Semliki Forest Virus-specific Non-structural Protein nsP3 Is a Phosphoprotein

Peränen, Johan; Takkinen, Kristiina; Kalkkinen, Nisse; Kääriäinen, Leevi

doi:10.1099/0022-1317-69-9-2165

Cited by 87 publications

(95 citation statements)

References 70 publications

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“…2A, lane 1) [16]. nsP1, nsP3 and nsP4 migrated close to each other in the 67-kDa region and could best be identified after immunoprecipitation [27]. The other minor protein bands were probably products from premature termination.…”

Section: Resultsmentioning

confidence: 83%

“…The in vitro translated precursors and the processing products were further identified by immunoprecipitations using antibodies raised against Escherichiu coli-expressed fusion proteins consisting of P-galactosidase and partial amino acid sequences of SFV nsP3 (aa 24-170) and nsP4 (aa 265-413) [27]. As controls we immunoprecipitdted the translational products of in-vitro-transcribed mRNAs coding for full-sized nsP3 (Fig.…”

Section: Resultsmentioning

confidence: 99%

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The Semliki‐Forest‐virus‐specific nonstructural protein nsP4 is an autoproteinase

Takkinen

Peränen

Keränen

et al. 1990

European Journal of Biochemistry

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The Semliki-Forest-virus-specific nonstructural proteins are translated as a large polyprotein (2431 amino acid residues), from which the mature polymerase components nsP1, nsP2, nsP3 and nsP4 are released by proteolytic cleavages. The complete ns polyprotein (P1234) can be cleaved in two alternative ways yielding either PI23 (with sequences of nsP1, nsP2 and nsP3) and nsP4 or P12 (nsP1 plus nsP2) and P34 (nsP3 plus nsP4). We studied the possible autoproteolytic role of nsP4 involved in the cleavage between nsP3 and nsP4 in an in vitro transcriptiontranslation system. cDNAs encoding P34 precursor and shorter precursor protein segments covering the nsP3-nsP4 cleavage region, were cloned under the T7 RNA polymerase promoter. The mRNAs synthesized in vitro were capped and translated in rabbit reticulocyte lysates. The translational products were analyzed by SDS/ PAGE. The precursor proteins containing nsP4 sequences were cleaved yielding the products with expected sizes, indicating that the cleavage took place at the nsP3-nsP4 junction. By deleting and truncating the cDNA coding for nsP4, the proteolytic activity was mapped within the 102 amino-terminal amino acids of nsP4. The cleavage between nsP3 and nsP4 can be inhibited by pepstatin A and probably takes place in cis, since exogenously added nsP4 was unable to mediate it.In many positive-strand RNA viruses and in retroviruses the primary translation product of the genome is a large polyprotein, which is proteolytically processed by one or more viral or host proteinases (for review see [l]).The translation of Semliki Forest virus (SFV), like other avirus-specific proteins, involves several proteolytic cleavages. The structural proteins (capsid protein C and envelope proteins El, E2 and E3) are translated as a polyprotein in the following order; NH2-C-E3-E2-6K-E1-COOH from a subgenomic 26s mRNA [2,3]. The cleavage of the amino-terminal capsid protein is specified by the virus genome, whereas cleavages of the membrane glycoprotein precursor are carried out by host proteinases [4 -71.A lot of evidence has accumulated which shows that the capsid protein is responsible for its own cleavage from the nascent polyprotein. At the carboxy terminus of the capsid protein there is a well-preserved sequence GDSG common to the active site of serine proteinases [8]. Amino acid changes in the capsid protein of temperature-sensitive mutants of Sindbis virus, an a-virus, defective in the processing of the structural polyprotein, map close to the GDSG sequence [9]. Insertion of an amino acid next to the putative 'active site' by oligonucleotide mutagenesis of cDNA abolished capsid-protein cleavage from a precursor containing the whole capsid protein and 38 amino-terminal amino acids of E3 protein of SFV in an in vitro transcription-translation system [lo].Correspondence to L. Kaariainen, Institute of Biotechnology, University of Helsinki, Valimotie 7, SF-00380 Helsinki, FinlandAbbreviatiuns. PhMeS02F, phenylmethylsulfonyl fluoride; SFV, Semliki Forest virus.Enzymes. Restriction end...

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Section: Resultsmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 99%

The Semliki‐Forest‐virus‐specific nonstructural protein nsP4 is an autoproteinase

Takkinen

Peränen

Keränen

et al. 1990

European Journal of Biochemistry

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“…nsP4 has been identified as the viral RNA-dependent RNA polymerase (RDRP) (4,5). The function of nsP3, the only SV nsP that is phosphorylated (6,7), is not known. nsP2 has an RNA helicase domain (8,9) and possesses protease activity.…”

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confidence: 99%

Identification of the amino acid sequence in Sindbis virus nsP4 that binds to the promoter for the synthesis of the subgenomic RNA

Stollar

2004

Proc. Natl. Acad. Sci. U.S.A.

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A gel mobility-shift assay was used to demonstrate the binding of the Sindbis virus transcriptase to the promoter for the synthesis of subgenomic (SG) RNA. The assay made use of a P15 fraction (the cell fraction that is pelleted at 15,000 ؋ g) from cells infected with recombinant vaccinia virions expressing various Sindbis virus nonstructural proteins (nsPs) and a 32 P-labeled 24-mer oligoribonucleotide representing the minimal sequence with SG promoter activity. By itself, nsP4, the viral RNA-dependent RNA polymerase, did not bind to the SG promoter; rather, all four nsPs were required for the binding of the transcriptase to the promoter. UV crosslinking of the transcriptase to a thiouridine-containing SG promoter, followed by V8 protease digestion of the complex, generated a peptide fragment that was bound to the SG promoter. This peptide fragment contained a sequence that corresponded to residues 329 -334 of nsP4. This peptide may be in the fingers domain of nsP4. The peptide that was identified contained Arg residues at positions 331 and 332. Another Arg is present at position 327. By changing each of the Arg residues to Ala, we demonstrated that only the Arg residues at positions 331 and 332 were required for binding nsP4 to the SG promoter. Sindbis virus (SV) is the prototype virus of the family Togaviridae, genus Alphavirus. Its positive-strand 11,703-nt RNA genome has a type 0 cap at the 5Ј end (1), and the 3Ј end is polyadenylated. The four nonstructural proteins (nsPs) nsP1, nsP2, nsP3, and nsP4 are encoded by the 5Ј terminal two-thirds of the viral genome, and the three structural proteins are encoded by a subgenomic (SG) RNA, the sequence of which is identical to the 3Ј terminal one-third of the viral genome (2, 3).Three species of viral RNA are made in SV-infected cells: (i) a positive-strand genomic RNA, (ii) a genome-length negativestrand RNA, and (iii) a positive-strand SG RNA. The genomic RNA serves as the template for the genome-length negativestrand RNA, which in turn serves as the template for both the genomic RNA and the positive-strand SG RNA. nsP4 has been identified as the viral RNA-dependent RNA polymerase (RDRP) (4, 5). The function of nsP3, the only SV nsP that is phosphorylated (6, 7), is not known. nsP2 has an RNA helicase domain (8, 9) and possesses protease activity. It is responsible for the processing of two large polyproteins, P123 and P1234, from which the four nsPs are derived. nsP1 has both guanylyl transferase and methyltransferase activity and is thus involved in the formation of the 5Ј cap on the positive-strand RNAs (10-12).As suggested by Kao et al. (13), we shall use the term ''replicase'' to denote the enzyme complex that is responsible for the replication of the genome, i.e., the synthesis of both the positive-and negative-strand genome-length RNAs. Because the SG RNA serves only as messenger RNA, the complex that is responsible for its synthesis will be referred to as the ''transcriptase.'' Both the replicase and the transcriptase are thought to contain the RDRP (i.e...

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“…The C-terminal domain of nsP3 is highly variable, both in sequence and length, among alphaviruses and is heavily phosphorylated at serine/threonine (Li et al, 1990;Peranen et al, 1988;Vihinen & Saarinen, 2000). This domain is not essential for SINV replication, but plays a host-celldependent role in SINV minus-strand RNA synthesis De et al, 2003;LaStarza et al, 1994a).…”

Section: Introductionmentioning

confidence: 99%

“…nsP3 interacts with a variety of cellular proteins that are likely to play essential roles in the formation or function of virus replication complexes (Cristea et al, 2006;Despres et al, 1995;Frolova et al, 2006;Gorchakov et al, 2008), and a primary function for nsP3 may be to recruit the required cellular proteins to sites of SINV replication. However, the role of the different domains of nsP3 and their posttranslational modifications in recruitment of cellular proteins are unknown.The C-terminal domain of nsP3 is highly variable, both in sequence and length, among alphaviruses and is heavily phosphorylated at serine/threonine (Li et al, 1990;Peranen et al, 1988;Vihinen & Saarinen, 2000). This domain is not essential for SINV replication, but plays a host-celldependent role in SINV minus-strand RNA synthesis De et al, 2003;LaStarza et al, 1994a).…”

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confidence: 99%

Interaction of Sindbis virus non-structural protein 3 with poly(ADP-ribose) polymerase 1 in neuronal cells

Park¹,

Griffin²

2009

Journal of General Virology

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The alphavirus non-structural protein 3 (nsP3) has a conserved N-terminal macro domain and a variable highly phosphorylated C-terminal domain. nsP3 forms complexes with cellular proteins, but its role in virus replication is poorly understood and protein interaction domains have not been defined. As the N-terminal macro domain can bind poly(ADP-ribose) (PAR), and PAR polymerase-1 (PARP-1) is activated and autoribosylated during Sindbis virus (SINV) infection, it was hypothesized that PARP-1 and nsP3 may interact. Co-immunoprecipitation studies showed that PARP-1 interacted with nsP3 during SINV infection of NSC34 neuronal cells and was most abundantly present in replication complexes that contained plus-and minus-strand SINV RNAs 10-14 h after infection, prior to PARP-1 activation or automodification with PAR. Treatment with an inhibitor of PARP enzymic activity did not affect the interaction between nsP3 and PARP-1 or SINV replication. Co-expression of individual domains of nsP3 with PARP-1 showed that nsP3 interacted with PARP-1 through the C-terminal domain, not the N-terminal macro domain, and that phosphorylation was not required. It was concluded that PARP-1 interacts with the Cterminal domain of nsP3, is present in replication complexes during virus amplification and may play a role in regulating virus RNA synthesis in neuronal cells. INTRODUCTIONSindbis virus (SINV) is the prototype of the genus Alphavirus, family Togaviridae, and has a positive-strand RNA genome. In humans, alphaviruses can cause encephalitis, and SINV causes arthritis and a rash (Calisher, 1994;Griffin, 2007;Laine et al., 2004). In mice, SINV causes encephalomyelitis, and neurons are the main target cells for infection in the central nervous system. SINV nonstructural proteins (nsPs) are translated as polyproteins (P123 and P1234) from the 59 two-thirds of the genome and are cleaved in a regulated fashion into four individual nsPs (nsP1-4) (de Groot et al., 1990). Within replication complexes, the polyproteins and individual nsPs have different functions in genomic and subgenomic plus-and minus-strand synthesis (Lemm et al., 1994(Lemm et al., , 1998Shirako & Strauss, 1994). nsP1 plays a role in capping virus RNAs (Mi et al., 1989;Scheidel et al., 1987), nsP2 is a multifunctional protein with helicase and 59 triphosphatase activities and is the protease that cleaves the non-structural polyprotein (Gomez de Cedró n et al., 1999;Ding & Schlesinger, 1989;Hardy & Strauss, 1989;Vasiljeva et al., 2000) and nsP4 is the RNA-dependent RNA polymerase and terminal adenyltransferase (Kamer & Argos, 1984;Tomar et al., 2006).The function of nsP3 is least understood. The protein has a conserved N-terminal macro domain, an intermediate linker domain and a variable highly phosphorylated Cterminal domain. nsP3 associates with cellular membranes (Peranen & Kaariainen, 1991) and plays a role in regulating RNA synthesis (De et al., 1996;LaStarza et al., 1994b;Wang et al., 1994), but its exact function is unknown. nsP3 interacts with a variety of cellular p...

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Semliki Forest Virus-specific Non-structural Protein nsP3 Is a Phosphoprotein

Cited by 87 publications

References 70 publications

The Semliki‐Forest‐virus‐specific nonstructural protein nsP4 is an autoproteinase

The Semliki‐Forest‐virus‐specific nonstructural protein nsP4 is an autoproteinase

Identification of the amino acid sequence in Sindbis virus nsP4 that binds to the promoter for the synthesis of the subgenomic RNA

Interaction of Sindbis virus non-structural protein 3 with poly(ADP-ribose) polymerase 1 in neuronal cells

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