Abstract
Semliki Forest virus (SFV) non-structural protein 1 (nsP1) is a major component of the virus replicase complex. It has previously been studied in cells infected with virus or using transient or stable expression systems. To extend these studies, tetracycline-inducible stable cell lines expressing SFV nsP1 or its palmitoylation-negative mutant (nsP16D) were constructed. The levels of protein expression and the subcellular localization of nsP1 in induced cells were similar to those in virus-infected cells. The nsP1 expressed by stable, inducible cell lines or by SFV-infected HEK293 T-REx cells was a stable protein with a half-life of approximately 5 h. In contrast to SFV infection, induction of nsP1 expression had no detectable effect on cellular transcription, translation or viability. Induction of expression of nsP1 or nsP16D interfered with multiplication of SFV, typically resulting in a 5–10-fold reduction in virus yields. This reduction was not due to a decrease in the number of infected cells, indicating that nsP1 expression does not block virus entry or initiation of replication. Expression of nsP1 interfered with virus genomic RNA synthesis and delayed accumulation of viral subgenomic RNA translation products. Expression of nsP1 with a mutation in the palmitoylation site reduced synthesis of genomic and subgenomic RNAs and their products of translation, and this effect did not resolve with time. These results are in agreement with data published previously, suggesting a role for nsP1 in genomic RNA synthesis.
Highlights
Semliki Forest virus (SFV) belongs to the genus Alphavirus
We studied the properties of non-structural protein 1 (nsP1) and its effect on host cells and on SFV infection by combining two powerful approaches, tetracycline-inducible stable cell lines and recombinant SFV genomes carrying marker genes in the replicase or the structural open reading frame (ORF). nsP1 expressed alone in the absence of the other virus replicase proteins was a stable protein with a half-life of about 5 h; stability was not affected by virus infection
A WST-1 cell viability assay revealed that, whilst SFV4 infection considerably reduced viability of HEK293 T-REx cells at 24 h p.i., induction of nsP1 or nsP16D expression had no effect on cell viability (Fig. 3c). These results showed that SFV nsP1 expression causes significant morphological changes in cells but on its own has no effect on transcription, translation or viability
Summary
Semliki Forest virus (SFV) belongs to the genus Alphavirus (family Togaviridae). Along with Sindbis virus (SV), the prototype member of the genus, SFV is one of the most studied alphaviruses. The 59 two-thirds of the genome encodes the non-structural (ns) polyprotein P1234. P1234 is autocatalytically cleaved into four individual non-structural proteins designated nsP1, nsP2, nsP3 and nsP4 (Kaariainen et al, 1987). The first cleavage takes place at the nsP3/4 cleavage site and releases mature nsP4, which together with the remaining P123 polyprotein forms the early minusstrand RNA replicase. The late replicase complex consists of fully processed non-structural proteins and synthesizes positive-sense genomic and subgenomic (sg) RNA molecules (Lemm et al, 1994; Shirako & Strauss, 1994; Vasiljeva et al, 2003; Kim et al, 2004)
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