Xueying Qiao

In vitro packaging of the bacteriophage φ6 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.

Characterization of Φ2954, a newly isolated bacteriophage containing three dsRNA genomic segments

BackgroundBacteriophage Φ12 is a member of the Cystoviridae and is distinct from Φ6, the first member of that family. We have recently isolated a number of related phages and five showed high similarity to Φ12 in the amino acid sequences of several proteins. Bacteriophage Φ2954 is a member of this group.ResultsΦ2954 was isolated from radish leaves and was found to have a genome of three segments of double-stranded RNA (dsRNA), placing it in the Cystoviridae. The base sequences for many of the genes and for the segment termini were similar but not identical to those of bacteriophage Φ12. However, the host specificity was for the type IV pili of Pseudomonas syringae HB10Y rather than for the rough LPS to which Φ12 attaches. Reverse genetics techniques enabled the production of infectious phage from cDNA copies of the genome. Phage were constructed with one, two or three genomic segments. Phage were also produced with altered transcriptional regulation. Although the pac sequences of Φ2954 show no similarity to those of Φ12, segment M of Φ2954 could be acquired by Φ12 resulting in a change of host specificity.ConclusionsWe have isolated a new member of the bacteriophage family Cystoviridae and find that although it shows similarity to other members of the family, it has unique properties that help to elucidate viral strategies for genomic packaging and gene expression.

The role of host protein YajQ in the temporal control of transcription in bacteriophage Φ6

Bacteriophage Φ6 contains three dsRNA genomic segments L, M, and S. The RNA is located inside a core particle composed of multiple copies of a major structural protein, an RNA-dependent RNA polymerase, a hexameric NTPase, and an auxiliary protein. The virion RNA polymerase in the core particle transcribes segments M and S in vitro. Yet early in infection, the transcription of L is highly active. Late in infection, transcription of L is low, and that of M and S is high. A host protein encoded by yajQ is responsible for the activation of L transcription. Knockout mutants of yajQ do not support the replication of Φ6, although they do support the replication of distantly related members of the Cystoviridae. Φ6 can mutate to independence of YajQ. This requires two mutations in the gene for the RNA-dependent RNA polymerase. YajQ acts indirectly on the polymerase by binding to P1, the major structural protein of the core. Previous studies have shown that the activity of the polymerase in the core is controlled by the conformation of the core particle structure.

Role of host protein glutaredoxin 3 in the control of transcription during bacteriophage Φ2954 infection

Bacteriophage Φ2954 contains three dsRNA genomic segments, designated L, M, and S. The RNA is located inside a core particle composed of multiple copies of a major structural protein, an RNA-dependent RNA polymerase, a hexameric NTPase, and an auxiliary protein. The core particle is covered by a shell of protein P8, and this structure is enclosed within a lipid-containing membrane. We have found that normal infection of the host Pseudomonas syringae is dependent on the action of a host protein, glutaredoxin 3 (GrxC). GrxC removes the P8 shell from the infecting particle and binds to the inner core. Removal of P8 activates the transcription of segments S and M, whereas binding of GrxC to the core particle activates the transcription of segment L. The differences in transcription behavior are due to the preference of the polymerase for G as the first base of the transcript. Transcripts of segments S and M begin with GCAA, whereas those of segment L begin with ACAA. The binding of GrxC to the particle results in changes in polymerase activity. Mutations resulting in independence of GrxC are found in the gene for protein P1, the major structural protein of the inner core particle.

Temporal control of message stability in the life cycle of dsRNA bacteriophage φ8

ABSTRACT The cystoviruses have genomes of three double-stranded RNA segments. The genes of the L transcript are expressed early in infection, while those of M and S are expressed late. In all cystovirus groups but one, the quantity of the L transcript late in infection is lower than those of the other two because of transcriptional control. In bacteriophage Φ8 and its close relatives, transcription of L is not controlled; instead, the L transcript is turned over rapidly late in infection. The three messages are produced in approximately equal amounts early in infection, but the amount of L is less than 10% of the amounts of the others late in infection. The decay of the Φ8 L message depends upon the production of protein Hb, which is encoded in segment L. It also depends upon a target site within the H gene region. Phage mutants lacking either the Hb gene or the target region do not show the late control of L message quantity. In addition to having a role as a negative regulator, Hb functions to neutralize the activity of protein J, encoded by segment S, which causes the degradation of all viral transcripts.

Temporal Control of Message Stability in the Life Cycle of Double-Stranded RNA Bacteriophage Φ8

ABSTRACT The cystoviruses have genomes of three double-stranded RNA segments. The genes of the L transcript are expressed early in infection, while those of M and S are expressed late. In all cystovirus groups but one, the quantity of the L transcript late in infection is lower than those of the other two because of transcriptional control. In bacteriophage Φ8 and its close relatives, transcription of L is not controlled; instead, the L transcript is turned over rapidly late in infection. The three messages are produced in approximately equal amounts early in infection, but the amount of L is less than 10% of the amounts of the others late in infection. The decay of the Φ8 L message depends upon the production of protein Hb, which is encoded in segment L. It also depends upon a target site within the H gene region. Phage mutants lacking either the Hb gene or the target region do not show the late control of L message quantity. In addition to having a role as a negative regulator, Hb functions to neutralize the activity of protein J, encoded by segment S, which causes the degradation of all viral transcripts.