Structural and functional studies of reovirus RNA-dependent RNA polymerase complex
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Date
1997-09-01T00:00:00Z
Authors
Yin, Peng
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Abstract
The reovirus core, formed by five different structural proteins and 10 segments of dsRNA, is a multienzyme complex. It contains all necessary components for transcription, methylation, and capping of progeny mRNA. The reovirus transcriptase has an unusual temperature profile, with optimum transcription occurring at approximately 50$\sp\circ$C, and little activity occurring below 30$\sp\circ$C or above 60$\sp\circ$C. Purified type 1 Lang cores transcribed most efficiently at 48$\sp\circ$C. The transcriptase temperature optimum of purified type 3 Dearing cores was 52$\sp\circ$C. In addition, T1L cores produced more mRNA per particle than did T3D cores at their respective temperature optima. Core particles were purified from inter-typic reassortants and used to map these differences. The M1 gene, which encodes minor core protein $\mu$2, was uniquely associated with the difference in temperature optimum of transcription (P =.0003). The L1 gene, which encodes minor core protein $\lambda$3, previously implicated as the RNA polymerase, and the M1 gene were associated with the difference in absolute amount of transcript produced (P $\le$ 0.01 and P $\le$ 0.0002 respectively). These data suggest that minor core protein $\mu$2 also plays a role in reovirus transcription. I also found that reovirus transcriptase within core has little activity occurring above 60$\sp\circ$C. However, cores preheated at 65$\sp\circ$C can still transcribe after return to 48$\sp\circ$C, suggesting a reversible alteration in the transcriptase complex, while the enzymatic activities of cores heated at 70$\sp\circ$C are irreversibly lost. Morphologically, cores heated at 65$\sp\circ$C or 70$\sp\circ$C have significantly fewer, or no, $\lambda$2 spike structures. Approximately 50% of the spike-defective cores treated at 65$\sp\circ$C have genomes, but none of the cores heated at 70$\sp\circ$C have genomes. The transcripts synthesized by cores preheated at temperature up to 65$\sp\circ$C, then re-incubated at 37 or 48$\sp\circ$C for 1 hour, are viral ssRNA molecules as shown by hybridization assays. Protein analyses and protease sensitivity assays of heated cores indicated that the $\lambda$2 spikes have undergone dramatic conformational alterations; the spike structures and $\lambda$2 proteins were completely removed by the heat and protease digestion treatments. In vitro translation assays demonstrated that viral proteins can be translated from the ssRNAs produced by 65$\sp\circ$C heated cores, as well as by the cores treated with heat (65$\sp\circ$C) and chymotrypsin treatments. These observations suggest that the spike-deficient cores still possess transcriptase activity. Since previous attempts to partially disassemble the core structure completely destroyed its enzymatic activities, these data represent the first system capable of partially dissociating reovirus cores without destroying their transcriptase activities and provide a novel system for better understanding of an important enzymatic complex.