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L Mindich

Publications and source records attributed to L Mindich.

At least 37 records · Page 2Linked to original sources
PubMed1994

Reconstitution of active replicase in procapsids of the segmented dsRNA bacteriophage phi 6.

Bacteriophage phi 6 has a genome of three segments of double-stranded RNA enclosed in a procapsid composed of four different proteins. The preformed procapsid is capable of packaging plus strand transcripts of the genomic segments in an in vitro reaction. The plus strands then serve as templates for in vitro minus strand synthesis. Procapsids that are missing protein P2 are incapable of minus strand synthesis. In this report we show that incubation with a cell extract containing P2 results in particles with normal amounts of attached P2 and with packaging and replicase activity. Particles lacking P7 have reduced replicase activity which can be augmented by incubation with extracts containing P7, but the amount of attached P7 is small.

Bacteriophage phi 6↗
PubMed1994

Plasmid-directed assembly of the lipid-containing membrane of bacteriophage phi 6.

The nucleocapsid of bacteriophage phi 6 is enveloped within a lipid-containing membrane. The membrane is composed of proteins P3, P6, P9, P10, and P13 and phospholipids. The relationship between membrane protein P9 and morphogenetic protein P12 was studied in the absence of phage infection. cDNA copies of genes 9 and 12 were expressed on plasmids in Pseudomonas syringae pv. phaseolicola. Immunoblotting demonstrated the presence of protein P9 in strains carrying both gene 9 and gene 12 but not in strains with gene 9 alone. In the absence of P12, P9 was found to be unstable. Simultaneous synthesis of proteins P9 and P12 led to the formation of a low-density P9 particle having a buoyant density similar to that of precursor structures composed of phospholipid and proteins isolated from phi 6-infected cells. These results are consistent with results of previous genetic experiments suggesting that P9 and P12 are necessary and sufficient for the formation of the phi 6 envelope. Extensions of P9 at the C terminus do not impair particle formation; however, N-terminal extensions or C-terminal deletions that extend into the hydrophobic region of P9 do impair particle formation.

Amino Acid Sequence↗
PubMed1994

Isolation and characterization of nonsense mutations in gene 10 of bacteriophage phi 6.

Nonsense mutants of bacteriophage phi 6 were isolated by a procedure that involved directed mutagenesis of a cDNA copy of genomic segment M, transcription of this segment, in vitro packaging into procapsids, and transfection of spheroplasts to form viable mutant phage. Recombinant phi 6 viruses that contained amber mutations in two open reading frames, ORF 10 and ORF D, of genomic segment M were isolated. We show that phi 6 protein P10 is the gene product of ORF 10. Further characterization of the phi 6 ORF 10(Am) mutant revealed that phi 6 membrane-associated protein P10 is not required to make enveloped phage particles in infected cells. Enveloped phage particles isolated from a phi 6 ORF 10(Am) infection contained extremely low levels of phi 6 membrane-associated proteins P6 and P3. The low abundance is due to the very low level of P6 synthesis in phi 6 ORF 10(Am)-infected cells. The results suggest that P10 might play a role in regulating the translation of gene 6. Protein P10 was found to be required for host lysis.

Bacteriophage phi 6↗
PubMed1993

RNA structure and heterologous recombination in the double-stranded RNA bacteriophage phi 6.

Bacteriophage phi 6 has a genome of three segments of double-stranded RNA, designated L, M, and S. A 1.2-kbp kanamycin resistance gene was inserted into segment M but was shown to be genetically unstable because of a high recombination rate between segment M and the 3' ends of segments S and L. The high rate of recombination is due to complementary homopolymer tracts bounding the kan gene. Removal of one arm of this potential hairpin stabilizes the insertion. The insertion of a 241- or 427-bp lacZ' gene into segment M leads to a stable Lac+ phage. The insertion of the same genes bounded by complementary homopolymer arms leads to recombinational instability. A stable derivative of this phage was shown to have lost one of the homopolymer arms. Several other conditions foster recombination. The truncation of a genomic segment at the 3' end prevents replication, but such a damaged molecule can be rescued by recombination. Similarly, insertion of the entire 3-kb lacZ gene prevents normal formation of virus, but the viral genes can be rescued by recombination. It appears that conditions leading to the retardation or absence of replication of a particular genomic segment facilitate recombinational rescue.

Bacteriophage T7↗
PubMed1992

Bacteriophage phi 6 envelope elucidated by chemical cross-linking, immunodetection, and cryoelectron microscopy.

Bacteriophage phi 6 is an enveloped dsRNA virus which infects the plant pathogenic Pseudomonas syringae bacterium. Using low dose cryoelectron microscopy we show that the nucleocapsid, spikeless virion, and intact virion have radii of 29, 35, and 43 nm, respectively. Thus, the membrane is 6 nm thick and the surface spikes of the receptor binding protein P3 extend 8 nm from the membrane surface. Cross-linking, immunological, and complementation evidence suggest that the spikes are formed of multimeric P3 molecules and that P3 is associated with membrane-bound protein P6. We observe that the envelope can accommodate up to 400 molecules of P3 but that the average virion contains less than one-fourth of this amount. Assembly of a very small number of P3 or truncated P3 molecules onto inactive virions restores infectivity, showing that only a few spikes are necessary for receptor binding and membrane fusion.

Antibodies, Monoclonal↗
PubMed1992

Construction of a transducing virus from double-stranded RNA bacteriophage phi6: establishment of carrier states in host cells.

Bacteriophage phi 6 contains three double-stranded RNA (dsRNA) genomic segments. We have constructed a plasmid that contains a cDNA copy of the middle (M) segment, with a gene for kanamycin resistance (kan) inserted into the PstI site. A transcript of this cDNA was incorporated in vitro into procapsids along with natural transcripts of the S and L segments. The procapsids were coated with nucleocapsid surface protein P8 and transfected into Pseudomonas syringae pv. phaseolicola. The resulting infectious virus, phi 6 K1, was found to contain an M segment that was 1.2 kbp larger than the normal 4.1 kbp. K1 formed small, turbid plaques, and its genome was unstable. Preparations of K1 contained from about 0.1 to 10% large, clear-plaque forms of the virus which were usually missing the kan gene, and in some cases, the resulting segment M was smaller than its normal size. Cells picked from lawns of host cells infected with K1 yielded colonies that were resistant to kanamycin (Kan). These colonies could be passaged on kanamycin-containing medium. The cells were found to contain large amounts of dsRNA corresponding to the viral genomic segments. Some strains continued to produce viable phage, while others lost this ability. One strain completely lost the small genomic segment S. Approximately 1 in 10,000 infected cells acquired the carrier state with the original phage isolate K1. However, we isolated a viral mutant that was able to induce the carrier state in 10 to 20% of the infected cells. The ability to use drug resistance as a test for the carrier state makes this system very useful for the study of the mechanisms of induction of persistent infections.

Bacteriophages↗
PubMed1992

Protein P4 of the bacteriophage phi 6 procapsid has a nucleoside triphosphate-binding site with associated nucleoside triphosphate phosphohydrolase activity.

Bacteriophage phi 6 contains three segments of double-stranded RNA. The procapsid consists of proteins P1, P2, P4, and P7, which are encoded by the viral L segment. cDNA copies of this segment have been cloned into plasmids that direct the production of these proteins, which assemble into polyhedral procapsids. These procapsids are capable of packaging plus-sense phi 6 RNA in the presence of nucleoside triphosphate and synthesizing the complementary minus strand to form double-stranded RNA. In this article, we report the presence of a nucleotide-binding site in protein P4. The viral procapsid and nucleocapsid exhibit a nucleoside triphosphate phosphohydrolase activity that converts nucleoside triphosphates into nucleoside diphosphates.

Adenosine Triphosphate↗
PubMed1992

Heterologous recombination in the double-stranded RNA bacteriophage phi 6.

Bacteriophage phi 6 contains three double-stranded RNA genomic segments. We have constructed a virus with an insertion of a kanamycin resistance gene in genomic RNA segment M. The virus forms small, turbid plaques, and its genome is unstable. Virus from a single plaque contained from about 0.1 to 10% large clear-plaque forms of the virus; these were usually missing the kanamycin resistance gene, and in many cases, the resulting segment M was larger or smaller than its normal size. Sequence analysis of the genomic RNA of the apparent deletions showed that they were formed by recombination events between segment M and either segment S or L. These heterologous recombination events resulted in the loss of the kanamycin resistance gene from segment M and the replacement of the 3' end of segment M with the 3' end of segment S or L. Although the 3' ends of the single-stranded RNA transcripts of the genomic segments appear to have extensive secondary structure, the sequences at the 3' ends are not involved in the specificity of genomic packaging.

Base Sequence↗
PubMed1992

In vitro packaging and replication of individual genomic segments of bacteriophage phi 6 RNA.

The genome of bacteriophage phi 6 contains three segments of double-stranded RNA. Procapsid structures whose formation was directed by cDNA copies of the large genomic segment are capable of packaging the three viral message sense RNAs in the presence of ATP. Addition of UTP, CTP, and GTP results in the synthesis of minus strands to form double-stranded RNA. In this report, we show that procapsids are capable of taking up any of the three plus-strand single-stranded RNA segments independently of the others. In manganese-containing buffers, synthesis of the corresponding minus strand takes place. In magnesium-containing buffers, individual message sense viral RNA segments were packaged, but minus-strand replication did not take place unless all three viral single-stranded RNA segments were packaged. Since the conditions of packaging in magnesium buffer more closely resemble those in vivo, these results indicated that there is no specific order or dependence in packaging and that replication is regulated so that it does not begin until all segments are in place.

Capsid↗
PubMed1992

Dependence of minus-strand synthesis on complete genomic packaging in the double-stranded RNA bacteriophage phi 6.

Bacteriophage phi 6 has a segmented genome consisting of three pieces of double-stranded RNA (dsRNA). The viral procapsid is the structure that packages plus strands, synthesizes the complementary negative strands to form dsRNA, and then transcribes dsRNA to form plus-strand message. The minus-strand synthesis of a particular genomic segment is dependent on prior packaging of the other segments. The 5' end of the plus strand is necessary and sufficient for packaging, while the normal 3' end is necessary for synthesis of the negative strand. We have now investigated the ability of truncated RNA segments which lack the normal 3' end of the molecules to stimulate the synthesis of minus strands of the other segments. Fragments missing the normal 3' ends were able to stimulate the minus-strand synthesis of intact heterologous segments. Minus-strand synthesis of one intact segment could be stimulated by the presence of two truncated nonreplicating segments. The 5' fragments of each single-stranded genomic segment can compete with homologous full-length single-stranded genomic segments in minus-strand synthesis reactions, suggesting that there is a specific binding site in the procapsid for each segment.

Bacteriophages↗
PubMed1991

In vitro packaging of the bacteriophage phi 6 ssRNA genomic precursors.

Bacteriophage phi 6 contains three segments of double-stranded RNA within a nucleocapsid. Plasmids containing cDNA copies of the large genomic segment direct the synthesis of viral proteins that assemble into procapsids in Escherichia coli or Pseudomonas phaseolicola. These structures are dodecahedral assemblages of proteins P1, P2, P4, and P7. We report in this paper that these particles are capable of packaging viral single-stranded plus-sense RNA in vitro. The packaging reaction requires the presence of ATP or dATP. Synthesis of minus strands takes place within this filled procapsid in the presence of all four nucleoside triphosphates. Packaged ssRNA is found to be protected from added ribonuclease.

Adenosine Triphosphate↗
PubMed1990

In vitro assembly of infectious nucleocapsids of bacteriophage phi 6: formation of a recombinant double-stranded RNA virus.

A system is described for assembling infectious bacteriophage phi 6 nucleocapsids in vitro. Procapsids encoded by cDNA copies of genomic segment L in Escherichia coli were used to package and replicate viral RNA segments. The resulting filled particles were shown to be capable of infecting host cell spheroplasts after incubation with purified nucleocapsid shell protein P8. The infected spheroplasts yielded infectious virions. A modified cDNA-derived RNA segment was inserted into virions by this method. The resulting infectious virions contained the same 4-base-pair deletion as the modified cDNA. These findings support the contention that the preformed procapsids are the "machine" that replicates the phi 6 genome, by showing that the cDNA-derived procapsids are competent to package and replicate RNA properly.

Bacteriophages↗
PubMed1990

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.

Bacteriophages↗
PubMed1988

Nucleotide sequence of the middle dsRNA segment of bacteriophage phi 6: placement of the genes of membrane-associated proteins.

The genome of the lipid-containing bacteriophage phi 6 contains three segments of double-stranded RNA. We have determined the nucleotide sequence of cDNA derived from the middle-size RNA segment. The coding sequences of three proteins on this segment were identified on the basis of size and the correlation of predicted N-terminal amino acid sequences with those found through the analysis of isolated proteins. In contrast to our results with the small phi 6 dsRNA segment, the open reading frames are not tightly clustered. The homologous terminal noncoding regions between the middle and small dsRNA segments are found to be more extensive than RNA sequencing had previously indicated.

Amino Acid Sequence↗
PubMed1988

Production of a polyhedral particle in Escherichia coli from a cDNA copy of the large genomic segment of bacteriophage phi 6.

A polyhedral particle that resembles in composition and structure the procapsid of bacteriophage phi 6 was produced in Escherichia coli containing cDNA copies of the entire large genomic segment inserted into expression vector plasmids under the control of lac or tac promoters. The particles were composed of proteins P1, P2, P4, and P7 in the same stoichiometry as in the intact virion. In electron micrographs of negatively stained samples, the particles appeared as hexagons, stars, or rings of 10 knobs, which are characteristic of the five-, three-, and twofold axes of symmetry characteristic of phi 6 procapsids. Stable particles were also produced from cDNA deletions that produce only P1 and P4. Other cDNA deletions producing P1 and P7 and P1 alone resulted in unstable particles which could only be visualized in electron micrographs of thin sections of E. coli transformed by the recombinant plasmids. Our results indicate that the assembly of the phi procapsid is independent of other phage proteins and of normal phage RNA.

Bacteriophages↗
PubMed1988

Nucleotide sequence of the large double-stranded RNA segment of bacteriophage phi 6: genes specifying the viral replicase and transcriptase.

The genome of the lipid-containing bacteriophage phi 6 contains three segments of double-stranded RNA. We determined the nucleotide sequence of cDNA derived from the largest RNA segment (L). This segment specifies the procapsid proteins necessary for transcription and replication of the phi 6 genome. The coding sequences of the four proteins on this segment were identified on the basis of size and the correlation of predicted N-terminal amino acid sequences with those found through analysis of isolated proteins. This report completes the sequence analysis of phi 6. This constitutes the first complete sequence of a double-stranded RNA genome virus.

Amino Acid Sequence↗
PubMed1986

Nucleotide sequence of the small double-stranded RNA segment of bacteriophage phi 6: novel mechanism of natural translational control.

The lipid-containing bacteriophage phi 6 has a genome composed of three segments of double-stranded RNA. We determined the nucleotide sequence of a cDNA copy of the smallest RNA segment. The coding sequences of the four proteins on this segment were identified. These sequences were clustered. Three of the genes had overlapping initiation-termination codons. All noncoding sequences were at the ends of the molecule. The genes of the small double-stranded RNA segment comprised two translational polarity groups. We propose that the translational coupling is the result of an inability of ribosomes to bind independently to two of the four genes. Translation of these genes occurred when ribosomes were delivered to them by translation of an upstream gene.

Bacteriophages↗