Volume 30, number 3 FEBS LETTERS March. 1973 THE TRANSLATION OF VACCINIA VIRUS MESSENGER RNA IN .ANIMAL CELL-FREE SYSTEMS F. FOURNIER*, D.R. TOVELL*, M. ESTEBAN, D.H. METZ*, L.A- BALL and lan M. KERR iVational Institute [or Medical Research, Mill Hill, L~Tndon NW7 IA.,I, England Peceived 29 January 1973 I. Introduction Cell-tree .,';ystems from animal cells in which added viral messenger RN,*t's (mRNA's)are translated have proved useful in studies on eukaryotic protein synthe- sis and its control. Of the v/ruses which contain their own transcrip~ases for the synti~e:ds of viral mRNA, however, o:dy with reovirus has it been possible to synthesise in vitro sufficient intact mRNA for its translation to be stt~died in this way [1-3]. Vaccinia is a large DNA virus which contains an RNA polymer- ase activity associated with the virus cores [4]. The core polymerase: prepared in vitro from purified virus, ~-itl effect the synthesis of RNA having properties similar to the viral mRNA present at early times in vaccinia-infected cells [4]. This RNA is a mixture of species, the sizes of which are consistent with their coding predominantly l'or individual viral proteins rather than for very. large precursor molecules as is the case with the picornavirus RNA's [5,61. Furthermore these RNA species unlike the reovirus mRNA's have poty A at their 3'-ends [7]. Ttms these presumptive vaccinia mRNA's appear to resemble animal cell mes- sengers more than do either of the other viral mRNA * Pre~nt addresses: F.F., Institute National de la Sante et de la Recherche M6dicale, Unit6 de Redterches star les Virus U. 43, Hopital Saint-V/.ncent-de-Paul, Paris Y.IV. D.R.T., Dept. of Micr,Jbiology an, d Immunology, Queens University, Kingston, Ontario, Canada, D.H.M., Research Division of Infectious Diseases, Childrcns liospit',tl Medical Center, 300 Longwood Avenue, Boston, Mass. 62115, USA. species most readily available for study - those of reo- and picornaviruses. As yet, however, no direct demonstration of messenger funetion has been re- ported for *.his vaccinia RNA. Ht:re we describe its translation in two mouse cell-free systems. 2. Materials and methods Vaccinia virus (strain WR) was grown and purified essentially according to Joklik [8] as previously de- scribed [91- "file method of Kates and Beeson was used in the preparation of the virus cores [4]. The reaction mLxture for RNA synthesir, contained: 2 X 10 I1 cores; 10/amoles ATP; 5 vmoles each of CTP, GTP and UTP; I 0/.tCi [3H I UTP (40 Ci/mmole); 20 ~umoles MgCI 2; 40 btmoles 2-mercaptoethanol; I00 vmoles phosphoenolpytuvate; 100 gg pyruvate kinase; 250 ~g Macaloid [lOI ; 200tamoles Tris-HCt, pH 8.5; in a total volume of 4.0 ml. Incorporation of radioactivity from [3H I UTP into an acid-insolub~.c, alkali and RNAase-labile product was completely de- pendent upon the presence of ATP, GTP and CTP. The mixture was incubated at 33 ° for 3 hr during which incorporation was almost linear, then centri- fuged at 30,OOOg for 30 rain to remove tile cores ann the Macaloid. The supematant was treated with I 0 mM EDTA and 1% (w/v) sodium dodecyl sulphate (SDS) at 37 ° for 5 min and the rnixture was layered onto a i 5-30% sucrose (w/v) gradient in ! 0 mM Tris- HCI pH 7.0, 100 mM NaCI, 10 mM EDTA, 0.5% SDS and centrifuged in the SW 27 rotor at 70,000g for 268 North-Holland Publishing Company - Amxterdam