Abstract:P2, an RNA-directed RNA polymerase (RdRP), is encoded on the largest of the three segments of the double-stranded RNA genome of cystoviruses. P2 performs the dual tasks of replication and transcription de novo on single-stranded RNA templates, and plays a critical role in the viral life-cycle. Work over the last few decades has yielded a wealth of biochemical and structural information on the functional regulation of P2, on its role in the spatiotemporal regulation of RNA synthesis and its variability across t… Show more
“…It is clear from the recent isolations of Cystoviridae from multiple environments, with only a single member infecting a Gram-positive host, that there are many more RNA phages yet to be discovered. Recently, Alphonse and Ghose (2017) examined known Cystoviridae using their encoded RdRp [ 32 ]. While ssRNA phage genomes have high mutation rates [ 74 ], RdRp appears to be conserved amongst RNA phage genomes and thus might be a good candidate as a genetic signature to identify further RNA phage sequences.…”
Section: Cystoviridaementioning
confidence: 99%
“…See text regarding gene information. (This figure was reproduced based on other images [ 28 , 32 , 33 ]).…”
The number of novel bacteriophage sequences has expanded significantly as a result of many metagenomic studies of phage populations in diverse environments. Most of these novel sequences bear little or no homology to existing databases (referred to as the “viral dark matter”). Also, these sequences are primarily derived from DNA-encoded bacteriophages (phages) with few RNA phages included. Despite the rapid advancements in high-throughput sequencing, few studies enrich for RNA viruses, i.e., target viral rather than cellular fraction and/or RNA rather than DNA via a reverse transcriptase step, in an attempt to capture the RNA viruses present in a microbial communities. It is timely to compile existing and relevant information about RNA phages to provide an insight into many of their important biological features, which should aid in sequence-based discovery and in their subsequent annotation. Without comprehensive studies, the biological significance of RNA phages has been largely ignored. Future bacteriophage studies should be adapted to ensure they are properly represented in phageomic studies.
“…It is clear from the recent isolations of Cystoviridae from multiple environments, with only a single member infecting a Gram-positive host, that there are many more RNA phages yet to be discovered. Recently, Alphonse and Ghose (2017) examined known Cystoviridae using their encoded RdRp [ 32 ]. While ssRNA phage genomes have high mutation rates [ 74 ], RdRp appears to be conserved amongst RNA phage genomes and thus might be a good candidate as a genetic signature to identify further RNA phage sequences.…”
Section: Cystoviridaementioning
confidence: 99%
“…See text regarding gene information. (This figure was reproduced based on other images [ 28 , 32 , 33 ]).…”
The number of novel bacteriophage sequences has expanded significantly as a result of many metagenomic studies of phage populations in diverse environments. Most of these novel sequences bear little or no homology to existing databases (referred to as the “viral dark matter”). Also, these sequences are primarily derived from DNA-encoded bacteriophages (phages) with few RNA phages included. Despite the rapid advancements in high-throughput sequencing, few studies enrich for RNA viruses, i.e., target viral rather than cellular fraction and/or RNA rather than DNA via a reverse transcriptase step, in an attempt to capture the RNA viruses present in a microbial communities. It is timely to compile existing and relevant information about RNA phages to provide an insight into many of their important biological features, which should aid in sequence-based discovery and in their subsequent annotation. Without comprehensive studies, the biological significance of RNA phages has been largely ignored. Future bacteriophage studies should be adapted to ensure they are properly represented in phageomic studies.
“…ϕ6 consists of two protein capsids surrounded by a lipid coat . The inner capsid, also known as the polymerase complex, encloses the three segments of a double-stranded RNA (dsRNA) genome and is assembled from four distinct protein components (Figure S1): 120 copies of P1, the major capsid protein, are arranged as asymmetric dimers on an icosahedral T = 1 lattice; 72 copies of the NTPase P4 assemble as hexamers on the 5-fold axes; ∼10–12 copies of the RNA-directed RNA polymerase P2 are located on the 3-fold axes; and ∼60 copies of the accessory protein P7 are most likely located near the 3-fold axes in the proximity of P2.…”
While solid-state nuclear magnetic resonance (ssNMR) has emerged as a powerful technique for studying viral capsids, current studies are limited to capsids formed from single proteins or single polyproteins. The ability to selectively label individual protein components within multiprotein viral capsids and the resulting spectral simplification will facilitate the extension of ssNMR techniques to complex viruses. In vitro capsid assembly by combining individually purified, labeled, and unlabeled components in NMR quantities is not a viable option for most viruses. To overcome this barrier, we present a method that utilizes sequential protein expression and in cell assembly of component-specifically labeled viral capsids in amounts suitable for NMR studies. We apply this approach to purify capsids of bacteriophage ϕ6 isotopically labeled on only one of its four constituent protein components, the NTPase P4. Using P4-labeled ϕ6 capsids and the sensitivity enhancement provided by dynamic nuclear polarization, we illustrate the utility of this method to enable ssNMR studies of complex viruses.
“…However, while for some virus families polymerase structure and function has been extensively studied (e.g. poliovirus ( Peersen, 2017 ), flavivirus ( Lu and Gong, 2017 ), cystovirus ( Alphonse and Ghose, 2017 ) and retrovirus ( Menéndez-Arias et al, 2017 )), there is an important lack of structural information in the case of alphavirus ( Pietilä et al, 2017 ), nidovirus ( Posthuma et al, 2017 ) or poxvirus ( Czarnecki and Traktman, 2017 ). Fig.…”
mentioning
confidence: 99%
“…The polymerases of double-stranded RNA viruses (dsRNA) are represented by P2, the RdRp of the Pseudomonas phage ϕ6 ( Alphonse and Ghose, 2017 ). Cystoviruses such as ϕ6 have segmented genomes and their virions contain the polymerase P2.…”
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