In situ structures of rotavirus polymerase in action and mechanism of mRNA transcription and release

Abstract

Transcribing and replicating a double-stranded genome require protein modules to unwind, transcribe/replicate nucleic acid substrates, and release products. Here we present in situ cryo-electron microscopy structures of rotavirus dsRNA-dependent RNA polymerase (RdRp) in two states pertaining to transcription. In addition to the previously discovered universal “hand-shaped” polymerase core domain shared by DNA polymerases and telomerases, our results show the function of N- and C-terminal domains of RdRp: the former opens the genome duplex to isolate the template strand; the latter splits the emerging template-transcript hybrid, guides genome reannealing to form a transcription bubble, and opens a capsid shell protein (CSP) to release the transcript. These two “helicase” domains also extensively interact with CSP, which has a switchable N-terminal helix that, like cellular transcriptional factors, either inhibits or promotes RdRp activity. The in situ structures of RdRp, CSP, and RNA in action inform mechanisms of not only transcription, but also replication.

New Insights Into the Effect of Residue Mutation on the Rotavirus VP1 Function Using Molecular Dynamic Simulations

“…Residues 584-586 are located close to the NSP2-binding sites on VP1 [8] and their decrease in accessibility may affect this binding. The decrease in residue usage of residues 453, 922 and 991-999 of K419W mutant, as shown by the average of ΔrBC analysis, may affect the function of VP1 as residue 453 is located to NSP2-binding sites on VP1 (aa 442 to 458) [8] whereas residues 922 and 991-999 are located in the C-terminal domain, responsible for splitting the dsRNA product during transcription [7]. The decrease of interactions of Trp419 (K419W mutant) with Ser399 and His423, as shown by contact map analysis, is the result of the bottleneck displacement.…”

“…We focus in the present study on rotavirus RNA-directed RNA polymerase (RdRp), named VP1 protein and coded by VP1 gene, as one of the most important proteins in the viral replication; it is involved in both transcription and genome replication/packaging [4,5] in the presence of other proteins, such as VP2 protein; its N-terminal tether domain extends around VP1 and forms cradle that helps stabilize the enzyme in position [6], suggesting to regulate VP1 transitions between transcription and replication states [7].…”

“…The three dimensional structure of proteins is not static, the different residues can move, and thus, stabilize or break non-covalent interactions. To better understand the effect of the generated mutants, structural data need to be linked to the temporal context of the virus replication/transcription due to the presence of a recently published structure for a catalytically active form of VP1 protein [7,9].…”

“…The structural analysis of RVA proteins upon mutations may enhance our ability to better understand their role in viral replication.We focus in the present study on rotavirus RNA-directed RNA polymerase (RdRp), named VP1 protein and coded by VP1 gene, as one of the most important proteins in the viral replication; it is involved in both transcription and genome replication/packaging [4,5] in the presence of other proteins, such as VP2 protein; its N-terminal tether domain extends around VP1 and forms cradle that helps stabilize the enzyme in position [6], suggesting to regulate VP1 transitions between transcription and replication states [7].From the structural point of view, VP1 protein is organized as three distinct domains: an Nterminal domain (residues 1 to 332); a central polymerase domain with canonical fingers (residues 333 to 488 and residues 524 to 595), palm (residues 489 to 523 and residues 596 to 685), and thumb subdomains (residues 686 to 778); and a C-terminal "bracelet" domain (residues 779 to 1089) [8].Together, the N-and C-terminal domains enclose the central polymerase domain to create a cagelike enzyme with a buried active site. Although the crystal structures of the RVA VP1 have been determined and fitted into their capsid structures [7][8][9], the structures of the viral particle-associated VP1 and cofactor proteins, such as NSP2, NSP5 and VP3, are still unknown.The importance of amino acid residues that affect the activity of VP1 protein has been demonstrated [7,10-12] and mutant VP1 proteins in specific positions were engineered in order to assess their capacity to synthesize dsRNA in vitro [10,11]. Authors reported that, based on their effect on the replication level, the induced mutations can be classified into three groups: (i) mutations with no significant effect on replication level; (ii) mutations significantly lower levels of dsRNA product; and (iii) mutation that enhanced the initiation capacity and product elongation rate of VP1 protein.…”

“…This mutant shows diminished viral growth, RNA synthesis, protein synthesis, and virion morphogenesis at 39°C compared to that at 31°C [13][14][15].The three dimensional structure of proteins is not static, the different residues can move, and thus, stabilize or break non-covalent interactions. To better understand the effect of the generated mutants, structural data need to be linked to the temporal context of the virus replication/transcription due to the presence of a recently published structure for a catalytically active form of VP1 protein [7,9].We sought to determine how VP1 residues mutations at the RNA entry tunnel contribute to the decrease of the dsRNA synthesis, based on previous experimental findings. Toward this end, molecular dynamics (MD) simulation were used to simulate protein movements at atomic resolution.…”