Transcription vaccinia virus

The second method for capping mRNA takes advantage of the activity of the vaccinia virus capping enzyme, also known as guanylyltransferase. This enzyme is commercially available. In the presence of GTP and S-adenosyl methionine (SAM), it can add a natural Cap structure (7-methylguanosine) to the 5 triphosphate of a RNA molecule. As it is an enzymatic reaction, it can bring a correct Cap to all mRNA molecules, and thus, it is optimal compared with in vitro transcription in the presence of standard Cap but similar theoretically to in vitro transcription in the presence of ARCA Cap. 

Distamycin A (309) is an oligopeptide, isolated from Streptomyces distallicus that inhibits transcription and replication of DNA viruses along with its other related semisynthetic analogs [18]. Example of DNA viruses inhibited by this group are vaccinia virus and HSV-1. Distamycin A also shows inhibitory activity for RT of retroviruses [18]. 

Moss B. Regulation of vaccinia virus transcription. Annu Rev Biochem. 1990 59 661-668. 

Keck JC, Baldick CJ, Moss B. Role of DNA replication in vaccinia virus gene expression A naked template is required for transcription of three late trans-activator genes. Cell. 1990 61 80-89. 

Kates, J. 1970. Transcription of the vaccinia virus genome and the occurrence of polyriboadenyhc acid sequences in messenger RNA. Cold Spring Harbor Symp. Quant. Biol. (In Press.)... 

Alkaline hydrolysates of mRNA from vaccinia virus (Kates, 1970) and mouse sarcoma 180 cells (Mendecki et al, 1972) show that the poly (A) tracts, 100-200 nucleotides in length, are located at the 3 -ter-minal end of the RNA molecules. Analysis of the reaction products of highly purified exoribonuclease specific for 3 -OH termini also indicates that most (and possibly all) of the poly (A) sequences are at the 3 -terminal end of the mRNA s (Molloy et al, 1972). This is supported by the fact that the time course of poly( A) labeling in polysomes indicates that this sequence is assembled after the rest of the RNA molecule has been completed (Mendecki et al, 1972). Poly (A) synthesis is sensitive to actinomycin D but to an extent less than that of the rest of the RNA molecule which further argues that the poly (A) segment is added after transcription is completed (Darnell et al, 1971b Mendecki et al, 1972). 

Bertholet, C., Drillien, R., and Wittek, R. (1985) One hundred base pairs of 5 flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription. Proc. Natl. Acad. Set. USA 82, 2096-2100. 

Cooper, J. A., and Moss, B., 1978, Transcription of vaccinia virus mRNA coupled to translation in vitro, Virology 88 149. 

Double-stranded RNA (dsRNA) has been implicated in picor-navirus-induced shut-off (Ehrenfeld and Hunt, 1971) however, the inhibition of protein synthesis caused by poliovirus double-stranded RNA is nondiscriminatory with respect to host or viral mRNAs (Celma and Ehrenfeld, 1974). Double-stranded RNA is obtained during extraction of RNA from infected cells and can also be found in in vitro transcription. It has been proposed that this dsRNA is produced by vaccinia virus both in vivo (Duesberg and Colby, 1969) and in vitro (Colby et al., 1971) as a result of symmetrical transcription. The role of vaccinia virus double-stranded RNA in shut-off has been investigated. To determine the relation of in v/vo-synthesized vaccinia virus-induced double-stranded RNA to shut-off, we measured the percentage and amount of double-strand RNA formed in infected L929 cells treated with cycloheximide conditions where severe inhibition occurs and a large amount of viral RNA is produced (Schrom... 

The notion that vaccinia virus-induced RNA may be involved in shut-off was independently proposed by Rosemond-Hombeak and Moss (1975) and Bablanian (1975). Rosemond-Hornbeak and Moss (1975) infected HeLa cell suspension cultures in the presence of 5-10 xg/ml of actinomycin D and showed that short poly(A)-rich RNAs were transcribed which sedimented a 5 S. Normal vaccinia virus transcripts sediment at 10-14 S (Becker and Joklik, 1964 Salzman et al.. 

Role of Vaccinia Virus in Vitro Transcripts in Selective Inhibition... 

On the basis of our previous results discussed above, early viral transcripts were primary candidates in the inhibition of host protein synthesis. Since in vitro transcription produces only the early species of viral transcripts (Kates and Beeson, 1970), we acquired these early species of viral RNA by means of in vitro transcription by viral cores and tested their effect on the translation of various exogenous mRNAs in in vitro cell-free systems rendered messenger dependent. The results from such experiments (Coppola and Bablanian, 1983) revealed that transcripts prepared in vitro of either 8-10 S or 4-7 S size classes inhibit globin, HeLa, and hamster cell mRNA translation in a reticulocyte cell-free protein-synthesizing system. Inhibition was observed not only under conditions where in vitro viral transcripts by themselves were capable of producing viral polypeptides, but also when low concentrations of in v/7ro-synthesized transcripts were used which were incapable of synthesizing polypeptides as determined by polyacrylamide-gel electrophoresis. In contrast, the transcripts synthesized in vitro by vaccinia virus cores had no inhibitory effect on the translation of cytoplasmic RNA obtained from vaccinia virus-infected cells at early times after infection. Therefore, this inhibitory effect, like that seen in vaccinia virus-infected cells, was selective. The vaccinia virus transcripts, synthesized in vitro, also inhibited encephalomyocarditis virus mRNA translation in the cell-free reticulocyte lysate system indicating that this inhibition does not require... 

In order to demonstrate that the inhibitory property of vaccinia virus in vitro transcripts is RNA, we subjected these preparations to alkaline hydrolysis, and micrococcal nuclease digestion. The resulting hydrolysate and digest no longer had an inhibitory effect on HeLa cell mRNA function indicating that the integrity of the transcripts is necessary for this inhibition (Coppola and Bablanian, 1983). 

Bossart, W., Nuss, D. L., and Paoletti, E., 1978, Effect of UV-irradiation on the expression of vaccinia virus gene products synthesized in a cell-free system coupling transcription and translation, J. Virol. 26 673. 

Coppola, G., and Bablanian, R., 1983, Discriminatory inhibition of protein synthesis in cell-free systems by vaccinia virus transcripts, Proc. Natl. Acad. Sci. USA 80 75. 

---