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Viruses 2017, 9(10), 292; doi:10.3390/v9100292

Polyprotein Processing as a Determinant for in Vitro Activity of Semliki Forest Virus Replicase

1
Department of Food and Environmental Sciences, University of Helsinki, Viikinkaari 9 PO Box 56, 00014 Helsinki, Finland
2
Department of Medical Microbiology, Leiden University Medical Center PO Box 9600, 2300 RC, Leiden, The Netherlands
*
Authors to whom correspondence should be addressed.
Received: 7 September 2017 / Revised: 29 September 2017 / Accepted: 3 October 2017 / Published: 7 October 2017
(This article belongs to the Special Issue Advances in Alphavirus Research)
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Abstract

Semliki Forest virus (SFV) is an arthropod-borne alphavirus that induces membrane invaginations (spherules) in host cells. These harbor the viral replication complexes (RC) that synthesize viral RNA. Alphaviruses have four replicase or nonstructural proteins (nsPs), nsP1–4, expressed as polyprotein P1234. An early RC, which synthesizes minus-strand RNA, is formed by the polyprotein P123 and the polymerase nsP4. Further proteolytic cleavage results in a late RC consisting of nsP1–4 and synthesizing plus strands. Here, we show that only the late RCs are highly active in RNA synthesis in vitro. Furthermore, we demonstrate that active RCs can be isolated from both virus-infected cells and cells transfected with the wild-type replicase in combination with a plasmid expressing a template RNA. When an uncleavable polyprotein P123 and polymerase nsP4 were expressed together with a template, high levels of minus-strand RNA were produced in cells, but RCs isolated from these cells were hardly active in vitro. Furthermore, we observed that the uncleavable polyprotein P123 and polymerase nsP4, which have previously been shown to form spherules even in the absence of the template, did not replicate an exogenous template. Consequently, we hypothesize that the replicase proteins were sequestered in spherules and were no longer able to recruit a template. View Full-Text
Keywords: alphavirus; Semliki Forest virus; nonstructural protein; polymerase; replication complex; in vitro replication; RNA synthesis alphavirus; Semliki Forest virus; nonstructural protein; polymerase; replication complex; in vitro replication; RNA synthesis
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MDPI and ACS Style

Pietilä, M.K.; Albulescu, I.C.; Hemert, M.J.; Ahola, T. Polyprotein Processing as a Determinant for in Vitro Activity of Semliki Forest Virus Replicase. Viruses 2017, 9, 292.

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