Location of the structural genes for glycoproteins gD and gE and for other polypeptides in the S component of herpes simplex virus type 1 DNA.

To map the structural genes for the gD and gE polypeptides and for other viral products encoded in the S component of herpes simplex virus type 1 DNA, we selected mRNAs capable of hybridizing to cloned viral DNA fragments and translated the mRNAs in vitro to determine which polypeptides were encoded therein. The gD and gE polypeptides were identified by immunoprecipitation with appropriate monoclonal and monospecific antibodies, whereas the other polypeptides were characterized only by their electrophoretic mobilities in polyacrylamide gels. We found that gD mRNA hybridized to a single SacI subfragment of BamHI fragment J, whereas gE mRNA hybridized to an adjacent SacI subfragment of BamHI fragment J and also to BamHI fragment X. These and other results permit the conclusion that the structural gene for gD is located between map coordinates 0.911 and 0.924, and the gene for gE is between map coordinates 0.924 and 0.951. We also found that mRNAs for polypeptides of 55,000, 42,000, 33,000, and 22,000 molecular weight hybridized to DNA fragments spanning the regions from map coordinates 0.911 to 0.924, 0.897 to 0.911, 0.939 to 0.965, and 0.939 to 0.965, respectively. Finally, in accord with the results of others, we found that mRNA for a 68,000-molecular-weight polypeptide hybridized to the two noncontiguous BamHI fragments N and Z, which share a reiterated DNA sequence.     

The translational map of the Autographa californica nuclear polyhedrosis virus (AcNPV) genome.

We have mapped early and late viral gene products expressed in Autographa californica nuclear polyhedrosis virus ( AcNPV )-infected Spodoptera frugiperda cells by cell-free translation of virus-specific RNA which was selected by hybridization to cloned restriction endonuclease fragments of AcNPV DNA. Proteins synthesized in vitro were labeled with [35S]methionine and analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography. At least four early AcNPV -specific polypeptides were found which mapped in two regions of the genome (9-25 and 43-59 map units). These early mRNAs are also synthesized at late times in the infection cycle. Cell-free translation of restriction fragment-selected late AcNPV -specific RNA (24 h post-infection) resulted in the identification and mapping of 24 viral proteins. Curiously, the region between approximately 70 and 80 map units on the viral genome has been found silent with respect to mRNA which is translatable in a cell-free system. However, there may be RNA transcribed from this viral DNA segment.     

Identification of polypeptide components of the Epstein-Barr virus early antigen complex with monoclonal antibodies.

Three monoclonal antibodies were produced against the Epstein-Barr virus-induced early antigen complex. These antibodies were shown to be specific for the early antigen complex by the fact that they only reacted with cells supporting a permissive or abortive Epstein-Barr virus infection and their synthesis was not affected by inhibitors of viral DNA synthesis. One monoclonal antibody, designated R3, was directed against a diffuse component of the early antigen complex since it reacted by immunofluorescence with cells fixed in acetone or methanol. The other two monoclonal antibodies, designated K8 and K9, reacted with a methanol-sensitive restricted component of this complex. The appearance of the R3 antigen in P3HR-1 superinfected Raji cells occurred approximately 4 h earlier than the antigen detected by K8. By both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioimmunoelectrophoresis, it was determined that the R3 monoclonal antibody recognized two major polypeptides with molecular weights of approximately 50,000 to 52,000, whereas K8 and K9 precipitated a protein of approximately 85,000. The R3 monoclonal antibody also immunoprecipitated an in vitro primary translation product. It was, therefore, possible to map this product to the Epstein-Barr virus DNA BamH1 M fragment. These in vitro products were slightly smaller than the in vivo proteins, suggesting that these proteins probably undergo posttranslational modification during the virus replication cycle.     

Hybridization selection and cell-free translation of mRNA''s encoded within the inverted terminal repetition of the vaccinia virus genome

Early polypeptides encoded within the 10,000-base pair terminally repeated region of the vaccinia virus genome were mapped by cell-free translation of mRNA that was selected by hybridization to restriction fragments and to separated strands of a recombinant lambda phage. The results, which were confirmed by hybrid arrest of translation, indicated that polypeptides of 7,500 (7.5K), 19,000 (19K), and 42,000 (42K) daltons mapped at approximately 3.2 to 4.3, 6.5 to 7.2, and 7.2 to 8.3 kilobase pairs from the end of the genome, respectively. mRNA's for the 42K and 7.5K polypeptides were transcribed towards the end of the genome, whereas mRNA for the 19K polypeptide was transcribed in the opposite direction. Including polyadenylic acid tails, the lengths of the mRNA's for the 7.5K, 19K, and 42K polypeptides, determined by gel electrophoresis of denatured RNA, hybridization selection, and cell-free translation, were approximately 1,200, 680, and 1,280 nucleotides, respectively. mRNA's for the 42K and 19K polypeptides were only about 100 nucleotides longer than the minimums required to code for their respective polypeptides, whereas mRNA for the 7.5K polypeptide contained 900 nucleotides of untranslated sequence. This long untranslated portion of the latter mRNA was probably located near the 3' end, because this gene was only inactivated by high doses of UV irradiation. This small target size also excluded certain models for RNA processing involving formation of the mRNA's for the 42K and 7.5K polypeptides from a common promoter. Rabbitpox virus, which has an inverted terminal repetition approximately half that of vaccinia virus, was also shown to encode mRNA's that hybridized to the cloned terminal segment of vaccinia virus DNA.     

Internal initiation of translation on the vesicular stomatitis virus phosphoprotein mRNA yields a second protein.

In vitro translation of a mixture of the vesicular stomatitis virus (VSV) polyadenylated mRNAs yielded a previously undetected protein with a molecular weight of approximately 7,000 (7K protein). Hybrid-arrested translation demonstrated that both the 7K protein and the VSV phosphoprotein (P protein) were encoded by the P protein message. Immunoprecipitation of the 7K protein with monoclonal antiserum directed against the P protein indicated that the two products were encoded in the same open reading frame. A protein of approximately the same size was immunoprecipitated from cytoplasmic extracts of VSV-infected cells by both the polyclonal and monoclonal antisera, and it is likely that it was a previously unrecognized viral gene product. Translational mapping of the P protein mRNA in vitro indicated that the 7K protein was encoded in the 3' one-third of the sequence. The synthesis of the 7K protein in vitro was unaffected by hybrid arrest conditions which blocked the 5' two-thirds of the mRNA and inhibited synthesis of the P protein. These results imply that the ribosomes bind and initiate translation internally on the P protein mRNA at a site located hundreds of nucleotides downstream from the capped 5' end.     

Characterization of Measles Virus-Specific Proteins Synthesized In Vivo and In Vitro from Acutely and Persistently Infected Cells

Measles virus protein synthesis has been analyzed in acutely and persistently infected cells. To assess the role of measles in subacute sclerosing panencephalitis (SSPE), measles viral proteins synthesized in vivo or in vitro were tested for reactivity with serum from a guinea pig(s) immunized with measles virus and sera from patients with SSPE. Guinea pig antimeasles virus serum immunoprecipitates the viral polypeptides of 78,000 molecular weight (glycosylated [G]), 70,000 molecular weight (phosphorylated [P]), 60,000 molecular weight (nucleocapsid [N]), and 35,000 molecular weight (matrix [M]) from cells acutely infected with measles virus as well as from chronically infected cells, but in the latter case, immunoprecipitated M protein has a reduced electrophoretic migration. Sera of SSPE patients immunoprecipitated all but the G protein in acutely infected cells and only the P and N proteins from chronically infected cells. In immunoprecipitates of viral polypeptides synthesized in a reticulocyte cell-free translation system, in response to mRNA from acutely or persistently infected cells, the 78,000-molecular-weight form of the G protein was not detected among the cell-free products of either mRNA. Guinea pig antimeasles virus serum immunoprecipitated P, N, and M polypeptides from the products of either form of mRNA, whereas SSPE serum immunoprecipitated the P and N polypeptides but not the M polypeptide. The differences in immunoreactivity of the antimeasles virus antiserum and the SSPE serum are discussed in terms of possible modifications of measles virus proteins in SSPE.     

Varicella-zoster viral glycoproteins analyzed with monoclonal antibodies.

Monoclonal antibodies to varicella-zoster virus were used to study viral glycoproteins by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on the viral glycoproteins immunoprecipitated, the five monoclonal antibodies fell into three groups. Two antibodies, 4B7 and 8G9 (group 1), immunoprecipitated a single glycoprotein of molecular weight (MW) 118,000 (118K glycoprotein) and had high neutralizing activity in the absence of complement. One antibody, 3C7 (group 2), which lacked neutralizing activity, immunoprecipitated two glycoproteins of MWs 120,000 and 118,000 and a glycoprotein giving a diffuse band in the region of 64,000 to 65,000. Pulse-chase experiments and experiments with monensin as an inhibitor of glycosylation suggested that the 120K polypeptide was derived by glycosylation of the 118K polypeptide and that a 43K antigen was processed into the 64 to 65K glycoprotein. Two antibodies, 3G8 and 4E6 (group 3), both had neutralizing activity only in the presence of complement, and both immunoprecipitated at least five polypeptides, with MWs ranging from 50,000 to 90,000. Antibody 3G8 was isotype immunoglobulin G2b (IgG2b), and its immunoprecipitating activity was stronger than that of 4E6, which was isotype IgG1. Pulse-chase experiments with antibody 3G8 showed that lower-MW glycopeptides chased into three polypeptides of MWs 90,000, 80,000, and 60,000 by 24 h. Immunoprecipitation experiments with antibody 3G8 on infected cells treated with glycosylation inhibitors 2-deoxyglucose, monensin, and tunicamycin, suggested that a prominent, early-appearing 70K polypeptide may have been processed into the glycoproteins of higher MWs and that the 60K polypeptide may have been derived by glycosylation of polypeptides of lower MWs.     

Two developmentally regulated messenger RNAs differing in their coding region may exist for the myelin-associated glycoprotein

Mouse and rat brain RNA were translated in vitro in a rabbit reticulocyte lysate translation system, and myelin-associated glycoprotein-related polypeptides were immunoprecipitated. Two products of Mr = 72,000 (p72MAG) and Mr = 67,000 (p67MAG) were specifically immunoprecipitated. These polypeptides were characterized as follows: 1) exogenously added purified myelin-associated glycoprotein (MAG) competitively eliminates their immunoprecipitation; 2) peptide maps obtained from them are very similar; 3) they are both destined to become glycoproteins, as assayed by insertion into dog pancreas microsomes and their subsequent sensitivity to endoglycosidase H. Translatable mRNA for the two polypeptides have different developmental expressions. p72MAG mRNA appears at an earlier age than p67MAG mRNA and remains the dominant MAG mRNA species throughout the period of rapid myelination. As the rate of myelination decreases, p67MAG mRNA becomes the dominant MAG mRNA species. Finally, two endoglycosidase H-sensitive polypeptides were specifically immunoprecipitated from mouse brain microsomes. Therefore, there exist two MAG proteins that differ slightly in their polypeptide structure and that are developmentally regulated. The possibility that the two polypeptides are synthesized de novo from two coordinately regulated mRNAs that differ in their coding region is discussed.     

Characterization of the major immediate-early polypeptides encoded by murine cytomegalovirus.

The immediate-early (IE) infected cell proteins induced by the murine cytomegalovirus (Smith strain) were studied. These polypeptides were identified as IE proteins by their synthesis in the presence of actinomycin D after removal from a protein synthesis block mediated by cycloheximide. By using a murine antiserum against murine cytomegalovirus, three abundant polypeptides of 89, 84, and 76 kilodaltons (kd) were immunoprecipitated. The three major proteins are phosphorylated but not glycosylated and share antigenic determinants recognized by monoclonal antibodies. The 84 and 76-kd polypeptides represent post-translational modification products of the 89-kd protein. Accordingly, in vitro translation of IE infected cell RNA revealed only the 89-kd polypeptide. The viral origin of the RNA species directing the synthesis of the major 89-kd IE polypeptide was verified by hybrid selection of IE RNA with DNA fragments representing the region from 0.769 to 0.815 map units of the murine cytomegalovirus genome. IE polypeptides were found to be located in the nuclei and the cytoplasm of infected cells. Studies on the kinetics of IE polypeptide synthesis revealed negative regulatory effects on IE gene expression correlated with the synthesis of early proteins.     

Application of denatured, electrophoretically separated, and immobilized lysates of herpes simplex virus-infected cells for detection of monoclonal antibodies and for studies of the properties of viral proteins

We report the use of herpes simplex virus type 1 (HSV-1)- and HSV-2-infected cell polypeptides (ICPs) separated by electrophoresis in polyacrylamide gels and transferred to nitrocellulose to (i) detect monoclonal antibodies to viral polypeptides and to (ii) study the properties of the proteins with the monoclonal antibodies. Our results were as follows. (i) When the antigens were electrophoretically separated in denaturing gels and then immobilized on nitrocellulose strips, we detected a greater diversity of monoclonal antibodies to viral proteins than when we used the technique of immune precipitation of soluble, nondenatured viral antigens. The primary advantage of the technique is in the detection of nonprecipitating antibody and of antibody to poorly soluble antigens not available for reaction in preparations cleared by high-speed centrifugation before immune reaction. (ii) Studies of the viral polypeptides reactive with three monoclonal antibodies indicated that the technique can be used to investigate several properties of the antigens. Specifically, monoclonal antibody to ICP 4 confirmed the accumulation of viral protein in the nucleus and the mapping of the gene in the S component. The results showed, however, that HSV-1 and HSV-2 ICP 4 do have common antigenic determinants. The reaction of a nonprecipitating monoclonal antibody with electrophoretically separated, immobilized polypeptides contained in cytoplasmic and nuclear fractions, those chemically deglycosylated, or those specified by specific HSV-1 x HSV-2 intertypic recombinants identified the antigens reactive with the second monoclonal antibody as various forms of glycoprotein gC. Of particular interest was a set of four antigens, 39,000 to 46,500 in apparent molecular weight, reactive with each of several monoclonal antibodies. These studies showed that two polypeptides partition in the cytoplasm and two in the nucleus and that all comap with the previously mapped ICPs 35 and 37 in the region of the genome defined by the viral thymidine kinase gene on the left and the glycoprotein gA/B gene on the right. Unlike ICP 4 and gC, the four polypeptides are linked by intermolecular bisulfide bonds, inasmuch as the polypeptides were not at the expected locations upon denaturation and electrophoresis in the absence of reducing agents.     

Transcriptional and translational mapping and nucleotide sequence analysis of a vaccinia virus gene encoding the precursor of the major core polypeptide 4b

We prepared antiserum that reacted with a major core polypeptide of approximately 62,000 daltons (62K polypeptide), designated 4b, and its 74K precursor, designated P4b. A cell-free translation product of vaccinia virus late mRNA that comigrated with P4b was specifically immunoprecipitated. The late mRNA encoding P4b hybridized to restriction fragments derived from the left end of the HindIII A fragment and to a lesser extent from the right side of the HindIII D fragment. A polypeptide that comigrated with P4a, the precursor of another major core polypeptide, was synthesized by mRNA that hybridized to DNA segments upstream of the P4b gene. Complete nucleotide sequence analysis of the P4b gene revealed an open reading frame, entirely within the HindIII A fragment, that was sufficient to encode a 644-amino-acid polypeptide of 73K. The 5' end of the P4b mRNA was located at or just above the translational initiation site.     

牛病毒性腹泻病毒单克隆抗体的研究

牛病毒性腹泻——粘膜病是世界性广泛流行的奶牛和肉牛的传染病。其病原为牛病毒性腹泻病毒(BVDV),属于披膜病毒科的瘟病毒属,它的许多生物学特性至今还不很清楚。本试验建立了12株分泌抗BVDV的单克隆抗体(McAb)杂交瘤细胞株,并结合免疫转移电泳法和放射免疫沉淀法,初步研究了BVDV的多肽。