Mutations at the palmitoylation site of non-structural protein nsP1 of Semliki Forest virus attenuate virus replication and cause accumulation of compensatory mutations.
The replicase of Semliki Forest virus (SFV) consists of four non-structural proteins, designated nsP1-4, and is bound to cellular membranes via an amphipathic peptide and palmitoylated cysteine residues of nsP1. It was found that mutations preventing nsP1 palmitoylation also attenuated virus replication. The replacement of these cysteines by alanines, or their deletion, abolished virus viability, possibly due to disruption of interactions between nsP1 and nsP4, which is the catalytic subunit of the replicase. However, during a single infection cycle, the ability of the virus to replicate was restored due to accumulation of second-site mutations in nsP1. These mutations led to the restoration of nsP1-nsP4 interaction, but did not restore the palmitoylation of nsP1. The proteins with palmitoylation-site mutations, as well as those harbouring compensatory mutations in addition to palmitoylation-site mutations, were enzymically active and localized, at least in part, on the plasma membrane of transfected cells. Interestingly, deletion of 7 aa including the palmitoylation site of nsP1 had a relatively mild effect on virus viability and no significant impact on nsP1-nsP4 interaction. Similarly, the change of cysteine to alanine at the palmitoylation site of nsP1 of Sindbis virus had only a mild effect on virus replication. Taken together, these findings indicate that nsP1 palmitoylation as such is not the factor determining the ability to bind to cellular membranes and form a functional replicase complex. Instead, these abilities may be linked to the three-dimensional structure of nsP1 and the capability of nsP1 to interact with other components of the viral replicase complex.
Žusinaite
Eva
E
Institute of Molecular and Cell Biology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia.
Tints
Kairit
K
Estonian Biocentre, Riia Street 23, 51010 Tartu, Estonia.|Institute of Molecular and Cell Biology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia.
Kiiver
Kaja
K
Estonian Biocentre, Riia Street 23, 51010 Tartu, Estonia.|Institute of Molecular and Cell Biology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia.
Spuul
Pirjo
P
Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland.
Karo-Astover
Liis
L
Estonian Biocentre, Riia Street 23, 51010 Tartu, Estonia.|Institute of Molecular and Cell Biology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia.
Merits
Andres
A
Institute of Molecular and Cell Biology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia.
Sarand
Inga
I
Estonian Biocentre, Riia Street 23, 51010 Tartu, Estonia.
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D010169:Palmitic Acids / Q000737:chemistry; D012093:Replicon; D012672:Semliki forest virus / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology*; D013347:Subcellular Fractions / Q000821:virology; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014774:Virulence / Q000235:genetics; D014779:Virus Replication / Q000235:genetics
D010169:Palmitic Acids; D017361:Viral Nonstructural Proteins