Elsevier

Virus Research

Volume 101, Issue 1, April 2004, Pages 45-55
Virus Research

RNA-dependent RNA polymerases of dsRNA bacteriophages

https://doi.org/10.1016/j.virusres.2003.12.005Get rights and content

Abstract

Genome replication and transcription of riboviruses are catalyzed by an RNA-dependent RNA polymerase (RdRP). RdRPs are normally associated with other virus- or/and host-encoded proteins that modulate RNA polymerization activity and template specificity. The polymerase complex of double-stranded dsRNA viruses is a large icosahedral particle (inner core) containing RdRP as a minor constituent. In φ6 and other dsRNA bacteriophages from the Cystoviridae family, the inner core is composed of four virus-specific proteins. Of these, protein P2, or Pol subunit, has been tentatively identified as RdRP by sequence comparisons, but the role of this protein in viral RNA synthesis has not been studied until recently. Here, we overview the work on the Pol subunits of φ6 and related viruses from the standpoints of function, structure and evolution.

Introduction

In most dsRNA viruses, genomic RNA is delivered into infected cell inside a transcriptionally active icosahedral particle that can generate (+) sense RNA copies. These can either serve as templates for protein synthesis (mRNAs) or be sequestered into newly formed subviral particles, where they are converted back to the double-stranded form. Because many dsRNA viruses have segmented genomes with the number of segments ranging from two in Birnaviridae to three in Cystoviridae and up to 12 in Reoviridae, it is logistically remarkable how one copy of each segment can be specifically picked up by an individual particle to reconstitute a functional genome (Mindich, 1999, Mindich, 2003, Patton and Spencer, 2000, Mertens and Diprose, 2003). Another important feature of the life cycle of dsRNA viruses is RNA metabolism, catalyzed by viral RNA-dependent RNA polymerase (RdRP). Since each virus encodes only one protein species carrying RdRP motifs, the RdRP is expected to catalyze both transcription of genomic dsRNA into (+) sense copies and the replication of the (+)ssRNA back into the dsRNA form. This review summarizes recent work on RdRP from bacteriophage φ6 and other Cystoviridae discussing its implications for the dsRNA virus research and the RNA virology in general.

Section snippets

Experimental systems for studying RNA synthesis in dsRNA viruses

Several systems have been used to address different aspects of RNA metabolism of dsRNA viruses. Initially, in vitro transcription systems have been developed based on purified viruses or cores containing dsRNA genomes (particle-based transcription). These were reported for reovirus (Skehel and Joklik, 1969), bacteriophage φ6 (Partirdge et al., 1979, Van Etten et al., 1973), cypovirus (Shimotohno and Miura, 1973), infectious pancreatic necrosis virus (Cohen, 1975), yeast virus-like particles (

Functions of Pol subunit in cystovirus life cycle

Bacteriophage φ6 infects the plant pathogenic bacterium Pseudomonas syringae. The φ6 genome consists of three dsRNA segments L, M and S. In addition to Pol (P2 protein), the icosahedral polymerase complex of φ6 contains three other proteins that either form a structural framework of the particle (P1) or are needed for RNA packaging and particle stability (P4 and P7). For a long time, φ6 has been the only known species in the Cystoviridae family. However, the family has been recently updated

Structural studies on RNA-dependent RNA polymerases

The first known structure of a nucleic acid polymerase was that of the Klenow fragment of E. coli DNA-dependent DNA polymerase (DdDP) I (Ollis et al., 1985). The shape of the molecule resembled a right hand with ‘palm’, ‘fingers’ and ‘thumb’ domains. Despite the absence of sequence homology with DdDP, reverse transcriptase (RT) and single-subunit DNA-dependent RNA polymerases (DdRP) possess a similar right-hand architecture (Kohlstaedt et al., 1992, Sousa et al., 1993, Steitz et al., 1993).

The

Molecular mechanism for initiating RNA-dependent RNA polymerization de novo

The structures of φ6 Pol complexes with NTPs and/or template suggest a model leading to the formation of the initiation complex for RNA polymerization (Butcher et al., 2001). Fig. 4 shows the likely sequence of events that take place during formation of the initiation complex. Steps I–IV, the template enters the tunnel and is locked in place by interactions with the specificity pocket, S. Site I is occupied by NTPs, presumably in rapid exchange. Step V, the D1 GTP binds to the initiation

RNA virus evolution

Similar tertiary structures are often formed by distant amino acid sequences. As one possibility, this may be a result of convergent evolution, when two evolutionary unrelated proteins independently “invent” analogous, functionally justified folds. For example, mammalian Polβ (DdDP) contains some structural elements common for other polymerases and uses universal two-metal-ion mechanism of catalysis, but because of its unique topology this protein is thought to be a product of independent line

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    Present address: Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA.

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