Analysis of structural proteins and products of cell-free translation of vaccinia virus of mRNA using mono-specific antibodies

The monospecific antiserums to the major proteins p12, p19, p20, p42, p61 of the vaccinia virus coat were obtained and analyzed. The dynamics of the proteins accumulation in the infected cells has been studied. Products of the cell-free translation of the total viral mRNA precipitated by the obtained antiserums were identified. The p20 protein has been found to be a result of p12 protein reversible oligomerization under the conditions of electrophoresis. The antigenic relation of p12 and p20 proteins to a p42 protein, also a p12 oligomer, has been demonstrated. The possibility is discussed to use the obtained antiserums for mapping of the genes corresponding to structural proteins in the genome of the vaccinia virus.     

Mapping the genes of the vaccinia virus, coding membrane proteins p34 and p40, by mRNA hybridization selection]

The HindIII--J HindIII-F fragments of the vaccinia virus DNA strain Lister have been analysed by the technique of mRNA hybridization selection with the subsequent translation in cell-free protein synthesizing system from the rabbit reticulocytes. The viral mRNA hybridizable with the HindIII--J fragment was shown to direct the synthesis of 30 kDa polypeptide in the cell-free system. This polypeptide was demonstrated to react specifically with antiserum to plasma membrane protein p34. The viral mRNA hybridizable with the HindIII-F fragment was shown to direct the synthesis of 37 kDa polypeptide in the cell-free system. This polypeptide reacts specifically with antiserum to major membrane protein p40.     

In vitro translation of adenovirus type 12-specific mRNA isolated from infected and transformed cells.

The early and late gene products of human adenovirus type 12 (Ad12), as well as the viral proteins synthesized in an Ad12-transformed cell line, were identified by translation of viral mRNA in an in vitro protein-synthesizing system. Cytoplasmic RNA was isolated from permissive KB or nonpermissive BHK cells infected with Ad12 and from Ad12-transformed HA12/7 cells. Virus-specific RNA was selected by hybridization to Ad12 DNA covalently bound to cellulose. Viral RNA was then translated in a fractionated rabbit reticulocyte cell-free system or in wheat germ S-30 extracts. The proteins synthesized were characterized by immunoprecipitation and subsequent electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. RNA prepared from KB cells late after infection with Ad12 elicited the synthesis of most of the structural polypeptides of the virion and at least two presumably nonstructural Ad12 proteins. When viral RNA isolated early after infection of KB cells with Ad12 was translated in vitro, 10 polypeptides were observed: E-68K, E-50K, E-42K, E-39K, E-34K, E-21K, E-19K, E-13K, E-12K, and E-10K. Ad12-specific RNA was also isolated from the Ad12-transformed hamster cell line HA12/7, which contains several copies of the Ad12 genome integrated in the host genome. The RNA codes for at least seven polypeptides with molecular weights very similar to those of the early viral proteins.     

Isolation and characteristics of vaccinia virus proteins associated with cell membranes of infected cells]

The proteins of vaccinia virus associated with plasma membrane infected cells BHK-21, p60, p45, p42, p40,p35,p34,p28,p23 were isolated from plasma membranes using affinity chromatography, gel-electrophoresis and passive elution. An immunochemical characterization was carried out using specific antiserum to these proteins. Investigation of temporal regulation of proteins synthesis in infected cells showed that proteins p60, p45, p42, p40, p28 were late, and p35, p34, p23--early-late proteins. Immunochemical analysis of vaccinia virus mRNA cell-free translational products was carried out using specific antiserum. The polypeptide-precursors of viral proteins were identified.     

Mapping and identification of the vaccinia virus thymidine kinase gene

The thymidine kinase gene of vaccinia virus (VV) was mapped on the viral genome by using cloned fragments of the viral DNA to hybridize to early viral mRNA. Individual DNA fragments that represented about half of the viral genome were assayed, both for their ability to arrest the cell-free synthesis of active VV thymidine kinase and for their ability to select functional mRNA for the viral enzyme. Both activities were located in HindIII fragment J, which maps near the middle of VV DNA and contains about 2.6% of the genome (4,800 base pairs). This DNA fragment encodes four known early polypeptides, and to determine which of these was thymidine kinase, early VV mRNA was fractionated by sucrose gradient centrifugation and used to direct cell-free synthesis of the active enzyme. The thymidine kinase mRNA cosedimented with several species that encoded polypeptides in the molecular weight range 15,000 to 25,000. Hybridization of these mRNAs to HindIII-J DNA selected a message that directed the synthesis of thymidine kinase and a single polypeptide with an apparent molecular weight of 19,000. The native molecular weight of VV thymidine kinase is about 80,000, so these data indicate that, unlike thymidine kinase from several other sources, the active VV enzyme is probably a tetramer of 19,000-molecular-weight subunits.     

Viral Proteins Formed in a Cell-Free RABBIT Reticulocyte System Programmed with RNA from a Temperature-Sensitive Mutant of Sindbis Virus

Viral messenger RNA was isolated from BHK cells infected with a temperature-sensitive mutant of Sindbis virus and was further purified using an oligo(dT) column. Addition of this mRNA cell-free extracts from rabbit reticulocytes led to formation of discrete authentic viral capsid protein when the reaction was performed at 29 C. However, this same protein-synthesizing system failed to make discrete viral capsid when incubated with the viral RNA at 39 C. Instead, larger-molecular-weight polypeptides that contained the viral capsid peptide sequences were produced. The inability to make a separate viral capside protein in vitro at elevated temperatures by the mRNA from this mutant exactly mimics the phenotype of this ts mutant in viral-infected cells. Three mechanisms are discussed that might account for a temperature-sensitive release of capsid. One of these is based on a model in which there are multiple sites for initiation of translation of polypeptides on a polycistronic viral mRNA.     

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.     

Identification of the DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus.

The DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus were located on the viral genome. The formation of an enzyme-guanylate covalent intermediate labeled with [alpha-32P]GTP allowed the identification of the large subunit of the capping enzyme and was used to monitor the appearance of the enzyme during the infectious cycle. This assay confirmed that after vaccinia infection, a novel 84,000-molecular-weight polypeptide corresponding to the large subunit was rapidly synthesized before viral DNA replication. Hybrid-selected cell-free translation of early viral mRNA established that vaccinia virus encoded a polypeptide identical in molecular weight with the 32P-labeled 84,000-molecular-weight polypeptide found in vaccinia virions. Like the authentic capping enzyme, this virus-encoded cell-free translation product bound specifically to DNA-cellulose. A comparison of the partial proteolytic digestion fragments generated by V8 protease, chymotrypsin, and trypsin demonstrated that the 32P-labeled large subunit and the [35S]methionine-labeled cell-free translation product were identical. The mRNA encoding the large subunit of the capping enzyme was located 3.1 kilobase pairs to the left of the HindIII D restriction fragment of the vaccinia genome. Furthermore, the mRNA was determined to be 3.0 kilobases in size, and its 5' and 3' termini were precisely located by S1 nuclease analysis.     

In vitro morphogenesis of foot-and-mouth disease virus.

Foot-and-mouth disease virion RNA is translated efficiently and completely in a rabbit reticulocyte lysate cell-free system. Treatment of cell-free lysates with monospecific serum prepared against the individual viral structural proteins or with monoclonal antibodies prepared against the inactivated virus or against a viral structural protein precipitated all of the structural proteins, suggesting that structural protein complexes were formed in vitro. Sucrose gradient analysis of the cell-free lysate indicated that complexes sedimenting at 5, 14, 60 to 70, and ca. 110S were assembled in vitro. Structural proteins VP0, VP1, and VP3 were the major polypeptides found in these complexes. The material sedimenting at 110S, i.e., containing VP0, VP1, and VP3, was precipitated by a 140S-specific monoclonal antibody but not by a 12S subunit-specific monoclonal antibody, suggesting that this capsid structure contained at least one epitope present on the intact virus.     

Synthesis of the E-antigen of the hepatitis B virus (HBeAg) in eukaryotic cells by a recombinant strain of the vaccinia virus]

The recombinant plasmids containing the gene for hepatitis B viral core-antigen with the pre-core-sequence controlled by the early-late promoter of the 7.5' K protein gene were constructed. The recombinant strains of vaccinia virus were obtained on their basis (vHBe42-1 and vHBe42-3) selectively expressing HBeAg of hepatitis B virus. The kinetics of HBeAg synthesis was studied in infected cells as well as secretion of the protein into culturing medium. Three proteins were found by blotting technique in the cells infected by vHBe42-3 that react with the specific antiserum to HBeAg and have the mol. masses 25, 22 and 17 kD. The completely processed HBeAg 17 kD was found in the culturing medium. The rabbit serums from the animals immunized by recombinant vHBe42-3 contained antibodies to HBeAg but not to HBcAg. This makes it possible to study the structural and functional organization, immunological properties and role of this antigen in pathogenesis of hepatitis virus B and to construct the specific test systems for screening HBeAg and corresponding antibodies.     

gag-Related polypeptides encoded by replication-defective avian oncoviruses.

The content of viral structural (gag) protein sequences in polypeptides encoded by replication-defective avian erythroblastosis virus (AEV) and myelocytomatosis virus MC29 was assessed by immunological and peptide analyses. Direct comparison with gag proteins of the associated helper viruses revealed that MC29 110K polypeptide contained p19, p12, and p27, whereas the AEV 75K polypeptide had sequences related only to p19 and p12. Both of these polypeptides contained some information that was unrelated to gag, pol, or env gene products. In addition, no homology was detected between these unique peptides of MC29 110K and AEV 75K. The AEV 75K polypeptide shared strain-specific tryptic peptides with the p19 encoded by its naturally occurring helper virus; this observation suggests that gag-related sequences in 75K were originally derived from the helper viral gag gene. Digestion of oxidized MC29 110K and AEV 75K proteins with the Staphylococcus aureus V8 protease generated a fragment which comigrated with N-acetylmethionylsulfoneglutamic acid, a blocked dipeptide which is the putative amino-terminal sequence of structural protein p19 and gag precursor Pr76gag. This last finding is evidence that the gag sequences are located at the N-terminal end of the MC29 110K and AEV 75K polypeptides.     

Early and late functions in a bipartite RNA virus: evidence for translational control by competition between viral mRNAs.

It has been shown previously that Drosophila cells infected with black beetle virus synthesize an early viral protein, protein A, a putative element of the viral RNA polymerase. Synthesis of protein A declines sharply by 6 h postinfection, whereas synthesis of viral coat protein alpha continues for at least 14 h. The early shutoff in protein A synthesis occurred despite the presence of equimolar proportions of the mRNAs for proteins A and alpha, RNAs 1 and 2, respectively. We have now been able to mimic this translational discrimination in a cell-free protein-synthesizing system prepared from infected or uninfected Drosophila cells, thus allowing further analysis of the mechanism by which translation of RNA 1 is selectively turned off. The results revealed no evidence for control by virus-encoded proteins or by virus-induced modification of mRNAs by the cell-free system. Rather, with increasing RNA concentration, viral RNA 1 was outcompeted by its genomic partner, RNA 2. This suggests that the early shutoff in intracellular synthesis of protein A is due to decreasing ability of RNA 1 to compete for a rate-controlling translational factor(s) as the concentration of viral RNAs accumulates within the infected cell.     

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.     

Preproceruloplasmin is a primary product of cell-free translation of ceruloplasmin messenger RNA

Summary Biosynthesis of ceruloplasmin was studied in wheat germ extract programmed with polysomal RNA from rat liver. Optimal potassium concentration for the total protein-synthesizing activity and for the synthesis of immunoreactive ceruloplasmin was 96 and 186 mM respectively. 7-methylguanosine 5′-monophosphate caused two-fold inhibition of the cell-free synthesis of ceruloplasmin. Immunoprecipitated ceruloplasmin that was synthesized at optimal potassium concentration was a homogeneous polypeptide of a molecular weight about 84 kD. The addition of membrane fractions from rat liver to the incubation mixture caused the conversion of the 84 kD polypeptide into 80 kD and 65 kD polypeptides that are similar to proceruloplasmins synthesized in rat liver during in vivo pulse labelling. The suggestion is made that 84 kD polypeptide is a primary product of the translation of ceruloplasmin mRNA (preproceruloplasmin).     

Encephalomyocarditis Virus Infection of Mouse Plasmacytoma Cells I. Inhibition of Cellular Protein Synthesis

Mouse plasmacytoma ascites tumor cells (MOPC 460) were efficiently infected with encephalomyocarditis virus. Inhibition of host protein synthesis was evident after 2 h and complete by 4 h postinfection. The mechanism by which virus infection results in inhibition of host cell protein synthesis was studied in vitro. Cell-free protein-synthesizing systems, prepared from uninfected and infected cells, were found to be equally active with respect to their abilities to translate cellular and viral mRNAs. The plasmacytoma cell-free system was also shown to be insensitive to the addition of double-stranded viral RNA. Host cellular mRNA was isolated from uninfected and infected cells. No difference in the amount or size distribution of the mRNA was detected. However, the mRNA from infected cells was translated only 46 to 49% as actively as that from uninfected cells. mRNA isolated from cells in which initiation of protein synthesis was inhibited with pactamycin was similarly inactivated. Simultaneous addition of viral RNA and cellular mRNA to the plasmacytoma cell-free system resulted in a complete suppression of the translation of the cellular message, whereas viral RNA was translated normally.