RNA dependent RNA polymerase (RdRp) is one of the most versatile enzymes of RNA viruses that is indispensable for replicating the genome as well as for carrying out transcription. The core structural features of RdRps are conserved, despite the divergence in their sequences. The structure of RdRp resembles that of a cupped right hand and consists of fingers, palm and thumb subdomains. The catalysis involves the participation of conserved aspartates and divalent metal ions. Complexes of RdRps with substrates, inhibitors and metal ions provide a comprehensive view of their functional mechanism and offer valuable insights regarding the development of antivirals. In this article, we provide an overview of the structural aspects of RdRps and their complexes from the Group III, IV and V viruses and their structure-based phylogeny.

RNA Dependent RNA Polymerases: Insights from Structure, Function and Evolution

The first sequenced genome was that of the 3569-nucleotide single-stranded RNA (ssRNA) bacteriophage MS2. Despite the recent accumulation of vast amounts of DNA and RNA sequence data, only 12 representative ssRNA phage genome sequences are available from the NCBI Genome database (June 2019). The difficulty in detecting RNA phages in metagenomic datasets raises questions as to their abundance, taxonomic structure, and ecological importance. In this study, we iteratively applied profile hidden Markov models to detect conserved ssRNA phage proteins in 82 publicly available metatranscriptomic datasets generated from activated sludge and aquatic environments. We identified 15,611 nonredundant ssRNA phage sequences, including 1015 near-complete genomes. This expansion in the number of known sequences enabled us to complete a phylogenetic assessment of both sequences identified in this study and known ssRNA phage genomes. Our expansion of these viruses from two environments suggests that they have been overlooked within microbiome studies.

Expansion of known ssRNA phage genomes: From tens to over a thousand

https://www.cell.com/cell/pdf/S0092-8674(19)30902-X.pdf

Structure of the Respiratory Syncytial Virus Polymerase Complex

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.

RNA Phage Biology in a Metagenomic Era

In single-stranded RNA bacteriophages (ssRNA phages) a single copy of the maturation protein binds the genomic RNA (gRNA) and is required for attachment of the phage to the host pilus. For the canonical Allolevivirus Qβ the maturation protein, A 2 , has an additional role as the lysis protein, by its ability to bind and inhibit MurA, which is involved in peptidoglycan biosynthesis. Here, we determined structures of Qβ virions, virus-like particles, and the Qβ–MurA complex using single-particle cryoelectron microscopy, at 4.7-A, 3.3-A, and 6.1-A resolutions, respectively. We identified the outer surface of the β-region in A 2 as the MurA-binding interface. Moreover, the pattern of MurA mutations that block Qβ lysis and the conformational changes of MurA that facilitate A 2 binding were found to be due to the intimate fit between A 2 and the region encompassing the closed catalytic cleft of substrate-liganded MurA. Additionally, by comparing the Qβ virion with Qβ virus-like particles that lack a maturation protein, we observed a structural rearrangement in the capsid coat proteins that is required to package the viral gRNA in its dominant conformation. Unexpectedly, we found a coat protein dimer sequestered in the interior of the virion. This coat protein dimer binds to the gRNA and interacts with the buried α-region of A 2 , suggesting that it is sequestered during the early stage of capsid formation to promote the gRNA condensation required for genome packaging. These internalized coat proteins are the most asymmetrically arranged major capsid proteins yet observed in virus structures.

https://www.pnas.org/content/pnas/114/44/11697.full.pdf

Structures of Qβ virions, virus-like particles, and the Qβ–MurA complex reveal internal coat proteins and the mechanism of host lysis

Upon infection of Escherichia coli by bacteriophage Qβ, the virus-encoded β-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qβ replicase holoenzyme complex, which is responsible for amplifying the Qβ (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the β-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1-2) in the recognition of Qβ (+)-RNA by the Qβ replicase complex. We show how three basic residues of the β subunit form a patch located adjacent to the OB2 domain, and use NMR spectroscopy to demonstrate for the first time that OB2 is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Qβ replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the β-subunit and protein S1 cooperatively bind the (+)-stranded Qβ genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.

/pdf/structural-basis-for-rna-genome-recognition-during-3rect92s25.pdf

Structural basis for RNA-genome recognition during bacteriophage Qβ replication

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Structural basis for RNA-genome recognition during bacteriophage Qβ replication