The template specificity of bacteriophage Phi6 RNA polymerase. (65/87)

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Subunit folds and maturation pathway of a dsRNA virus capsid. (66/87)

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Electrophoretic mobility confirms reassortment bias among geographic isolates of segmented RNA phages. (67/87)

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Rescue of maturation off-pathway products in the assembly of Pseudomonas phage phi 6. (68/87)

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Double-stranded RNA bacteriophage phi 6 protein P4 is an unspecific nucleoside triphosphatase activated by calcium ions. (69/87)

Double-stranded RNA bacteriophage phi 6 has an envelope surrounding the nucleocapsid (NC). The NC is composed of a surface protein, P8, and proteins P1, P2, P4, and P7, which form a dodecahedral polymerase complex enclosing the segmented viral genome. Empty polymerase complex particles (procapsids) package positive-sense viral single-stranded RNAs provided that energy is available in the form of nucleoside triphosphates (NTPs). Photoaffinity labelling of both the NC and the procapsid has earlier been used to show that ATP binds to protein P4 and that the NC hydrolyzes NTPs. Using the NC and the NC core particles (NCs lacking surface protein P8) and purified protein P4, we demonstrate here that multimeric P4 is the active NTPase. Isolation of multimeric P4 is successful only in the presence of NTPs. The activity of P4 is the same in association with the viral particles as it is in pure form. P4 is an unspecific NTPase hydrolyzing ribo-NTPs, deoxy NTPs, and dideoxy NTPs to the corresponding nucleoside diphosphates. The Km of the reaction for ATP, GTP, and UTP is around 0.2 to 0.3 mM. The NTP hydrolysis by P4 absolutely requires residual amounts of Mg2+ ions and is greatly activated when the Ca2+ concentration reaches 0.5 mM. Competition experiments indicate that Mg2+ and Ca2+ ions have approximately equal binding affinities for P4. They might compete for a common binding site. The nucleotide specificity and enzymatic properties of the P4 NTPase are similar to the NTP hydrolysis reaction conditions needed to translocate and condense the viral positive-sense RNAs to the procapsid particle.  (+info)

The large genome segment of dsRNA bacteriophage phi6 is the key regulator in the in vitro minus and plus strand synthesis. (70/87)

Bacteriophage phi6 is a double-stranded RNA (dsRNA) virus that has a genome composed of three linear dsRNA segments (L, M, S). These are encapsidated into a dodecahedral procapsid particle consisting of proteins P1, P2, P4, and P7. Empty preformed procapsids are able to package the plus-sense single-stranded RNA (ssRNA) of each genome segment, to synthesize the corresponding minus strands ("replication") to form dsRNA segments, and to continue to the plus strand synthesis ("transcription") in which the dsRNA segments are used as templates in production of plus-sense ssRNA. In this study, we have investigated the requirements for the switch-on of minus and plus strand syntheses. We show that there exists an inverse relationship between regulation of the ssRNA packaging and minus strand synthesis. The packaging of single-stranded l, which has previously been shown to be packaged as the last, is the necessary signal for the onset of the minus strand synthesis. The absolute requirement for plus strand synthesis is minus strand synthesis of l, but in addition, the minus strand synthesis of m and the packaging of s segment are needed for efficient plus strand synthesis. Furthermore, the second nucleotide at the 5'-end of each segment regulates the extent of the transcription.  (+info)

Interference with bacteriophage phi 6 genomic RNA packaging by hairpin structures. (71/87)

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.  (+info)

Differentiation between minus- and plus-strand synthesis: polymerase activity of dsRNA bacteriophage phi 6 in an in vitro packaging and replication system. (72/87)

Empty procapsids of the segmented dsRNA virus phi 6, produced in Escherichia coli from a cloned L genome segment, package plus-strand phi 6 ssRNA genomic segments, synthesize minus strands, and transcribe the newly formed dsRNA templates. Procapsids can be restricted to minus-strand synthesis by high concentrations of CaCl2 or low concentrations of nucleotides, enabling us to separate the viral minus-strand (replication) and plus-strand (transcription) RNA-dependent RNA polymerase activities in vitro. Reaction conditions for minus-strand synthesis were optimized. Plus-strand synthesis by procapsids could be activated by binding of purine nucleoside triphosphates to a low-affinity NTP-binding site. The second 5'-terminal nucleotide of the phi 6 plus-sense ssRNA L genomic segment is important for determining the level of transcription of that segment and the generation of infectious procapsids.  (+info)