Spiroplasma virus 4: nucleotide sequence of the viral DNA, regulatory signals, and proposed genome organization

Abstract

The replicative form (RF) of spiroplasma virus 4 (SpV4) has been cloned in Escherichia coli, and the cloned RF has been shown to be infectious by transfection (M. C. Pascarel-Devilder, J. Renaudin, and J.-M. Bové, Virology 151:390-393, 1986). The cloned SpV4 RF was randomly subcloned and was fully sequenced by the dideoxy chain termination technique, using the M13 cloning and sequencing system. The nucleotide sequence of the SpV4 genome contains 4,421 nucleotides with a G+C content of 32 mol%. The triplet TGA is not a termination codon but, as in Mycoplasma capricolum (F. Yamao, A. Muto, Y. Kawauchi, M. Iwami, S. Iwagani, Y. Azumi, and S. Osawa, Proc. Natl. Acad. Sci. USA 82:2306-2309, 1985), probably codes for tryptophan. With these assumptions, nine open reading frames (ORFs) were identified. All nine are characterized by an ATG or GTG initiation codon, one or several termination codons, and a Shine-Dalgarno sequence upstream of the initiation codon. The nine ORFs are distributed in all three reading frames. One of the ORFs (ORF1) corresponds to the 60,000-dalton capsid protein gene. Analysis of codon usage showed that T- and A-terminated codons are preferably used, reflecting the low G+C content (32 mol%) of the SpV4 genome. The viral DNA contains two G+C-rich inverted repeat sequences. One could be involved in transcription termination and the other in initiation of cDNA strand synthesis. The SpV4 genome was found to contain at least three promoterlike sequences quasi-identical to those of eubacteria. These results fully support the bacterial origin of spiroplasmas.