In vitro replication, packaging, and transcription of the segmented double-stranded RNA genome of bacteriophage phi 6: studies with procapsids assembled from plasmid-encoded proteins

The genome of the lipid-containing bacteriophage phi 6 contains three segments of double-stranded RNA (dsRNA). We prepared cDNA copies of the viral genome and cloned this material in plasmids that replicate in Escherichia coli and Pseudomonas phaseolicola, the natural host of phi 6. These plasmids direct the formation of viral proteins and the assembly of structures similar to viral procapsids containing proteins P1, P2, P4, and P7. We found that these particles are capable of taking up viral single-stranded RNA and synthesizing the minus strands to produce dsRNA structures. Once the dsRNA is formed, it is then used as a template for the production of viral plus strands in a reaction that resembles normal transcription. The particles were also capable of directly transcribing exogenous dsRNA. The replicase reactions were specific for phi 6 RNA, were specific for procapsids, and resulted in substantial incorporation of product dsRNA into particles. These results offer strong support to a model in which genomic packaging is done by preformed procapsids.

[1]  J. Vieira,et al.  The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. , 1982, Gene.

[2]  M. Romantschuk,et al.  cDNA cloning of portions of the bacteriophage phi 6 genome , 1985, Journal of bacteriology.

[3]  D. Burbank,et al.  RNA polymerase activity associated with bacteriophage φ6 nucleocapsid , 1979 .

[4]  L. A. Day,et al.  The molecular weight of bacteriophage θ6 and its nucleocapsid , 1980 .

[5]  E. Koonin,et al.  Tentative identification of RNA‐dependent RNA polymerases of dsRNA viruses and their relationship to positive strand RNA viral polymerases , 1989, FEBS letters.

[6]  J. Patton,et al.  Structure and protein composition of the rotavirus replicase particle. , 1988, Virology.

[7]  H. Iba,et al.  Transcriptional regulation of three double-stranded RNA segments of bacteriophage phi 6 in vitro , 1983, Journal of virology.

[8]  R. Franklin Purification and properties of the replicative intermediate of the RNA bacteriophage R17. , 1966, Proceedings of the National Academy of Sciences of the United States of America.

[9]  R. Haselkorn,et al.  Displacement of parental RNA strands during in vitro transcription by bacteriophage φ6 nucleocapsids , 1980, Cell.

[10]  L. Black DNA packaging in dsDNA bacteriophages. , 1989, Annual review of microbiology.

[11]  L. Mindich Bacteriophage phi 6: a unique virus having a lipid-containing membrane and a genome composed of three dsRNA segments. , 1988, Advances in virus research.

[12]  J. V. Etten,et al.  Characterization of a segmented double-helical RNA from bacteriophage φ6 , 1973 .

[13]  R. Hamatake,et al.  Morphogenesis of phi X174: in vitro synthesis of infectious phage from purified viral components. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[14]  M. Ewen,et al.  In vitro replication and transcription of the segmented double-stranded RNA bacteriophage phi 6. , 1988, Virology.

[15]  N. Pagratis,et al.  Detection of bacteriophage phi 6 minus-strand RNA and novel mRNA isoconformers synthesized in vivo and in vitro, by strand-separating agarose gels. , 1990, Virology.

[16]  J. Miller,et al.  Specificity of mutations induced in transfected DNA by mammalian cells. , 1984, The EMBO journal.

[17]  A. Tsugita,et al.  Purification of viral RNA by means of bentonite. , 1961, Virology.

[18]  J. V. Van Etten,et al.  Semiconservative synthesis of single-stranded RNA by bacteriophage phi 6 RNA polymerase , 1980, Journal of Virology.

[19]  J. V. Van Etten,et al.  Bacteriophage φ6: a Lipid-Containing Virus of Pseudomonas phaseolicola , 1973, Journal of virology.

[20]  R. Wickner,et al.  Reconstitution of template-dependent in vitro transcriptase activity of a yeast double-stranded RNA virus. , 1989, The Journal of biological chemistry.

[21]  D. Bamford,et al.  Isolation of nonsense mutants of lipid-containing bacteriophage PRD1 , 1982, Journal of virology.

[22]  P. Kaesberg,et al.  Synthesis of complementary strands of heterologous RNAs with Qbeta replicase. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[23]  L. Mindich,et al.  The characterization of a 120 S particle formed during phi 6 infection. , 1980, Virology.

[24]  P. Gottlieb,et al.  Nucleotide sequence of the large double-stranded RNA segment of bacteriophage phi 6: genes specifying the viral replicase and transcriptase , 1988, Journal of virology.

[25]  J. Patton Synthesis of simian rotavirus SA11 double-stranded RNA in a cell-free system. , 1986, Virus research.

[26]  M. Obinata,et al.  Synthesis of probes for RNA using Qβ-replicase , 1975 .

[27]  G. Ditta,et al.  Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[28]  F. Studier,et al.  Analysis of bacteriophage T7 early RNAs and proteins on slab gels. , 1973, Journal of molecular biology.