Biomedical subjects

L Mindich

Publications and source records attributed to L Mindich.

At least 19 recordsLinked to original sources

PubMed2001

Reverse genetics and recombination in Phi8, a dsRNA bacteriophage.

Bacteriophage Phi8 has a genome of three dsRNA segments. It is able to acquire plasmid transcripts of cDNA copies of the genomic segments as replacements of its resident chromosomes. It is also able to effect recombination between the plasmid transcripts and the resident chromosomes. Depending upon the extent of sequence identity between the plasmid transcript and the resident chromosome, the recombination can be homologous or heterologous. Homologous recombination has not previously been reported for viruses with double-stranded RNA genomes.

Base Sequence↗

PubMed2000

Characterization of phi 13, a bacteriophage related to phi 6 and containing three dsRNA genomic segments.

The three dsRNA genomic segments of bacteriophage Phi 13 were copied as cDNA and the nucleotide sequences were determined. The organization of the genome is similar to that of Phi 6, and there is significant similarity in the amino acid sequences of the proteins of the polymerase complex and one of the membrane proteins, P6. There is little or no similarity in the nucleotide sequences. Several features of the viral proteins differ markedly from those of Phi 6. Although both phages are covered by a lipid-containing membrane, the protein compositions are different. The host attachment protein consists of two peptides rather than one and the phage attaches directly to the LPS of the host rather than to a Type IV pilus. Despite the differences in the structure of the membranes, the two viruses can successfully exchange the genes for host attachment proteins and thereby change their host specificities.

Bacteriophage phi 6↗

PubMed2000

Characterization of phi8, a bacteriophage containing three double-stranded RNA genomic segments and distantly related to Phi6.

The three double-stranded RNA genomic segments of bacteriophage Phi8 were copied as cDNA, and their nucleotide sequences were determined. Although the organization of the genome is similar to that of Phi6, there is no similarity in either the nucleotide sequences or the amino acid sequences, with the exception of the motifs characteristic of viral RNA polymerases that are found in the presumptive polymerase sequence. Several features of the viral proteins differ markedly from those of Phi6. Although both phages are covered by a lipid-containing membrane, the protein compositions are very different. The most striking difference is that protein P8, which constitutes a shell around the procapsid in Phi6, is part of the membrane in Phi8. The host attachment protein consists of two peptides rather than one and the phage attaches directly to the lipopolysaccharide of the host rather than to a type IV pilus. The host range of Phi8 includes rough strains of Salmonella typhimurium and of pseudomonads

Amino Acid Motifs↗

PubMed1999

A symmetry mismatch at the site of RNA packaging in the polymerase complex of dsRNA bacteriophage phi6.

The polymerase complex of the enveloped double-stranded RNA (dsRNA) bacteriophage phi6 fulfils a similar function to those of other dsRNA viruses such as Reoviridae. The phi6 complex comprises protein P1, which forms the shell, and proteins P2, P4 and P7, which are involved in RNA synthesis and packaging. Icosahedral reconstructions from cryo-electron micrographs of recombinant polymerase particles revealed a clear dodecahedral shell and weaker satellites. Difference imaging demonstrated that these weak satellites were the sites of P4 and P2 within the complex. The structure determined by icosahedral reconstruction was used as an initial model in an iterative reconstruction technique to examine the departures from icosahedral symmetry. This approach showed that P4 and P2 contribute to structures at the 5-fold positions of the icosahedral P1 shell which lack 5-fold symmetry and appear in variable orientations. Reconstruction of isolated recombinant P4 showed that it was a hexamer with a size and shape matching the satellite. Symmetry mismatch between the satellites and the shell could play a role in RNA packaging akin to that of the portal vertex of dsDNA phages in DNA packaging. This is the first example of dsRNA virus in which the structure of the polymerase complex has been determined without the assumption of icosahedral symmetry. Our result with phi6 illustrates the symmetry mismatch which may occur at the sites of RNA packaging in other dsRNA viruses such as members of the Reoviridae.

Bacteriophage phi 6↗

PubMed1999

Reverse genetics of dsRNA bacteriophage phi 6.

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Bacteriophage phi 6↗

PubMed1999

Isolation of additional bacteriophages with genomes of segmented double-stranded RNA.

Eight different bacteriophages were isolated from leaves of Pisum sativum, Phaseolus vulgaris, Lycopersicon esculentum, Daucus carota sativum, Raphanus sativum, and Ocimum basilicum. All contain three segments of double-stranded RNA and have genomic-segment sizes that are similar but not identical to those of previously described bacteriophage phi6. All appear to have lipid-containing membranes. The base sequences of some of the viruses are very similar but not identical to those of phi6. Three of the viruses have little or no base sequence identity to phi6. Two of the viruses, phi8 and phi12, contain proteins with a size distribution very different from that of phi6 and do not package genomic segments of phi6. Whereas phi6 attaches to host cells by means of a pilus, several of the new isolates attach directly to the outer membrane. Although the normal hosts of these viruses seem to be pseudomonads, those viruses that attach directly to the outer membrane can establish carrier states in Escherichia coli or Salmonella typhimurium. One of the isolates, phi8, can form plaques on heptoseless strains of S. typhimurium.

Bacteriophages↗

PubMed1999

Precise packaging of the three genomic segments of the double-stranded-RNA bacteriophage phi6.

Bacteriophage phi6 has a genome of three segments of double-stranded RNA. Each virus particle contains one each of the three segments. Packaging is effected by the acquisition, in a serially dependent manner, of the plus strands of the genomic segments into empty procapsids. The empty procapsids are compressed in shape and expand during packaging. The packaging program involves discrete steps that are determined by the amount of RNA inside the procapsid. The steps involve the exposure and concealment of binding sites on the outer surface of the procapsid for the plus strands of the three genomic segments. The plus strand of segment S can be packaged alone, while packaging of the plus strand of segment M depends upon prior packaging of S. Packaging of the plus strand of L depends upon the prior packaging of M. Minus-strand synthesis begins when the particle has a full complement of plus strands. Plus-strand synthesis commences upon the completion of minus-strand synthesis. All of the reactions of packaging, minus-strand synthesis, and plus-strand synthesis can be accomplished in vitro with isolated procapsids. Live-virus constructions that are in accord with the model have been prepared. Mutant virus with changes in the packaging program have been isolated and analyzed.

Bacteriophage phi 6↗

PubMed1998

Isolation of a mutant that changes genomic packaging specificity in phi6.

Bacteriophage phi6 has a genome of three segments of double-stranded RNA enclosed in a polyhedral procapsid. Plus strand transcripts of the segments are packaged in a serially dependent fashion in which S can package alone, M depends on S, and L depends on S and M. We have isolated a mutant form of the virus in the carrier state that has lost segment S. This finding presented an apparent anomaly with respect to the packaging program. Sequencing of gene 1 of segment L in this virus showed a translational change of arginine to glycine at the 14th position. Procapsids prepared from cDNA containing this mutation show behavior in in vitro packaging that is consistent with the phenotype of the mutant virus. The procapsids are able to package segment S alone, but this RNA is present in reduced amounts when the other segments are present. Segments M and L package without dependence on segment S. The mutant virus appears to produce procapsids that are at the second stage of the packaging program.

Bacteriophage phi 6↗

PubMed1998

Directed changes in the number of double-stranded RNA genomic segments in bacteriophage phi6.

Bacteriophage Phi6 has a genome of three segments of double-stranded RNA. The segments are designated S, M, and L. Each segment has a unique packaging site, pac, near the 5' end of the plus strand. The plus strands of the segments are normally packaged in the order S, M, L. Chimeras of segment M and S in which segment M is at the 5' end of the plus strand can be stably incorporated into the virion; however, an independent segment S must be included along with normal segment L, even if it contains no active genes. A chimera of segment M and S in which segment S is at the 5' end of the plus strand can be stably incorporated into the virion along with normal segment L to form a two-segment genome. A chimera of segments S, M, and L in which the packaging sequence is that of S can also form a stable nonsegmented genome. These findings are consistent with a model that we have proposed for the packaging of the Phi6 genome.

Bacteriophage phi 6↗

PubMed1998

Mutational analysis of the role of nucleoside triphosphatase P4 in the assembly of the RNA polymerase complex of bacteriophage phi6.

Bacteriophage phi6 is a complex enveloped double-stranded RNA virus with a segmented genome and replication strategy quite similar to that of the Reoviridae. An in vitro packaging and replication system using purified components is available. The positive-polarity genomic segments are translocated into a preformed polymerase complex (procapsid) particle. This particle is composed of four proteins: the shell-forming protein P1, the RNA polymerase P2, and two proteins active in packaging. Protein P7 is involved in stable packaging, and protein P4 is a homomultimeric potent nucleoside triphosphatase that provides the energy for the RNA translocation event. In this investigation, we used mutational analysis to study P4 multimerization and assembly. P4 is assembled onto a preformed particle containing proteins P2 and P7 in addition to P1. Only simultaneous production of P1 and P4 in the same cell leads to P4 assembly on P1 alone, whereas the P1 shell is incompetent for accepting P4 if produced separately. The C-terminal part of P4 is essential for particle assembly but not for multimerization or enzymatic activity. Altering the P4 nucleoside triphosphate binding site destroys the ability to form multimers.

Bacteriophage phi 6↗

PubMed1997

Stoichiometric packaging of the three genomic segments of double-stranded RNA bacteriophage phi6.

A model that explains the stoichiometric packaging of the chromosomes of phi6, a bacteriophage with a genome of three unique double-stranded RNA segments, is proposed and supported. Ordered switches in packaging specificity and RNA synthesis are determined by the amount of RNA within the procapsid. The plus strand of segment S binds to one of several sites on the outside of the empty procapsid. The RNA enters and the procapsid expands so that the S sites are lost and M sites appear. Packaging of segment M results in the loss of the M sites and the appearance of the L sites. Packaging of L readies the particle for minus-strand synthesis. If any of the segments is less than normal size, packaging of that class of segments continues until the normal content of RNA for that segment is packaged and the binding sites then change.

Bacteriophages↗

PubMed1997

An in vitro system for the investigation of heterologous RNA recombination.

Bacteriophage Phi6 has a genome of three segments of double-stranded RNA enclosed in a polyhedral procapsid. Purified procapsids are capable of the specific packaging of viral plus strands and the synthesis of their complementary minus strands. The genomic segments of Phi6 are capable of heterologous recombination. We have prepared an in vitro system containing purified procapsids that is capable of packaging plus strands of the genomic segments and synthesizing minus strands on these templates. The system generates heterologous recombination products when stimulated by having one of the plus strands incapable of serving as a template for minus strand synthesis. Recombinants were produced upon transfection of spheroplasts with the in vitro packaged and replicated RNA. Sites of recombination were not found to be localized in particular regions of either the donor or the recipient strands.

Bacteriophage phi 6↗

PubMed1995

Acquisition of a fourth genomic segment in bacteriophage phi 6, a bacteriophage with a genome of three segments of dsRNA.

Bacteriophage phi 6 has a genome of three segments of double-stranded RNA enclosed in a polyhedral procapsid. Packaging of individual segments is dependent upon unique packaging sequences near the 5' ends of the segments. We have prepared deletions in segments L and M that decrease their size by half. Phages with these deletions can be propagated on host strains carrying plasmids with complementing genes. The deletion segments are present in two copies per virion. Phage carrying a deletion segment can acquire the transcript of the complementing plasmid if the latter has a packaging sequence. If the packaging sequence is homologous to that of the deletion segment, acquisition occurs at high frequency. If it is heterologous, then recombination exchanges the heterologous packaging sequence for a homologous one or it attaches the transcript to one of the other genomic segments.

Bacteriophages↗

PubMed1995

Packaging of multiple copies of reduced-size genomic segments by bacteriophage phi 6.

Bacteriophage phi 6 has a genome of three segments of double-stranded RNA enclosed in a polyhedral procapsid. The preformed procapsid is capable of packaging plus-strand transcripts of the genomic segments in an in vitro reaction. Packaging of individual segments is dependent upon unique packaging sequences of about 300 nucleotides near the 5' ends of the segments. We have prepared segments L, M, and S with internal deletions that decrease their size by as much as sixfold without affecting either their packaging sequences or their 3' ends. Although packaging of genomic segments is normally very precise, with only one of each in a procapsid, these smaller segments are packaged in multiples such that the total number of nucleotides for each segment class approaches that of the normal genomic segment.

Bacteriophages↗

PubMed1995

In vitro packaging of individual genomic segments of bacteriophage phi 6 RNA: serial dependence relationships.

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 packaging of the three segments shows a strong order of dependence in that segment S packages alone, but segment M requires S and and segment L requires S and M for efficient packaging. Packaging of individual segments is dependent on unique packaging sequences of about 200 nucleotides near the 5' ends of the segments. Deletions that invade these regions destroy packaging competence for the particular segment and for the dependent segments as well. In the presence of 2 mM phosphate and at magnesium ion concentrations above 4 mM, packaging becomes progressively more independent and ultimately nonspecific with respect to phi 6 sequences.

Bacteriophage phi 6↗

PubMed1995

Interference with bacteriophage phi 6 genomic RNA packaging by hairpin structures.

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. Minus-strand synthesis within the procapsid then results in the production of the double-stranded RNA genome. When plus-strand transcripts contain strong hairpin structures near the 3' ends, they are subject to heterologous recombination to remove the hairpins. We now find that the sequences bounded by the hairpins as well as those 3' to them are excluded from particles in packaging reactions. This finding implies that packaging occurs from the 5' end and that the explanation for the facilitation of recombination by the hairpin structures is the lack of entry of the 3' ends rather than a difficulty of progressing through the hairpin by the phage polymerase. Packaging of segment M is dependent on the packaging of segment S. An S segment containing a strong hairpin is able to facilitate the packaging of segment M. This result implies that there is more than one entry pore into the procapsid.

Bacteriophage phi 6↗

PubMed1994

Identification of the packaging regions within the genomic RNA segments of bacteriophage phi 6.

Bacteriophage phi 6↗

PubMed1994

RNA structural requirements for stability and minus-strand synthesis in the dsRNA bacteriophage phi 6.

Bacteriophage phi 6 has a genome consisting of three segments of double-stranded RNA designated L, M, and S. Each virion contains one of each genomic segment. Empty procapsids can package plus-strand transcripts of the genomic segments if the 5' regions are intact. Minus-strand synthesis takes place if all three segments are packaged and if the 3' end of the segment is intact. The 3' ends of the segments contain four hairpin structures within a region of high sequence conservation. We now show that removal of parts of this region leads to progressive but limited loss of ability to support minus-strand synthesis. The defective 3' ends can be corrected by heterologous recombination with the termini of other segments. Segments that have small deletions in the conserved region and that support apparently normal minus-strand synthesis are highly recombinogenic.

Bacteriophage phi 6↗