Molecular cloning of the genome of poliovirus type 1.

Abstract

Poliovirus cDNA RNA hybrids were prepared from the Mahoney strain ofpoliovirus type 1 by using reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) and cloned in the Escherichia coliplasmidpBR322. Bacteria colonies carrying recombinant plasmids were selected by in situ hybridization with virus-specific RNase Ti-resistant oligonucleotides. Analysis of the cDNA inserts by restriction mapping and electron microscopy showed that the cloned cDNAs, the longest of which was 3.2 kIlobase pairs, originated from various parts of the viral RNA, covering at least 99% ofthe genome length. Due to overlapping ofthe clones, the restriction map of the poliovirus genome could be reconstructed. The complete 5' end of the genome was successfully cloned in at least one of the recombinant plasmids, pPVl-366.The genome of poliovirus is a polyadenylylated RNA of plusstrand polarity. Its 5' end is covalently attached to a small viral protein (VP) termed VPg (for review, see ref. 1). The complete sequence of poliovirus type 1 (PV-1) RNA has recently been determined (2). Poliovirus has a unique strategy ofgene expression; its primary translation product is a 220,000-dalton polyprotein, noncapsid VP (NCVP) NCVP00, that is processed into numerous polypeptides by proteinase(s) (3). Most of the viral polypeptides have been mapped; the capsid proteins are encoded in the 5'-terminal halfofthe genome in the order 5'-VP4-VP2-VP3-VP1, whereas nonstructural proteins such as NCVP X, a putative proteinase, and NCVP4, the viral RNA polymerase (4, 5), are encoded in the middle and the 3' part of the genome, respectively (2, 3). However, the functions of many viral polypeptides are still obscure. Also, little is known about the regulation of proteolytic processing or the mechanism of RNA replication.Recombinant DNA technology allows synthesis and cloning in Escherichia coli of double-stranded cDNA copies of a viral RNA genome. The availability of such clones would be helpful for detailed analysis of the organization of the poliovirus genome, including confirmation of the nucleotide sequence information obtained by other means. In addition, the expression of virus-specific proteins in E. coli may open the way to the development of new types of vaccine. This is desirable in view of the fact that vaccination with both killed and live poliovirus has met with difficulties, particularly in tropical countries. Finally, should expression of cloned poliovirus cDNA in eukaryotic cells prove possible, the system would offer a unique opportunity for in vitro site-directed mutagenesis and thus for genetic analyses of the virus.In this paper, we report the isolation of PV-1 cDNA clones that together cover 99% of the viral genome, and we present a restriction map of poliovirus-specific DNA. MATERIALS AND METHODSConstruction and Screening of Clones Containing Recombinant DNAs. The Mahoney strain of PV-1 was grown in suspension cultures of HeLa cells, and viral RNA was extracted as described (6). cDNA was prepared by using an oligo(dT)10 primer and ...

Complete nucleotide sequence of the attenuated poliovirus Sabin 1 strain genome.

“…The nucleotide sequence of this extended material was determined chemically (14). Comparison of the sequence with that of the previously established 5'-terminal sequences (15)(16)(17) indicated that the extension proceeded to the very 5' end of the viral genome. Sequence analysis of the EB1 fragment in a plasmid pVS(1)EB1 revealed that this insert contained the sequence corresponding to that ofthe first 220 bases of the viral genome.…”

“…1). van der Werf et al (15) have succeeded in the direct cloning of the 5' end ofpoliovirus RNA using RNADNA hybrids. Because we were unable to obtain directly 5'-terminal clones, we used a restriction fragment (positions 221-267) from clone pVS(1)BB3 that was 5'-end-labeled and employed as a primer to synthesize a primer extension fragment.…”

Structure and Expression of Genes for Flavivirus Immunogens.

“…In one instance it was possible to obtain a complete set of subgenomic clones of polio cDNA from a single cycle of cloning (van der Werf et al, 1981). Similarly, reverse transcriptions of in vitro polyadenylated Rift Valley virus RNA with oligo dT as primer yielded cDNA transcripts internal to the viral genome (J. Dalrymple and C. Schmaljohn, personal communication).…”

Localization of a poliovirus type 1 neutralization epitope in viral capsid polypeptide VP1.

“…To (17) and subeloned within the P-lactamase gene of pBR322. As the VP1 sequence contains no initiator AUG triplet, being located internally in the poliovirus genome (22)(23)(24), it was necessary to fuse it in phase behind the AUG of the f3-lactamase gene.…”

“…The minicell-producing strain GC26 (Bl-thrleu-) was kindly provided by G. Cesareni. Plasmids pPV1-366 and pSW1l9 have been described (15,17).Construction of Plasmids. Plasmid DNA was cleaved by restriction endonucleases following manuicturers' conditions.…”