The nsP3 protein of Semliki Forest virus is a phosphoprotein, which is processed from a large nonstructural polyprotein. The nsP3 gene was isolated from the large coding region by the polymerase chain reaction technique and cloned into a eukaryotic expression vector. Using the constructed pSVNS3 expression vector it was shown that nsP3 could be phosphorylated in the absence of other virus-specific proteins. This suggests that the formation of a complex with the other non-structural proteins is not required for the phosphorylation of nsP3. About half of the synthesized nsP3, in pSVN3-transfected COS cells, could be fractionated into the mitochondrial pellet fraction indicating that nsP3 is associated with large intracellular structures. Immunofluorescence microscopy of pSVNS3-transfected COS cells showed that nsP3 was found in the cytoplasm localized to vesicle-like structures. These results suggest that nsP3 contains information for specific interaction with large intracellular vesicular structures.
AhlquistP.,
StraussE. G.,
RiceC. M.,
StraussJ. H.,
HaseloffJ.,
ZimmernD.1985; Sindbis virus proteins nsPl and nsP2 contain homology to nonstructural proteins from several RNA plant viruses. Journal of Virology 53:536–542
DominguezG.,
WangC.-H.,
FreyT. L.1990; Sequence of the genome RINA of rubella virus: evidence for genetic rearrangement during togavirus evolution. Virology 177:225–238
FelgnerP. L.,
GadekT. R.,
HolmM.,
RomanR.,
ChanH. W.,
WenzM.,
NorthropJ. P.,
RingoldG. M.,
DanielsenM.1987; Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proceedings of the National Academy of Sciences, U.S.A. 84:7413–7417
FroshauerS.,
KartenbeckJ.,
HeleniusA.1988; Alphavirus RNA replication occurs on the cytoplasmic surface of endosomes and lysosomes. Journal of Cell Biology 107:2075–2086
GaroffH.,
FrischaufA.-M.,
SimonsK.,
LehrachH.,
DeliusH.1980a; The capsid protein of Semliki Forest virus has clusters of basic amino acids and prolines in its amino terminal region. Proceedings of the National Academy of Sciences, U.S.A. 77:6376–6380
HahnY. S.,
GrakouiA.,
RiceC. M.,
StraussE. G.,
StraussJ. H.1989a; Mapping of RNA-temperature-sensitive mutants of Sindbis virus: complementation group F mutants have lesions in nsp4. Journal of Virology 63:1194–1202
HahnY. S.,
StraussE. G.,
StraussJ. H.1989b; Mapping of RNA temperature-sensitive mutants of Sindbis virus: assignment of complementation groups A, B and G to nonstructural proteins. Journal of Virology 63:3142–3150
KeränenS.,
RuohonenI.1983; Nonstructural proteins of Semliki Forest virus: synthesis, processing and stability in infected cells. Journal of Virology 47:505–515
Kondor-KochC.,
BurkeB.,
GaroffH.1983; Expression of Semliki Forest virus proteins from cloned complementary DNA. I. The fusion activity of the spike protein. Journal of Cell Biology 97:644–651
LiG.,
RiceC.1989; Mutagenesis of the in-frame opal termination codon preceding nsP4 of Sindbis virus: studies of translational readthrough and its effects on virus replication. Journal of Virology 63:1326–1337
PeränenJ.,
TakkinenK.,
KalkkinenN.,
KääriäinenL.1988; Semliki Forest virus-specific non-structural protein nsP3 is a phosphoprotein. Journal of General Virology 69:2165–2178
RiceC. M.,
LevisR.,
StraussJ. H.,
HuangN. V.1987; Production of infectious RNA transcripts from Sindbis virus cDNA clones:mapping lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. Journal of Virology 61:3809–3819
SaikiR. K.,
GelfandD. H.,
StoffelS.,
ScharfS. F.,
HiguchiR.,
HornR. T.,
MullisK. B.,
ErlichH. A.1988; Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491
SangerF.,
NicklenS.,
CoulsonA. R.1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U.S.A 74:5463–5467
StraussE. G.,
RiceC. M.,
StraussJ. H.1988; Nonstructural proteins nsP3 and nsP4 of Ross River and O′Nyong-nyong viruses: Sequence comparison with those of other alphaviruses. Virology 164:265–274
TakkinenK.,
PeränenJ.,
KeraänenS.,
SöderlundH.,
KääriäinenL.1990; The Semliki Forest virus-specific nonstructural protein is an autoprotease. European Journal of Biochemistry 189:33–38
TowbinH.,
StaehelinT.,
GordonJ.1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, U.S.A.764350–4354