sequences of the genome were subcloned into M13 vectors, and sequences of up to 350 bases were determined from each of these sites. Amino acid sequences predicted for fragments of open reading frames defined by these sequences were compared with a library of the protein sequences of major open reading frames predicted from the complete DNA sequences of VZV and EBV. Of the 13 short amino acid sequences obtained from HVS, only 3 were recognizably homologous to proteins encoded by VZV, but all 13 HVS sequences were unambiguously homologous to gene products encoded by EBV. The HVS reading frames identified by this method included homologs of the major capsid polypeptides, glycoprotein H, the major nonstructural DNA-binding protein, thymidine kinase, and the homolog of the regulatory gene product of the BMLF1 reading frame of EBV. Locally as well as globally, the order and relative orientation of these genes resembled that of their homologs on the EBV genome. Despite the major differences in their nucleotide compositions and in the nature and arrangements of reiterated DNA sequences, the genomes of the lymphotropic herpesviruses HVS and EBV encode closely related proteins, and they share a common organization of these coding sequences which differs from that of the neurotropic herpesviruses, VZV and herpes simplex virus.     

Molecular characterization of a mucin-type antigen associated with human pancreatic cancer. The DU-PAN-2 antigen

This work describes the molecular characterization of a human pancreatic cancer-associated antigen defined by a murine monoclonal antibody (DU-PAN-2). DU-PAN-2 antigen was isolated from a pancreatic adenocarcinoma cell line (HPAF) or patient's ascitic fluid, and the antigenic activity was monitored by competitive inhibition radioimmunoassay. Affinity chromatography and CsCl/guanidine HCl density gradient centrifugation were employed to remove other populations of mucin-type glycoproteins and noncovalently associated proteins, respectively. Three electrophoretically distinct components were detected by 1% agarose gel electrophoresis and were resolved by chromatography on Sepharose CL-4B. The major fraction (FII) was subjected to carbohydrate and amino acid analyses. The sum of threonine, serine, proline, glycine, and alanine comprised more than 50% of the amino acid residues. The saccharide units, O-glycosidically linked to the peptide via GalNAc, contained fucose, galactose, GlcNAc, GalNAc, and sialic acid. The total carbohydrate content of FI and FII was 80.8% and 77.4% by weight, respectively. The molecular weight of FII antigen showed two species of molecules of 1.45 X 10(6) and 4.59 X 10(6) by analytical sedimentation equilibrium. DU-PAN-2 antigen was susceptible to neuraminidase, pepsin, Pronase, and papain digestion. These results suggest that both protein components and sialic acid residues may play important roles in the binding of DU-PAN-2 antibody.     

Map location of homologous regions between Marek''s disease virus and herpesvirus of turkey and the absence of detectable homology in the putative tumor-inducing gene.

The DNA region having homology between Marek's disease virus and herpesvirus of turkey was assigned to the restriction map of Marek's disease virus by Southern blot hybridization. Under moderate conditions at the level of 15% mismatching, homology was found to be distributed throughout the Marek's disease virus genome. The long inverted-repeat regions (TRL and IRL), which are considered to play a significant role in tumorigenicity, did not show any homology to herpesvirus of turkey DNA.     

Molecular cloning and characterization of form I antigen genes of Shigella sonnei.

Sau3AI-generated DNA fragments of the Shigella sonnei large plasmid encoding the form I antigen were cloned into Escherichia coli with cosmid vector pHSG262. One resulting plasmid, designated pJK1137, was studied further. Restriction endonuclease mapping and analysis of transposon Tn3 insertion mutants demonstrated that the form I antigen genes were located within a region of about 12.6 kb consisting of the two contiguous HindIII fragments of 1.26 kb and 12.4 kb. The results of complementation studies between Tn3 insertion mutants of pJK1137 and recombinant plasmids carrying different parts of the form I antigen genes indicated that the 12.6 kb DNA sequence contained at least four gene clusters, regions A, B, C and D. Analysis of radioactively labelled proteins in minicells demonstrated that the DNA sequence of about 12.6 kb coded for at least four specific proteins of 42, 23, 48 and 39 kDa. The former two were coded by region A, the latter two by region D.     

High-frequency antigens of human erythrocyte membrane sialoglycoproteins, IV. Molecular properties of the U antigen

The nature of the common erythrocyte antigen U, that is absent from S-s-U-cells, which lack glycophorin B (Ss sialoglycoprotein), was investigated using six different antisera. The molecular features of a U-like antigen (Duclos), detected by a hitherto unique serum, were also studied. The U and Duclos antigens are complex in that they exhibit relationships with the MNSs and Rh blood group systems. Various fractionation, cleavage, or modification products of normal erythrocyte membranes were used in hemagglutination inhibition assays. Both, the U and Duclos antigens were found to represent labile structures that require lipids, at least for optimum expression of antigen activity. The antigens could be solubilized using conditions of Triton X-100 extraction that release glycophorin B, but solubilize the Rh antigens only to a small extent. Anti-U and anti-Duclos were also inhibited, albeit weakly, by glycophorin B-containing fractions obtained by chromatographic separation of Triton X-100 extracts. The residues approx. 33-39 of glycophorin B represent essential parts of the U antigen, as judged from proteolytic digestion and chemical modification. Conversely, the expression of Duclos activity seems to require a region of glycophorin B (C-terminal of the positions approx. 34-36) that could not be cleaved by various proteinases. Data obtained with anti-Duclos have to be interpreted with caution, since there is evidence that this serum might contain a mixture of antibodies.