Cell-free translation of simian virus 40 16S and 19S L-strand-specific mRNA classes to simian virus 40 major VP-1 and minor VP-2 and VP-3 capsid proteins.

Simian virus 40 capsid proteins VP-1, VP-2, and VP-3 have been synthesized in wheat germ and reticulocyte cell-free systems in response to either poly(A)-containing mRNA from the cytoplasm of infected cells or viral RNA purified by hybridization to simian virus 40 DNA linked to Sepharose. All three viral polypeptides synthesized in vitro are specifically immunoprecipitated with anti-simian virus 40 capsid serum. VP-2 and VP-3 are related by tryptic peptide mapping to each other but not to VP-1. The most abundant class of L-strand-specific viral mRNA, the 16S species, codes for the major capsid protein. The relatively minor 19S class directs the cell-free synthesis of VP-1, VP-2, and VP-3. Whether the 19S RNA represents more than one distinct species of mRNA is not yet clear. VP-1 mRNA can be isolated from the cytoplasm, detergent-washed nuclei, and the nuclear wash fraction. The mRNA from the nuclear wash fraction is enriched for VP-2 mRNA when compared to other viral or cellular polypeptides.

Purification of SV-40 messenger RNA by hybridization to SV-40 DNA covalently bound to Sepharose.

SV-40 DNA sheared form was coupled in a stable covalent bond to cyanogen bromide activated Sepharose. Under the conditions used at least 80% of the DNA was bound to Sepharose. The T 1/2 of hybridization of 0.5 mug/ml of SV-40 cRNA to SV-40 DNA-Sepharose was 1 hr. This rate of hybridization is sufficiently rapid to purify SV-40 sequences from solutions containing as little as 0.05-0.1 mug/ml. Nonspecific hybridization of RNA is in the range of 0.1-0.2% of the total input RNA. The DNA-Sepharose is fairly stable and can be reused several times to purify RNA. The SV-40 DNA-Sepharose was used to select large quantities of virus specific RNA from SV-40 infected BS-C-1 cells. The virus specific RNA when added to cell-free extracts from wheat germ was shown to direct the synthesis of the major viral structural protein VP-1.

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.

Methylation of simian virus 40 Hpa II site affects late, but not early, viral gene expression.

DNA methylation has been correlated with reduced gene expression in a number of studies, although evidence for a casual link between the two events has been lacking. Because microinjection of simian virus 40 (SV40) DNA into the nucleus of Xenopus laevis oocytes results in the synthesis of both early and late viral gene products, it was possible to test whether a specific methylation event can affect gene expression. The single SV40 Hpa II site at 0.72 SV40 map units was specifically methylated with Hpa II methylase. When this DNA was injected into oocytes, there was a marked reduction in the synthesis of the major late viral capsid protein VP-1, relative to the synthesis by an unmethylated control. However, production of the early proteins (the large and small tumor antigens) was not affected by Hpa II methylation. Therefore, methylation at a single site on the viral DNA located near the 5' end of the late region can specifically repress late gene expression. The possible mechanisms by which this repression is mediated are discussed.

Cell-free translation of messenger RNA of simian virus 40: synthesis of the major capsid protein.

Extracts of wheat germ are capable of synthesizing the major capsid protein of simian virus 40. Poly(A)-containing RNA from BS-C-1 cells infected with simian virus 40 directed the synthesis of a novel polypeptide that migrates in polyacrylamide gels together with the major capsid polypeptide of simian virus 40, VP-1. The patterns of the major tryptic peptides of purified VP-1 and the novel polypeptide synthesized in vitro were identical after two-dimensional paper electrophoresis. The novel polypeptide was not synthesized in response to poly(A)-rich RNA from uninfected cells or from virus-infected cells treated with cytosine arabinoside. Messenger RNA from infected cells purified by selective hybridization to DNA of simian virus 40 directs the synthesis of a major polypeptide of electrophoretic mobility similar to that of VP-1 of simian virus 40. This approach should prove useful in identifying additional products specified by DNA tumor viruses.