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.
Highlights
Positive-strand RNA (+RNA) viruses are the most common type of RNA viruses, and include numerous human pathogens [1]
All four nonstructural proteins (nsPs) are required for viral RNA synthesis [6,7,8], and they form a membrane-associated replication complex (RC). nsP1 is the RNA-capping enzyme and the only viral protein responsible for anchoring the RC to membranes; nsP2 is the helicase and protease; nsP3 interacts with several host factors; and nsP4 is the core RNA-dependent RNA
To further isolate the RCs, post-nuclear supernatant (PNS) was separated into a P15 pellet and S15 supernatant by differential centrifugation at 15,000× g, and the distribution of viral nsPs as well as RNA was studied
Summary
Positive-strand RNA (+RNA) viruses are the most common type of RNA viruses, and include numerous human pathogens [1]. The recent re-emergence and outbreaks of chikungunya virus (CHIKV) highlight the medical significance of alphaviruses, which belong to the alphavirus-like superfamily containing several families of +RNA viruses from animals or plants [2,3,4]. All four nsPs are required for viral RNA synthesis [6,7,8], and they form a membrane-associated replication complex (RC). HA-affinity tag in the nonstructural protein 4B (NS4B) has been used to purify hepatitis C virus (HCV) RCs [38]. The results indicate that the minus-strand synthesis requires that RCs or spherules are assembled at the same time as the minus-strand synthesis is initiated, and this was not achieved in vitro because the replicase proteins had already formed spherules. The minus-strand synthesis seems to depend on the RC assembly, SFV spherule formation does not depend on viral
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