Cell-free coupling of influenza virus RNA transcription and translation

Abstract

A cell-free coupled system for the transcription and translation of fowl plague virus RNA is described. The system utilizes a new nuclease-preincubated rabbit reticulocyte lysate that has a high sensitivity to exogenous mRNA and a very low level of nuclease activity. Translation of the viral proteins in the coupled system is strictly dependent upon the viral transcriptase activity. In the coupled system the optimal concentration of magnesium is intermediate between the optimum for transcription and that for translation. Translation of the viral proteins seems faithful. The products represent the major viral peptides M and NP and two peptides with the same electrophoretic mobility as HA and P2. Virion NA is not resolved in the kind of polyacrylamide gels described. Proteins M and NP were immunoprecipitable with monospecific antisera. It is concluded that the virion-associated RNA polymerase transcribes the negative-stranded segments of the viral genome coding for these major structural proteins into fully functional mRNA's.

Differential effects of various double-stranded RNAs on protein synthesis in rabbit reticulocyte lysates

“…The nuclease treatment was stopped with EGTA [ethylene glycol bis(,8-aminoethyl etherf-A.A"tetraacetic acid] and the nuclease-preincubated lysates (NPL) were stored in liquid nitrogen. For the preparation of NPL, the procedure described by Pelham and Jackson (1976) was slightly modified (Content et al, 1977).…”