Cell-free translation of influenza virus mRNA

Abstract

Cytoplasmic poly(A)-rich RNA extracted from fowl plague virus-infected cells was found to program efficiently the translation of two major peptides in the wheat germ cell-free system. These peptides have the same electrophoretic mobility, on polyacrylamide gels, as the two major virion proteins M and NP. I35S]methionine tryptic peptide analysis by one-dimensional paper ionophoresis and finger printing by two-dimensional thin-layer ionophoresis and chromatography show a high degree of similarity between the two in vitro products and the authentic viral proteins M and NP. Although virion RNA is devoid of any poly(A) sequence, it is confirmed here that the viral complementary cytoplasmic RNA contains poly(A) stretches of varying lengths. Intact purified virion was found to promote the synthesis of very low amounts of the same NP and M proteins in this cell-free system. Quantitative aspects of the data would indicate that this is due to minute amounts of complementary viral RNA associated with the virion or with the virion RNA itself. In conclusion, it is shown directly by cell-free translation of authentic viral products that the influenza virion is "negative stranded" (Baltimore, 1971), at least for its two major structural proteins.

Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA

“…ox Poly(A) of in vitro synthesized RNA. Influt enza viral cRNA synthesized in the infected cell C contains poly(A) (10,13,17). To determine whether the RNA synthesized in vitro also cont tains poly(A), the RNA products were tested for their ability to bind to oligo(dT)-cellulose (Tag ble 3).…”

Cell-free synthesis of measles virus proteins

“…No in vitro polypeptides were seen to have electrophoretic mobilities near that of the viral glycoprotein. Other investigators have reported an inability to detect synthesis of glycosylated proteins in the wheat germ system (2,4,13). Furthermore, 78K protein was not detected in immunoprecipitates of viral polypeptides synthesized in a reticulocyte cell-free translation system (15).…”

Cell-free coupling of influenza virus RNA transcription and translation

“…We routinely used 6 x 108 cpm of [35S]methionine per ml. Nevertheless, to keep the specific radioactivity of the methionine as high as possible, we diluted it to 160,000 cpm/pmol, with a final concentration of L-methionine at 4 ,uM.…”

“…Influenza viruses are enveloped animal viruses whose genome is composed of a segmented single-stranded RNA, probably entirely "negative stranded" (15,19). This finding is based on several lines of evidence, as follows: (i) the virion RNA is not a template for viral protein synthesis in the wheat germ cell-free system (20), whereas in similar conditions the cytoplasmic polyadenylic acid [poly(A)]-rich RNA from influenza virus-infected cells promotes the synthesis of the major virus structural proteins (4,6,16); (ii) poly(A)-containing mRNA isolated from infected-cell polysomes is entirely complementary to the virion RNA (5,14); and (iii) the virion contains an RNA-dependent RNA polymerase (19).…”