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Role of host protein glutaredoxin 3 in the control of transcription during bacteriophage Phi2954 infection.

ABSTRACT: Bacteriophage Phi2954 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.

SUBMITTER: Qiao J

PROVIDER: S-EPMC2851929 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Role of host protein glutaredoxin 3 in the control of transcription during bacteriophage Phi2954 infection.

Qiao Jian J Qiao Xueying X Sun Yang Y Mindich Leonard L

Proceedings of the National Academy of Sciences of the United States of America 20100315 13

Bacteriophage Phi2954 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 ...[more]

PMID: 20231437

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Project description:fRSL1 is a jumbo myovirus stably and lytically infecting the phytopathogenic bacterium Ralstonia solanacearum. In this study, we investigate the infection cycle of fRSL1 and provide a genomic, proteomic and transcriptomic view of this phage. Its 231-kbp genome sequence showed many genes lacking detectable homologs in the current databases and was vastly different from previously studied phage genomes. In addition to these orphan proteins, fRSL1 was found to encode several enzymes that are unique among known viruses. These include enzymes for the salvage pathway of NAD+ and for the biosynthetic pathways of lipid, carbohydrate and homospermidine. A chitinase-like protein was found to be a potential lysis enzyme. Our proteomics analysis suggests that fRSL1 virions contain at least 25 distinct proteins. We identified six of them including a tail sheath protein and a topoisomerase IB by N-terminal sequencing. Based on a DNA microarray analysis, we identified two transcription patterns. Total RNA was isolated from 3 ml of fRSL1-infected MAFF730138 cells (containing 1 x 108 CFU infected at moi 0.5 ~ 4) at 0, 10, 30, 90 min, and 30 h post infection (p.i.) using an RNAprotect Bacteria Reagent kit (Qiagen K.K., Tokyo, Japan) according to the manufacturer’s protocol. The integrity and concentration of total RNA were measured using a bioanalysis unit (Agilent 2100 Bioanalyser, Aligent Tech. Palo Alto, CA). Fluorescence-labeled antisense RNA was synthesized by direct incorporation of Cy5-UTP (GE Healthcare Bio-Science Corp., Piscataway, NJ), using each RNA sample and an RNA Transcript SureLABEL Core kit (Takara Bio Inc.). The labeled antisense RNAs were hybridized simultaneously to the microarray chip. DNA microarray preparation, hybridization, processing, scanning, and analyses were performed by Filgen Inc. (Nagoya Japan). For each fRSL1 ORF, 1 to 5 coding sequences of 35 to 40 nucleotide-long were synthesized. A total of 1,100 sequences were put in duplicate on a 40 x 55 array chip. The fluorescence images of hybridized microarrays were obtained with a GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA). The Array-Pro Analyzer Ver4.5 (Media Cybernetics, Inc., Silver Spring, MD) was used to determine the signal intensity of each spot and its local background. Scan data images were alalyzed using Microarray Data Analysis Tool Ver3.0 software (Filgen Inc.)

Dataset Information

Role of host protein glutaredoxin 3 in the control of transcription during bacteriophage Phi2954 infection.

Publications

Role of host protein glutaredoxin 3 in the control of transcription during bacteriophage Phi2954 infection.

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