Direction of synthesis of the message for a 35 000 polypeptide of herpes simplex virus type 2 ERIC FROST, ' MARHO FHLION, AND MARTHA SUH~ lnstifut du Cancer cle 1WontrPai, Centre Hosgittrbier Notre-Dame, 1.560 est, rue Sherbrooke, Mon~rk(11 (Quk.), Cuuada H2L $MI Received September 12, 1983 Frost, E., Filion, M. & Suh, M. (1984) Direction of synthesis of the message for a 35 000 polypeptide of herpes simplex virus type 2. Can. J. Biochem. Cell Bioi. 62, 266-269 A restriction endonuclease map of the coding sequences for the transformation-related 35 000 polypeptide of herpes simplex virus type 2 is reported, using 11 enzymes that cut DNA infrequently. Hybrid-arrested translation with small DNA fragments was used to locate the 5' terminus of these coding sequences. A navel procedure involving translation of the 35 000 mRNA selected with cloned viral DNA that had been digested with exonuclease was employed to confirm that this message is transcribed from left to right. The 35 008 polypeptide can thus be assigned to a 1 .2-kilobase nnRNA recently reported with the same starting point and direction. Frost? E., Filion, M. & Suh, M. (1 984) Direction of synthesis of the mcssage for a 35 000 polypeptide of herpes simplex virus type 2. Can. J. Biochem. Cell BioO. 62, 264-269 Utilisant 1 I enzymes qui ne coupent pas frkquernment la DNA, nous avrms dress6 une carte des sites d'action d'endonuclkases de restriction sur les stquences du DNA codant pour le polypeptide 35 000 impliqd dans la transfornaation par le virus herpes simplex de type 2. Nous avons employ6 la technique d9am&t de Ia traduction du nmRNA par hybridation avec de petits fragments de DNA pour localiser l7extr6mit6 5' des s6quences sodant pour la protCine 35 000. Une technique nouvelle, baske sur la traduction du mRNA du polypeptide 35 000, mRNA sklectionne par ie DNA viral clone et digtr6 par des exonuclCases, nous a permis de confirmer que ce message est transcrit de gauche li droite. Le polypeptide de 35 000 est donc traduit a partir d'un mRNA de 1,2 kilobase rkcemment dkcrit et qui posskde le 1n6me point dc dipart et la meme direction de lecture. [Traduib par la revue] Introduction Such an activity could be instrumental in the initiation of Heves simplex (HSV) type 2 has been linked transformation. It has also been suggested that the with cervical cancer in seroepiderniological studies (1, continued Presence of an HSV-2 polypeptide is not 2). In vitro, it has been shown to transform rodent cells required in transfmmed cells, but that it serves only to to a malignant state (3, 4). The present epidemic of initiate transformation by a "hit-and-run" mechanism genital HSV infections has biochemical ex- (18). A ribonucleotide reductase could initiate transfor- plorition of putative transformation-related poly- mation by a hit-and-run peptides . The sequencing of the proposed coding region for this Although several viral proteins have been reported to P ~ ~ Y P ~ P ~ ~ ~ ~ has ver). been reported (18, 191, be present in turnours (5-7) or vansformed cells (8- 12), following identification of the approximate location of the evidence is strongest for a polypeptide of molecular the coding sequences by hybridization selection of RNA weight 35 OM) - 38 000 (which we call 35 ~QQ), that can and in vitr0 translation (20-22). A 1.2-kb mRNA has be immunoprecipitated from most HSV-2 transformed been identified in viiro as a transcriptional product of hamster ( 12, 13) and mouse (unpublished) lines. It is this region ( 19). In this repofi we complete the proof that one of the two HSV-2 proteins that can be immunopreci- this 1 -2-kb &NA indeed codes for the 35 000 protein, pitated significantly more often by cervical cancer by demonstrating that this polypeptide is Vanslated patient as opposed to control sera (14). Furthermore, it is a message that starts near the beginning of the the major early translation product from an HSV-2 DNA 02-kb mRNA and is read in the same left to right fragment capable of inducing transformation in rodents direction- (1% 16). I< has recently been proposed (17) that this plypeptide has a ribonucleotide-reductass activity. ABBREVIATIONS: HSV, herpes simplex virus; kb, kilo- base(s); HART, hybrid-mested translation; PIPES, piper- azine-N,N1-bis(2-ethanesulfonk acid); bg, base pair(s); SDS , sodium dodecyl sulfate. '~esermt address: Centre International de Rccherches Mkdicales de Franseville, B . P. 769, Franceville, RCpublique Gabsnaise. 2~uthor to whom all correspondence should be addressed. Methods Cells and virus BHM-21 clone I3 cells were grown in a 5% C02 atmosphere in a-medium (Gibco Laboratories) containing 10% fetal calf serum in plastic tissue culture flasks (Coming). Plaque- purified HSV-2 strain 333 was used for all infections. RNA exfraction WNA preparations were made from BHK-21 cells infected with HSV-2 at a multiplicity of 20 plaque forming unitsjcell. The cells were inoculated with virus for 60 min in a-medium Can. J. Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by Nanjing University of Posts and Telecommunications on 06/05/13 For personal use only.