Nucleic Acids Research Production of infectious poUovirus from doited cDNA b dramatically increased by SV40 transcription and replication signals

Sub-genomic cDNA clones representing the entire genomic RNA of poliovirus Type 1 (Mahoney) have been isolated in E. coll. Construction of a complete cDNA copy of the poliovirus genome in the EcoRI site of plasmid vector pBR325 from these clones is described. Introduction of plasmid DNA containing the complete cDNA copy of polio RNA into cultured primate cells by transfection produces infectious poliovirus. The virus produced by such a transfection appears to be identical to wild type poliovirus. Isolation of a polio recombinant plasmid containing SV40 transcription and replication signals is also described. Transfection of COS-1 cells with this plasmid yields greater than 1,600 plaque-forming units (PFU) per ug of input DNA. INTRODUCTION The molecular biology of poliovirus, a member of the picornavirus group, has been well-characterized with respect to details of the virus life cycle and the primary structure and genetic organization of the viral RNA. The complete nucleotide sequence of the nearly 7.5 kb plus strand genomic RNA that is 3*-polyadenylated and 5'-linked to a small polypeptide (VPg) was reported more than 2 years ago (1,2). Further structural studies based on this sequence elucidated the precise genomic encoding locations of nearly 20 viral-specific polypeptides as well as the amino acid pairs cleaved in the giant polyprotein precursor (the only translation product of poliovirus mRNA) to generate these polypeptides (3,4,5,6,7). Yet, to date, the functions of only two nonstructural polypeptides specified by the viral RNA have been elucidated. Protein P3-4b (also called p63) has been shown to be a primer-dependent viral RNA polymerase (8,9). Protein P3-7c is the viral proteinase that cleaves at nearly all of the glutamine-glycine pairs used in the processing of most precursor polypeptides (10). The functions of the majority of the viral-specific proteins found in infected cells (e.g., P2-3b, P2-5b, P3-2, VPg) are largely unknown. In order to determine the functions of these nonstructural polio proteins, we have initiated experiments to genetically manipulate the poliovirus genome. To this end we and others (2,11,12,13) have generated cDNA copies of poliovirus RNA and have cloned these copies in E. coll. In this paper, we describe the isolation of three sub-genomic cDNA clones of © IRL Press Limited, Oxford, England. 5123 Nucleic Acids Research poliovirus RNA that, together, represent the entire viral genome. One of these clones, pAD337, contains the 5' proximal sequences of polio RNA, including the 5' canonical nucleotides. A second clone, pKE195, contains the 3 terminal sequences of the poliovirus genome, including a poly(A) tract of 84 adenylate residues. The third clone, pKE88, has sequences originating from the middle region of the viral RNA. The above three clones were used to construct a complete copy of the poliovirus genome in the EcoRI site of the plasmid vector pBR325. We show that when DNA from a plasmid containing the full-length cDNA copy of poliovirus RNA (pDS303) is introduced into cultured primate cells, infectious poliovirus is produced. Our results are thus in agreement with those published previously by Racaniello and Baltimore (14). In addition, we show that simian virus 40 (SV40) transcription and/or replication signals increase the production of poliovirus from cloned plasmid DNAs by 18to 250-fold, depending upon the cell line used for transfection. MATERIALS AND METHODS Bacterial Strains Routine propagation of plasmids was carried out in E. eoli HB101. Transformation with plasmid DNA was carried out in E^coli HB101 or JA221 lpp"/F' lacl^. The latter strain is an E^ eoli K-12 derivative (15) that takes up DNA very efficiently (K. Nakamura and M. Inouye, personal communication). Synthesis of Poliovirus Complementary DNA (cDNA) Synthesis of single strand cDNA was carried out with purified poliovirus RNA, oligo dT(i2-18)' a n ^ ovifln myoblastosis virus reverse transcriptase (Life Sciences, Inc) as previously described (16,17). We also included 500 units/ml human placental RNase Inhibitor (RNAsin; Biotech) in our cDNA synthesis reactions. After synthesis at 37°C or 42°C for 2 hr, the reaction mixture was phenolrchloroform (1:1) extracted and the nucleic acids were recovered by ethanol precipitation. The sample was then dissolved in 250 ul of 20 mM EDTA, 0.1 N NaOH and incubated for 1 hr at 60°C. The reaction mixture was neutralized with HC1, chromatographed on a small (0.5 cm x 10 cm) Sephadex G-200 column to separate the polio cDNA from free nucleotides, and the purified cDNA was recovered by ethanol precipitation. Second Strand Synthesis and Tailing Reactions for 5' cDNA Clones Approximately 2 ug of cDNA (from above) was tailed with dCMP at the 3' end using terminal deoxynucleotidyl transferase (P. L. Biochemicals) as described by Deng and Wu (18). After incubation for 20 min at 30°C, the reaction mixture was extracted with phenohchloroform (1:1) and precipitated with ethanol. The resulting pellet was resuspended in 76 mM TRIS-HC1, pH 8.3, 90 mM KC1, and 23 ug/ml ollgo (dG)12.1g (Collaborative Research). Hybridization was at 56°C for 5 min and 50°C for 10 min