Characterization of purified Sindbis virus nsP4 RNA-dependent RNA polymerase activity in vitro

Journal Article · Thu Feb 05 04:00:00 EST 2009 · Virology

DOI:https://doi.org/10.1016/j.virol.2008.10.030· OSTI ID:21182802

Rubach, Jon K ^[1]; Wasik, Brian R; Rupp, Jonathan C ^[2]; Kuhn, Richard J ^[3]; Hardy, Richard W ^[2]

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1. Life Sciences Institute and Department of Biological Chemistry, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI 48109 (United States)
2. Department of Biology, Indiana University, 1001 E. Third Street, Bloomington, IN 47405 (United States)
3. Markey Center for Structural Biology and Department of Biological Sciences, Purdue University, 915 W. State St., West Lafayette, IN 47907 (United States)

The Sindbis virus RNA-dependent RNA polymerase (nsP4) is responsible for the replication of the viral RNA genome. In infected cells, nsP4 is localized in a replication complex along with the other viral non-structural proteins. nsP4 has been difficult to homogenously purify from infected cells due to its interactions with the other replication proteins and the fact that its N-terminal residue, a tyrosine, causes the protein to be rapidly turned over in cells. We report the successful expression and purification of Sindbis nsP4 in a bacterial system, in which nsP4 is expressed as an N-terminal SUMO fusion protein. After purification the SUMO tag is removed, resulting in the isolation of full-length nsP4 possessing the authentic N-terminal tyrosine. This purified enzyme is able to produce minus-strand RNA de novo from plus-strand templates, as well as terminally add adenosine residues to the 3' end of an RNA substrate. In the presence of the partially processed viral replicase polyprotein, P123, purified nsP4 is able to synthesize discrete template length minus-strand RNA products. Mutations in the 3' CSE or poly(A) tail of viral template RNA prevent RNA synthesis by the replicase complex containing purified nsP4, consistent with previously reported template requirements for minus-strand RNA synthesis. Optimal reaction conditions were determined by investigating the effects of time, pH, and the concentrations of nsP4, P123 and magnesium on the synthesis of RNA.

• OSTI ID: 21182802
• Journal Information: Virology, Journal Name: Virology Journal Issue: 1 Vol. 384; ISSN VIRLAX; ISSN 0042-6822
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
ADENOSINE
BIOSYNTHESIS
IN VITRO
MAGNESIUM
MUTATIONS
PH VALUE
PURIFICATION
RNA
RNA POLYMERASES
TYROSINE
VIRUSES