Site-specific Protease Activity of the Carboxyl-terminal Domain of Semliki Forest Virus Replicase Protein nsP2
2001; Elsevier BV; Volume: 276; Issue: 33 Linguagem: Inglês
10.1074/jbc.m104786200
ISSN1083-351X
AutoresLidia Vasiljeva, Leena Valmu, Leevi Kääriäinen, Andres Merits,
Tópico(s)Insect Resistance and Genetics
ResumoThe virus-specific components (nsP1–nsP4) of Semliki Forest virus RNA polymerase are synthesized as a large polyprotein (P1234), which is cleaved by a virus-encoded protease. Based on mutagenesis studies, nsP2 has been implicated as the protease moiety of P1234. Here, we show that purified nsP2 (799 amino acids) and its C-terminal domain Pro39 (amino acids 459–799) specifically process P1234 and its cleavage intermediates. Analysis of cleavage products ofin vitro synthesized P12, P23, and P34 revealed cleavages at sites 1/2, 2/3, and 3/4. The cleavage regions of P1/2, P2/3, and P3/4 were expressed as thioredoxin fusion proteins (Trx12, Trx23, and Trx34), containing ∼20 amino acids on each side of the cleavage sites. After exposure of these purified fusion proteins to nsP2 or Pro39, the reaction products were analyzed by SDS-polyacrylamide gel electrophoresis, mass spectrometry, and amino-terminal sequencing. The expected amino termini of nsP2, nsP3, and nsP4 were detected. The cleavage at 3/4 site was most efficient, whereas cleavage at 1/2 site required 5000-fold more of Pro39, and 2/3 site was almost resistant to cleavage. The activity of Pro39 was inhibited byN-ethylmaleimide, Zn2+, and Cu2+, but not by EDTA, phenylmethylsulfonyl fluoride, or pepstatin, in accordance with the thiol proteinase nature of nsP2. The virus-specific components (nsP1–nsP4) of Semliki Forest virus RNA polymerase are synthesized as a large polyprotein (P1234), which is cleaved by a virus-encoded protease. Based on mutagenesis studies, nsP2 has been implicated as the protease moiety of P1234. Here, we show that purified nsP2 (799 amino acids) and its C-terminal domain Pro39 (amino acids 459–799) specifically process P1234 and its cleavage intermediates. Analysis of cleavage products ofin vitro synthesized P12, P23, and P34 revealed cleavages at sites 1/2, 2/3, and 3/4. The cleavage regions of P1/2, P2/3, and P3/4 were expressed as thioredoxin fusion proteins (Trx12, Trx23, and Trx34), containing ∼20 amino acids on each side of the cleavage sites. After exposure of these purified fusion proteins to nsP2 or Pro39, the reaction products were analyzed by SDS-polyacrylamide gel electrophoresis, mass spectrometry, and amino-terminal sequencing. The expected amino termini of nsP2, nsP3, and nsP4 were detected. The cleavage at 3/4 site was most efficient, whereas cleavage at 1/2 site required 5000-fold more of Pro39, and 2/3 site was almost resistant to cleavage. The activity of Pro39 was inhibited byN-ethylmaleimide, Zn2+, and Cu2+, but not by EDTA, phenylmethylsulfonyl fluoride, or pepstatin, in accordance with the thiol proteinase nature of nsP2. Semliki Forest virus nonstructural protein matrix-assisted laser desorption/ionization time-of-flight high performance liquid chromatography polyacrylamide gel electrophoresis N-ethylmaleimide l-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane phenylmethylsulfonyl fluoride amino acid(s) polymerase chain reaction short long thioredoxin N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Sindbis virus The genomes of many positive strand RNA viruses are expressed as polyproteins in order to achieve the expression of multiple proteins from a single message, unlike the mRNAs of their eukaryotic host cells, which mostly code for single proteins. Thus, proteolyses of the polyprotein precursors are essential events in the regulation of the replication and morphogenesis of these RNA viruses. In picornaviruses and flaviviruses, the entire RNA genome is translated as a single polyprotein, from which the structural and nonstructural proteins are processed by proteolysis. In picornavirus-infected cells, the processing is carried out by virus-encoded proteases within the polyprotein, whereas the processing of flavivirus polyprotein is assisted by host proteases (1Ryan D.M. Flint M. J. Gen. Virol. 1997; 78: 699-723Crossref PubMed Scopus (189) Google Scholar, 2Ryan D.M. Monaghan S. Flint M. J. Gen. Virol. 1998; 79: 947-959Crossref PubMed Scopus (66) Google Scholar). The large RNA genomes of coronaviruses (approximately 30 kilobases) and arteriviruses (12.7–15.7 kilobases), together classified as Nidovirales, use in addition to the polyprotein strategy also a set of subgenomic mRNAs (3Ziebuhr J. Snijder J.E. Gorbalenya A.E. J. Gen. Virol. 2000; 81: 853-879Crossref PubMed Scopus (741) Google Scholar). Alphaviruses and rubella virus, members of the Togaviridae family, express two polyproteins. The nonstructural polyprotein is expressed directly from the RNA genome, whereas the structural polyprotein is synthesized from a subgenomic mRNA (4Strauss J.H. Strauss E.G. Microbiol. Rev. 1994; 58: 491-562Crossref PubMed Google Scholar, 5Schlesinger M.J. Granoff A. Webster R.G. Encyclopedia of Virology. 2nd Ed. Oxford University Press, Oxford1999: 1656-1663Crossref Google Scholar, 6Frey T.K. Wolinsky J.S. Granoff A. Webster R.G. Encyclopedia of Virology. 2nd Ed. Oxford University Press, Oxford1999: 1592-1601Crossref Google Scholar).Semliki Forest virus (SFV)1is a typical alphavirus with a lipoprotein envelope surrounding the nucleocapsid. The 5′ two-thirds of the 11.5-kilobase 42Kim J.L. Morgenstern K.A. Lin C. Fox T. Dwyer M.D. Landro J.A. Chambers S.P. Markland W. Lepre C.A. O'Malley E.T. Harbeson S.L. Rice C.M. Murcko M.A. Caron P.R. Thomson J.A. Cell. 1996; 87: 343-355Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar S RNA genome codes for the nonstructural polyprotein (P1234) of 2432 aa, which is autocatalytically cleaved to finally yield the virus-specific components of the RNA polymerase complex, nsP1–nsP4 (4Strauss J.H. Strauss E.G. Microbiol. Rev. 1994; 58: 491-562Crossref PubMed Google Scholar, 5Schlesinger M.J. Granoff A. Webster R.G. Encyclopedia of Virology. 2nd Ed. Oxford University Press, Oxford1999: 1656-1663Crossref Google Scholar, 7Kääriäinen L. Takkinen K. Keränen S. Söderlund H. J. Cell Sci. Suppl. 1987; 7: 231-250Crossref PubMed Google Scholar). The processing of the nonstructural polyproteins P1234 and P123 of Sindbis virus (SIN), another alphavirus, has been studied in detail (4Strauss J.H. Strauss E.G. Microbiol. Rev. 1994; 58: 491-562Crossref PubMed Google Scholar, 8Strauss J.H. Strauss E.G. Semin. Virol. 1990; 1: 347-356Google Scholar, 9ten Dam E. Flint M. Ryan M.D. J. Gen. Virol. 1999; 80: 1879-1888Crossref PubMed Scopus (23) Google Scholar). Using mostly in vitro translation and site-directed mutagenesis as tools, autocatalytic protease activity was detected in the polyprotein and its cleavage intermediates. The protease activity was localized to nsP2, and more precisely, to its carboxyl-terminal part (10Hardy W.R. Strauss J.H. J. Virol. 1989; 63: 4653-4664Crossref PubMed Google Scholar, 11Ding M.X. Schlesinger M.J. Virology. 1989; 171: 280-284Crossref PubMed Scopus (79) Google Scholar). Cysteine 481 and histidine 558 were identified as essential residues for the autoprotease activity (12Strauss E.G. de Groot R.J. Levinson R. Strauss J.H. Virology. 1992; 191: 932-940Crossref PubMed Scopus (102) Google Scholar), supporting the view that the protease is a thiol proteinase belonging to the papain superfamily. The same conclusion was reached by sequence comparisons (13Gorbalenya A.E. Koonin E.V. Lai M.-C. FEBS Lett. 1991; 288: 201-205Crossref PubMed Scopus (270) Google Scholar). The respective mutation of Cys-478 in SFV nsP2 also inactivates the autocatalytic processing of P1234, P123, and P23 (14Merits A. Vasiljeva L. Ahola T. Kääriäinen L. Auvinen P. J. Gen. Virol. 2001; 82: 765-773Crossref PubMed Scopus (60) Google Scholar).The amino-terminal domain of SFV nsP2 (residues 1–470) has been shown to house several enzymatic activities including RNA triphosphatase (15Vasiljeva L. Merits A. Auvinen P. Kääriäinen L. J. Biol. Chem. 2000; 275: 17281-17287Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), nucleoside triphosphatase (16Rikkonen M. Peränen J. Kääriäinen L. J. Virol. 1994; 68: 5804-5810Crossref PubMed Google Scholar), and RNA helicase (17Gomez de Cedrón M. Ehsani N. Mikkola M.L. Garcı́a J.A. Kääriäinen L. FEBS Lett. 1999; 448: 19-22Crossref PubMed Scopus (132) Google Scholar). Interestingly, a significant amount of nsP2, synthesized during infection, is transported into the nucleus (18Peränen J. Rikkonen M. Liljeström P. Kääriäinen L. J. Virol. 1990; 64: 1888-1896Crossref PubMed Google Scholar, 19Peränen J. Rikkonen M. Kääriäinen L. J. Histochem. Cytochem. 1993; 41: 447-454Crossref PubMed Scopus (56) Google Scholar). The core of the nuclear localization signal was mapped to a short sequence P647RRR (20Rikkonen M. Peränen J. Kääriäinen L. Virology. 1992; 189: 462-473Crossref PubMed Scopus (77) Google Scholar). nsP2 mutant PRDR is not lethal for the virus in cell culture, but SFV carrying this mutation is apathogenic for mouse (21Rikkonen M. Virology. 1996; 218: 352-361Crossref PubMed Scopus (66) Google Scholar). In addition, the carboxyl-terminal domain of SFV nsP2 has been implicated in the regulation of the synthesis of the subgenomic mRNA (22Keränen S. Kääriäinen L. J. Virol. 1979; 47: 505-515Crossref Google Scholar, 23Sawicki D.L. Sawicki S.G. Virology. 1985; 144: 20-34Crossref PubMed Scopus (39) Google Scholar, 24Suopanki J. Sawicki D.L. Sawicki S.G. Kääriäinen L. J. Gen. Virol. 1998; 79: 309-319Crossref PubMed Scopus (51) Google Scholar).Taken together, these data suggest that alphavirus nsP2 consists of two structurally independent domains, each possessing a distinct set of biological activities. However, direct proof that purified nsP2 or its carboxyl-terminal part has protease activity has been lacking. To this end, recombinant nsP2 and a set of its amino-terminally truncated variants were expressed in Escherichia coli, purified by metal-chelate chromatography, and assayed for the presence of protease activity. Both full-length nsP2 and its soluble carboxyl-terminal fragment Pro39 (aa 458–799) catalyzed site-specific proteolysis of SFV P1234 in vitro. Furthermore, both nsP2 and Pro39 could specifically cleave purified recombinant fusion proteins containing short (∼40 aa) SFV-specific peptides, which span protease cleavage sites. Using this newly devised in vitro assay, we show that Pro39 was inactivated by N-ethylmaleimide, in accordance with the catalytic mechanism of cysteine proteases.DISCUSSIONPrevious work on Semliki Forest virus showed that early in infection the synthesis of the negative strand RNA was strictly dependent on protein synthesis and ceased in about 15 min after addition of cycloheximide (31Sawicki D.L. Sawicki S.G. J. Virol. 1980; 34: 108-118Crossref PubMed Google Scholar), whereas late in infection the synthesis of positive strand RNAs could continue for several hours in the absence of protein synthesis. Solution to this dilemma came from findings with Sindbis virus, another alphavirus, where the processing intermediate P123 of the nonstructural polyprotein together with nsP4 was shown to be responsible for the synthesis of the negative strand RNA (32Lemm J.A. Rice C.M. J. Virol. 1993; 67: 1905-1915Crossref PubMed Google Scholar, 33Lemm J.A. Rice C.M. J. Virol. 1993; 67: 1916-1926Crossref PubMed Google Scholar, 34Shirako Y. Strauss J.H. J. Virol. 1994; 68: 1874-1885Crossref PubMed Google Scholar). Cleavage of P123 is essential for the synthesis of the positive RNA strands. Thus, the regulated processing of the nonstructural polyprotein controls the early events of virus infection. An overactive processing mutant of Sindbis virus nsP2 (N614D) cannot replicate, as the P123 intermediate is too short-lived to enable the necessary synthesis of the complementary RNA (35Lemm J.A. Rümenapf T. Strauss E.G. Strauss J.H. Rice C.M. EMBO J. 1994; 13: 2925-2934Crossref PubMed Scopus (231) Google Scholar).Our knowledge of the processing of alphavirus nonstructural polyprotein(s) is based mostly on experiments of in vitrotranslation of Sindbis virus RNA. Ingenious constructions by which the cleavage sites were mutated alone and in different combinations, together with constructs coding for enzymatically inactive polyprotein as substrates, have been used to analyze this complex process (4Strauss J.H. Strauss E.G. Microbiol. Rev. 1994; 58: 491-562Crossref PubMed Google Scholar, 8Strauss J.H. Strauss E.G. Semin. Virol. 1990; 1: 347-356Google Scholar,9ten Dam E. Flint M. Ryan M.D. J. Gen. Virol. 1999; 80: 1879-1888Crossref PubMed Scopus (23) Google Scholar). These experiments showed that the polyprotein P1234 itself and all its cleavage intermediates containing nsP2 to be active proteases. The cleavability of the different sites varied and was dependent on the order of removal of different nsPs from the polyprotein substrate. Particularly interesting was the finding where the cleavage of site 2/3 in P1234 or P123 was only possible after the cleavage of nsP1 (36Shirako Y. Strauss J.H. Virology. 1990; 177: 54-64Crossref PubMed Scopus (65) Google Scholar). Evidently, conformational changes in P1234 and its cleavage products affect the interactions between the cleavage sites and the protease domain of nsP2 in a complex manner. To understand the processes better, we have characterized purified SFV nsP2 and its carboxyl-terminal fragment Pro39 as proteolytic enzymes. As substrates we used in vitro synthesized polyproteins P1234, P123, P23 and P34, as well as recombinant thioredoxin fusion proteins, which contain short SFV-specific fragments, spanning the polyprotein processing sites 1/2, 2/3, and 3/4.We show for the first time that purified nsP2 has proteolytic activity, which cleaves readily the 3/4 site of P1234 and P34. Deletion series of nsP2 resulted in a soluble, active carboxyl-terminal fragment consisting of amino acid residues 459–799, which was designated as Pro39. It was purified to near homogeneity by metal-affinity chromatography. The specificities of Pro39 and nsP2 were identical, indicating that the amino-terminal half of nsP2 does not affect the fidelity of the protease. According to sequence alignments with the thiol protease superfamily, Pro39 contains a conserved protease domain (459), but also almost 200 carboxyl-terminal extra amino acids (9ten Dam E. Flint M. Ryan M.D. J. Gen. Virol. 1999; 80: 1879-1888Crossref PubMed Scopus (23) Google Scholar,13Gorbalenya A.E. Koonin E.V. Lai M.-C. FEBS Lett. 1991; 288: 201-205Crossref PubMed Scopus (270) Google Scholar). Our attempts to delete 40–120 amino acids from the carboxyl terminus of nsP2 resulted in insoluble or inactive proteins (Fig. 1). Experiments with temperature-sensitive mutants of SIN and SFV nsP2 have shown that amino acid replacements N700K in SIN ts133, K736S in SIN ts24, and M781T in SFV ts4 result in inhibition of protease activity at 39 °C, suggesting that the extreme carboxyl terminus of nsP2 participates somehow in the protease function (24Suopanki J. Sawicki D.L. Sawicki S.G. Kääriäinen L. J. Gen. Virol. 1998; 79: 309-319Crossref PubMed Scopus (51) Google Scholar).Establishment of a biochemical assay system consisting of isolated Pro39 and thioredoxin attached cleavage regions of the SFV nonstructural polyprotein allowed characterization of the viral protease under defined experimental conditions. Pro39 was inactivated by N-ethylmaleimide but not with pepstatin, EDTA, or PMSF. These are properties, which are in accordance with its classification as a thiol proteinase of the papain superfamily. However, Pro39 is not inhibited by E-64, which is a typical inhibitor of cysteine proteases (37Beynon R.J. Bond J.S. Beynon R.J. Bond J.S. Proteolytic Enzymes: A Practical Approach. Oxford University Press, Oxford, United Kingdom1989: 83-104Google Scholar). Sensitivity for NEM and resistance for E-64 have been previously reported for poliovirus 3C thiol proteinase (38Lawson M.A. Semler B.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9919-9923Crossref PubMed Scopus (53) Google Scholar, 39Yu F.Sh. Lloyd R.E. Virology. 1991; 182: 615-625Crossref PubMed Scopus (64) Google Scholar). Another interesting feature of Pro39 is the inhibition by zinc ions (Fig.8).When Pro39 (or nsP2) was added to the reaction mixture, after in vitro translation of P12 or P123, almost a quantitative release of nsP1 was observed. Similarly, when P34 or P1234 were used as substrates, quantitative release of nsP4 was seen, whereas only a small amount of nsP3 was released from P23, P123, or P1234 (Fig. 3). These results suggested that sites 1/2 and 3/4 were exposed to the added protease, whereas site 2/3 was not. To study this phenomenon under controlled conditions, in which the large protein domains would not interfere sterically with the proteolysis, we constructed fusion proteins with a different number of amino acid residues around the cleavage sites.Constructs with less than 10 amino acids on both sides of the cleavage site were not digested by Pro39 or nsP2 (data not shown). We ended up using thioredoxin fusion proteins with about 40 residues of each cleavage region (Trx12, Trx23, and Trx34). Isolation of the cleavage products and their mass spectrometric analysis, as well as amino-terminal sequencing showed that Pro39 cleavage products were derived exactly from the predicted cleavage sites, determined previously by radiosequence analysis (29Kalkkinen N. Laaksonen M. Söderlund H. Jörnvall H. Virology. 1981; 113: 188-195Crossref PubMed Scopus (10) Google Scholar, 30Kalkkinen N. Wittmann-Liebold B. Salnikow J. Erdmann V.A. Advanced Methods in Protein Microsequence Analysis. Springer-Verlag, Heidelberg1986: 194-206Crossref Google Scholar) (Table II). As controls we used thioredoxin fusion proteins with mutations close to the cleavage site, which were not digested by Pro39 or nsP2 (Fig. 4). Thus, we conclude that both proteases recognize specifically the three cleavage sites of the SFV nonstructural polyprotein. However, there were large differences in the sensitivity of the different cleavage sites, the 3/4 junction being most sensitive. Roughly 5000-fold more Pro39 was needed for complete cleavage of site 1/2 (Fig. 5). Under the same conditions, only a small amount of Trx23 was cleaved.The different sensitivities of the three cleavage sites may well reflect the different specificities of the protease associated with the polyproteins, in which the cleavage at site 2/3 of P123 or P1234 does not take place unless preceded by cleavage of nsP1. The fact that Pro39 can catalyze cleavage at 1/2 site of in vitro translated SFV P12, P123, and P1234, which normally undergoes cleavage incis (14Merits A. Vasiljeva L. Ahola T. Kääriäinen L. Auvinen P. J. Gen. Virol. 2001; 82: 765-773Crossref PubMed Scopus (60) Google Scholar), is better understood when realized that the estimated molar enzyme to substrate ratio represents an excess of 50 to 1, which is difficult to imagine to take place during virus infection. Thus, we cannot exclude the possibility that cleavages at sites 1/2 and 2/3 require cofactor(s), which might be derived from the other nsPs. Such a situation has been characterized thoroughly for the NS3 protease of hepatitis C virus. The site-specific proteolytic activity of NS3 protease was greatly increased by a short amino acid sequence of NS4 protein adjacently located in the polyprotein (40Failla C. Tomei L. De Francesco R. J. Virol. 1994; 68: 3753-3760Crossref PubMed Google Scholar, 41Lin C. Rice C.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7622-7626Crossref PubMed Scopus (84) Google Scholar, 43Love R.A. Parge H.E. Wickersham J.A. Hostomsky Z. Habuka N. Moomaw E.W. Adachi T. Hostomska Z. Cell. 1996; 87: 331-342Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar,44Shimizu Y. Yamaji K. Masuho Y. Yokota T. Inoue H. Sudo K. Satoh S. Shimotohno K. J. Virol. 1996; 70: 127-132Crossref PubMed Google Scholar).The processing intermediate P123, together with nsP4, enables the synthesis of the complementary RNA for a short time period, whereafter P123 is autocatalytically cleaved to yield the components of the stable RNA polymerase among them nsP2. The released nsP2 exercises its role in two different forms. As a part of the RNA polymerase complex (45Kujala P. Ikäheimonen A. Ehsani N. Vihinen H. Auvinen P. Kääriäinen L. J. Virol. 2001; 75: 3873-3884Crossref PubMed Scopus (177) Google Scholar), the amino-terminal domain provides RNA triphosphatase and RNA helicase activities (15Vasiljeva L. Merits A. Auvinen P. Kääriäinen L. J. Biol. Chem. 2000; 275: 17281-17287Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 17Gomez de Cedrón M. Ehsani N. Mikkola M.L. Garcı́a J.A. Kääriäinen L. FEBS Lett. 1999; 448: 19-22Crossref PubMed Scopus (132) Google Scholar). As "soluble nsP2," the carboxyl-terminal domain acts as a regulator of 26 S RNA synthesis (24Suopanki J. Sawicki D.L. Sawicki S.G. Kääriäinen L. J. Gen. Virol. 1998; 79: 309-319Crossref PubMed Scopus (51) Google Scholar) and as atrans-acting protease, which catalyzes the rapid cleavage of P1234 and P123, thus preventing the negative strand RNA synthesis late in infection. The genomes of many positive strand RNA viruses are expressed as polyproteins in order to achieve the expression of multiple proteins from a single message, unlike the mRNAs of their eukaryotic host cells, which mostly code for single proteins. Thus, proteolyses of the polyprotein precursors are essential events in the regulation of the replication and morphogenesis of these RNA viruses. In picornaviruses and flaviviruses, the entire RNA genome is translated as a single polyprotein, from which the structural and nonstructural proteins are processed by proteolysis. In picornavirus-infected cells, the processing is carried out by virus-encoded proteases within the polyprotein, whereas the processing of flavivirus polyprotein is assisted by host proteases (1Ryan D.M. Flint M. J. Gen. Virol. 1997; 78: 699-723Crossref PubMed Scopus (189) Google Scholar, 2Ryan D.M. Monaghan S. Flint M. J. Gen. Virol. 1998; 79: 947-959Crossref PubMed Scopus (66) Google Scholar). The large RNA genomes of coronaviruses (approximately 30 kilobases) and arteriviruses (12.7–15.7 kilobases), together classified as Nidovirales, use in addition to the polyprotein strategy also a set of subgenomic mRNAs (3Ziebuhr J. Snijder J.E. Gorbalenya A.E. J. Gen. Virol. 2000; 81: 853-879Crossref PubMed Scopus (741) Google Scholar). Alphaviruses and rubella virus, members of the Togaviridae family, express two polyproteins. The nonstructural polyprotein is expressed directly from the RNA genome, whereas the structural polyprotein is synthesized from a subgenomic mRNA (4Strauss J.H. Strauss E.G. Microbiol. Rev. 1994; 58: 491-562Crossref PubMed Google Scholar, 5Schlesinger M.J. Granoff A. Webster R.G. Encyclopedia of Virology. 2nd Ed. Oxford University Press, Oxford1999: 1656-1663Crossref Google Scholar, 6Frey T.K. Wolinsky J.S. Granoff A. Webster R.G. Encyclopedia of Virology. 2nd Ed. Oxford University Press, Oxford1999: 1592-1601Crossref Google Scholar). Semliki Forest virus (SFV)1is a typical alphavirus with a lipoprotein envelope surrounding the nucleocapsid. The 5′ two-thirds of the 11.5-kilobase 42Kim J.L. Morgenstern K.A. Lin C. Fox T. Dwyer M.D. Landro J.A. Chambers S.P. Markland W. Lepre C.A. O'Malley E.T. Harbeson S.L. Rice C.M. Murcko M.A. Caron P.R. Thomson J.A. Cell. 1996; 87: 343-355Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar S RNA genome codes for the nonstructural polyprotein (P1234) of 2432 aa, which is autocatalytically cleaved to finally yield the virus-specific components of the RNA polymerase complex, nsP1–nsP4 (4Strauss J.H. Strauss E.G. Microbiol. Rev. 1994; 58: 491-562Crossref PubMed Google Scholar, 5Schlesinger M.J. Granoff A. Webster R.G. Encyclopedia of Virology. 2nd Ed. Oxford University Press, Oxford1999: 1656-1663Crossref Google Scholar, 7Kääriäinen L. Takkinen K. Keränen S. Söderlund H. J. Cell Sci. Suppl. 1987; 7: 231-250Crossref PubMed Google Scholar). The processing of the nonstructural polyproteins P1234 and P123 of Sindbis virus (SIN), another alphavirus, has been studied in detail (4Strauss J.H. Strauss E.G. Microbiol. Rev. 1994; 58: 491-562Crossref PubMed Google Scholar, 8Strauss J.H. Strauss E.G. Semin. Virol. 1990; 1: 347-356Google Scholar, 9ten Dam E. Flint M. Ryan M.D. J. Gen. Virol. 1999; 80: 1879-1888Crossref PubMed Scopus (23) Google Scholar). Using mostly in vitro translation and site-directed mutagenesis as tools, autocatalytic protease activity was detected in the polyprotein and its cleavage intermediates. The protease activity was localized to nsP2, and more precisely, to its carboxyl-terminal part (10Hardy W.R. Strauss J.H. J. Virol. 1989; 63: 4653-4664Crossref PubMed Google Scholar, 11Ding M.X. Schlesinger M.J. Virology. 1989; 171: 280-284Crossref PubMed Scopus (79) Google Scholar). Cysteine 481 and histidine 558 were identified as essential residues for the autoprotease activity (12Strauss E.G. de Groot R.J. Levinson R. Strauss J.H. Virology. 1992; 191: 932-940Crossref PubMed Scopus (102) Google Scholar), supporting the view that the protease is a thiol proteinase belonging to the papain superfamily. The same conclusion was reached by sequence comparisons (13Gorbalenya A.E. Koonin E.V. Lai M.-C. FEBS Lett. 1991; 288: 201-205Crossref PubMed Scopus (270) Google Scholar). The respective mutation of Cys-478 in SFV nsP2 also inactivates the autocatalytic processing of P1234, P123, and P23 (14Merits A. Vasiljeva L. Ahola T. Kääriäinen L. Auvinen P. J. Gen. Virol. 2001; 82: 765-773Crossref PubMed Scopus (60) Google Scholar). The amino-terminal domain of SFV nsP2 (residues 1–470) has been shown to house several enzymatic activities including RNA triphosphatase (15Vasiljeva L. Merits A. Auvinen P. Kääriäinen L. J. Biol. Chem. 2000; 275: 17281-17287Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), nucleoside triphosphatase (16Rikkonen M. Peränen J. Kääriäinen L. J. Virol. 1994; 68: 5804-5810Crossref PubMed Google Scholar), and RNA helicase (17Gomez de Cedrón M. Ehsani N. Mikkola M.L. Garcı́a J.A. Kääriäinen L. FEBS Lett. 1999; 448: 19-22Crossref PubMed Scopus (132) Google Scholar). Interestingly, a significant amount of nsP2, synthesized during infection, is transported into the nucleus (18Peränen J. Rikkonen M. Liljeström P. Kääriäinen L. J. Virol. 1990; 64: 1888-1896Crossref PubMed Google Scholar, 19Peränen J. Rikkonen M. Kääriäinen L. J. Histochem. Cytochem. 1993; 41: 447-454Crossref PubMed Scopus (56) Google Scholar). The core of the nuclear localization signal was mapped to a short sequence P647RRR (20Rikkonen M. Peränen J. Kääriäinen L. Virology. 1992; 189: 462-473Crossref PubMed Scopus (77) Google Scholar). nsP2 mutant PRDR is not lethal for the virus in cell culture, but SFV carrying this mutation is apathogenic for mouse (21Rikkonen M. Virology. 1996; 218: 352-361Crossref PubMed Scopus (66) Google Scholar). In addition, the carboxyl-terminal domain of SFV nsP2 has been implicated in the regulation of the synthesis of the subgenomic mRNA (22Keränen S. Kääriäinen L. J. Virol. 1979; 47: 505-515Crossref Google Scholar, 23Sawicki D.L. Sawicki S.G. Virology. 1985; 144: 20-34Crossref PubMed Scopus (39) Google Scholar, 24Suopanki J. Sawicki D.L. Sawicki S.G. Kääriäinen L. J. Gen. Virol. 1998; 79: 309-319Crossref PubMed Scopus (51) Google Scholar). Taken together, these data suggest that alphavirus nsP2 consists of two structurally independent domains, each possessing a distinct set of biological activities. However, direct proof that purified nsP2 or its carboxyl-terminal part has protease activity has been lacking. To this end, recombinant nsP2 and a set of its amino-terminally truncated variants were expressed in Escherichia coli, purified by metal-chelate chromatography, and assayed for the presence of protease activity. Both full-length nsP2 and its soluble carboxyl-terminal fragment Pro39 (aa 458–799) catalyzed site-specific proteolysis of SFV P1234 in vitro. Furthermore, both nsP2 and Pro39 could specifically cleave purified recombinant fusion proteins containing short (∼40 aa) SFV-specific peptides, which span protease cleavage sites. Using this newly devised in vitro assay, we show that Pro39 was inactivated by N-ethylmaleimide, in accordance with the catalytic mechanism of cysteine proteases. DISCUSSIONPrevious work on Semliki Forest virus showed that early in infection the synthesis of the negative strand RNA was strictly dependent on protein synthesis and ceased in about 15 min after addition of cycloheximide (31Sawicki D.L. Sawicki S.G. J. Virol. 1980; 34: 108-118Crossref PubMed Google Scholar), whereas late in infection the synthesis of positive strand RNAs could continue for several hours in the absence of protein synthesis. Solution to this dilemma came from findings with Sindbis virus, another alphavirus, where the processing intermediate P123 of the nonstructural polyprotein together with nsP4 was shown to be responsible for the synthesis of the negative strand RNA (32Lemm J.A. Rice C.M. J. Virol. 1993; 67: 1905-1915Crossref PubMed Google Scholar, 33Lemm J.A. Rice C.M. J. Virol. 1993; 67: 1916-1926Crossref PubMed Google Scholar, 34Shirako Y. Strauss J.H. J. Virol. 1994; 68: 1874-1885Crossref PubMed Google Scholar). Cleavage of P123 is essential for the synthesis of the positive RNA strands. Thus, the regulated processing of the nonstructural polyprotein controls the early events of virus infection. An overactive processing mutant of Sindbis virus nsP2 (N614D) cannot replicate, as the P123 intermediate is too short-lived to enable the necessary synthesis of the complementary RNA (35Lemm J.A. Rümenapf T. Strauss E.G. Strauss J.H. Rice C.M. EMBO J. 1994; 13: 2925-2934Crossref PubMed Scopus (231) Google Scholar).Our knowledge of the processing of alphavirus nonstructural polyprotein(s) is based mostly on experiments of in vitrotranslation of Sindbis virus RNA. Ingenious constructions by which the cleavage sites were mutated alone and in different combinations, together with constructs coding for enzymatically inactive polyprotein as substrates, have been used to analyze this complex process (4Strauss J.H. Strauss E.G. Microbiol. Rev. 1994; 58: 491-562Crossref PubMed Google Scholar, 8Strauss J.H. Strauss E.G. Semin. Virol. 1990; 1: 347-356Google Scholar,9ten Dam E. Flint M. Ryan M.D. J. Gen. Virol. 1999; 80: 1879-1888Crossref PubMed Scopus (23) Google Scholar). These experiments showed that the polyprotein P1234 itself and all its cleavage intermediates containing nsP2 to be active proteases. The cleavability of the different sites varied and was dependent on the order of removal of different nsPs from the polyprotein substrate. Particularly interesting was the finding where the cleavage of site 2/3 in P1234 or P123 was only possible after the cleavage of nsP1 (36Shirako Y. Strauss J.H. Virology. 1990; 177: 54-64Crossref PubMed Scopus (65) Google Scholar). Evidently, conformational changes in P1234 and its cleavage products affect the interactions between the cleavage sites and the protease domain of nsP2 in a complex manner. To understand the processes better, we have characterized purified SFV nsP2 and its carboxyl-terminal fragment Pro39 as proteolytic enzymes. As substrates we used in vitro synthesized polyproteins P1234, P123, P23 and P34, as well as recombinant thioredoxin fusion proteins, which contain short SFV-specific fragments, spanning the polyprotein processing sites 1/2, 2/3, and 3/4.We show for the first time that purified nsP2 has proteolytic activity, which cleaves readily the 3/4 site of P1234 and P34. Deletion series of nsP2 resulted in a soluble, active carboxyl-terminal fragment consisting of amino acid residues 459–799, which was designated as Pro39. It was purified to near homogeneity by metal-affinity chromatography. The specificities of Pro39 and nsP2 were identical, indicating that the amino-terminal half of nsP2 does not affect the fidelity of the protease. According to sequence alignments with the thiol protease superfamily, Pro39 contains a conserved protease domain (459), but also almost 200 carboxyl-terminal extra amino acids (9ten Dam E. Flint M. Ryan M.D. J. Gen. Virol. 1999; 80: 1879-1888Crossref PubMed Scopus (23) Google Scholar,13Gorbalenya A.E. Koonin E.V. Lai M.-C. FEBS Lett. 1991; 288: 201-205Crossref PubMed Scopus (270) Google Scholar). Our attempts to delete 40–120 amino acids from the carboxyl terminus of nsP2 resulted in insoluble or inactive proteins (Fig. 1). Experiments with temperature-sensitive mutants of SIN and SFV nsP2 have shown that amino acid replacements N700K in SIN ts133, K736S in SIN ts24, and M781T in SFV ts4 result in inhibition of protease activity at 39 °C, suggesting that the extreme carboxyl terminus of nsP2 participates somehow in the protease function (24Suopanki J. Sawicki D.L. Sawicki S.G. Kääriäinen L. J. Gen. Virol. 1998; 79: 309-319Crossref PubMed Scopus (51) Google Scholar).Establishment of a biochemical assay system consisting of isolated Pro39 and thioredoxin attached cleavage regions of the SFV nonstructural polyprotein allowed characterization of the viral protease under defined experimental conditions. Pro39 was inactivated by N-ethylmaleimide but not with pepstatin, EDTA, or PMSF. These are properties, which are in accordance with its classification as a thiol proteinase of the papain superfamily. However, Pro39 is not inhibited by E-64, which is a typical inhibitor of cysteine proteases (37Beynon R.J. Bond J.S. Beynon R.J. Bond J.S. Proteolytic Enzymes: A Practical Approach. Oxford University Press, Oxford, United Kingdom1989: 83-104Google Scholar). Sensitivity for NEM and resistance for E-64 have been previously reported for poliovirus 3C thiol proteinase (38Lawson M.A. Semler B.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9919-9923Crossref PubMed Scopus (53) Google Scholar, 39Yu F.Sh. Lloyd R.E. Virology. 1991; 182: 615-625Crossref PubMed Scopus (64) Google Scholar). Another interesting feature of Pro39 is the inhibition by zinc ions (Fig.8).When Pro39 (or nsP2) was added to the reaction mixture, after in vitro translation of P12 or P123, almost a quantitative release of nsP1 was observed. Similarly, when P34 or P1234 were used as substrates, quantitative release of nsP4 was seen, whereas only a small amount of nsP3 was released from P23, P123, or P1234 (Fig. 3). These results suggested that sites 1/2 and 3/4 were exposed to the added protease, whereas site 2/3 was not. To study this phenomenon under controlled conditions, in which the large protein domains would not interfere sterically with the proteolysis, we constructed fusion proteins with a different number of amino acid residues around the cleavage sites.Constructs with less than 10 amino acids on both sides of the cleavage site were not digested by Pro39 or nsP2 (data not shown). We ended up using thioredoxin fusion proteins with about 40 residues of each cleavage region (Trx12, Trx23, and Trx34). Isolation of the cleavage products and their mass spectrometric analysis, as well as amino-terminal sequencing showed that Pro39 cleavage products were derived exactly from the predicted cleavage sites, determined previously by radiosequence analysis (29Kalkkinen N. Laaksonen M. Söderlund H. Jörnvall H. Virology. 1981; 113: 188-195Crossref PubMed Scopus (10) Google Scholar, 30Kalkkinen N. Wittmann-Liebold B. Salnikow J. Erdmann V.A. Advanced Methods in Protein Microsequence Analysis. Springer-Verlag, Heidelberg1986: 194-206Crossref Google Scholar) (Table II). As controls we used thioredoxin fusion proteins with mutations close to the cleavage site, which were not digested by Pro39 or nsP2 (Fig. 4). Thus, we conclude that both proteases recognize specifically the three cleavage sites of the SFV nonstructural polyprotein. However, there were large differences in the sensitivity of the different cleavage sites, the 3/4 junction being most sensitive. Roughly 5000-fold more Pro39 was needed for complete cleavage of site 1/2 (Fig. 5). Under the same conditions, only a small amount of Trx23 was cleaved.The different sensitivities of the three cleavage sites may well reflect the different specificities of the protease associated with the polyproteins, in which the cleavage at site 2/3 of P123 or P1234 does not take place unless preceded by cleavage of nsP1. The fact that Pro39 can catalyze cleavage at 1/2 site of in vitro translated SFV P12, P123, and P1234, which normally undergoes cleavage incis (14Merits A. Vasiljeva L. Ahola T. Kääriäinen L. Auvinen P. J. Gen. Virol. 2001; 82: 765-773Crossref PubMed Scopus (60) Google Scholar), is better understood when realized that the estimated molar enzyme to substrate ratio represents an excess of 50 to 1, which is difficult to imagine to take place during virus infection. Thus, we cannot exclude the possibility that cleavages at sites 1/2 and 2/3 require cofactor(s), which might be derived from the other nsPs. Such a situation has been characterized thoroughly for the NS3 protease of hepatitis C virus. The site-specific proteolytic activity of NS3 protease was greatly increased by a short amino acid sequence of NS4 protein adjacently located in the polyprotein (40Failla C. Tomei L. De Francesco R. J. Virol. 1994; 68: 3753-3760Crossref PubMed Google Scholar, 41Lin C. Rice C.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7622-7626Crossref PubMed Scopus (84) Google Scholar, 43Love R.A. Parge H.E. Wickersham J.A. Hostomsky Z. Habuka N. Moomaw E.W. Adachi T. Hostomska Z. Cell. 1996; 87: 331-342Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar,44Shimizu Y. Yamaji K. Masuho Y. Yokota T. Inoue H. Sudo K. Satoh S. Shimotohno K. J. Virol. 1996; 70: 127-132Crossref PubMed Google Scholar).The processing intermediate P123, together with nsP4, enables the synthesis of the complementary RNA for a short time period, whereafter P123 is autocatalytically cleaved to yield the components of the stable RNA polymerase among them nsP2. The released nsP2 exercises its role in two different forms. As a part of the RNA polymerase complex (45Kujala P. Ikäheimonen A. Ehsani N. Vihinen H. Auvinen P. Kääriäinen L. J. Virol. 2001; 75: 3873-3884Crossref PubMed Scopus (177) Google Scholar), the amino-terminal domain provides RNA triphosphatase and RNA helicase activities (15Vasiljeva L. Merits A. Auvinen P. Kääriäinen L. J. Biol. Chem. 2000; 275: 17281-17287Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 17Gomez de Cedrón M. Ehsani N. Mikkola M.L. Garcı́a J.A. Kääriäinen L. FEBS Lett. 1999; 448: 19-22Crossref PubMed Scopus (132) Google Scholar). As "soluble nsP2," the carboxyl-terminal domain acts as a regulator of 26 S RNA synthesis (24Suopanki J. Sawicki D.L. Sawicki S.G. Kääriäinen L. J. Gen. Virol. 1998; 79: 309-319Crossref PubMed Scopus (51) Google Scholar) and as atrans-acting protease, which catalyzes the rapid cleavage of P1234 and P123, thus preventing the negative strand RNA synthesis late in infection. Previous work on Semliki Forest virus showed that early in infection the synthesis of the negative strand RNA was strictly dependent on protein synthesis and ceased in about 15 min after addition of cycloheximide (31Sawicki D.L. Sawicki S.G. J. Virol. 1980; 34: 108-118Crossref PubMed Google Scholar), whereas late in infection the synthesis of positive strand RNAs could continue for several hours in the absence of protein synthesis. Solution to this dilemma came from findings with Sindbis virus, another alphavirus, where the processing intermediate P123 of the nonstructural polyprotein together with nsP4 was shown to be responsible for the synthesis of the negative strand RNA (32Lemm J.A. Rice C.M. J. Virol. 1993; 67: 1905-1915Crossref PubMed Google Scholar, 33Lemm J.A. Rice C.M. J. Virol. 1993; 67: 1916-1926Crossref PubMed Google Scholar, 34Shirako Y. Strauss J.H. J. Virol. 1994; 68: 1874-1885Crossref PubMed Google Scholar). Cleavage of P123 is essential for the synthesis of the positive RNA strands. Thus, the regulated processing of the nonstructural polyprotein controls the early events of virus infection. An overactive processing mutant of Sindbis virus nsP2 (N614D) cannot replicate, as the P123 intermediate is too short-lived to enable the necessary synthesis of the complementary RNA (35Lemm J.A. Rümenapf T. Strauss E.G. Strauss J.H. Rice C.M. EMBO J. 1994; 13: 2925-2934Crossref PubMed Scopus (231) Google Scholar). Our knowledge of the processing of alphavirus nonstructural polyprotein(s) is based mostly on experiments of in vitrotranslation of Sindbis virus RNA. Ingenious constructions by which the cleavage sites were mutated alone and in different combinations, together with constructs coding for enzymatically inactive polyprotein as substrates, have been used to analyze this complex process (4Strauss J.H. Strauss E.G. Microbiol. Rev. 1994; 58: 491-562Crossref PubMed Google Scholar, 8Strauss J.H. Strauss E.G. Semin. Virol. 1990; 1: 347-356Google Scholar,9ten Dam E. Flint M. Ryan M.D. J. Gen. Virol. 1999; 80: 1879-1888Crossref PubMed Scopus (23) Google Scholar). These experiments showed that the polyprotein P1234 itself and all its cleavage intermediates containing nsP2 to be active proteases. The cleavability of the different sites varied and was dependent on the order of removal of different nsPs from the polyprotein substrate. Particularly interesting was the finding where the cleavage of site 2/3 in P1234 or P123 was only possible after the cleavage of nsP1 (36Shirako Y. Strauss J.H. Virology. 1990; 177: 54-64Crossref PubMed Scopus (65) Google Scholar). Evidently, conformational changes in P1234 and its cleavage products affect the interactions between the cleavage sites and the protease domain of nsP2 in a complex manner. To understand the processes better, we have characterized purified SFV nsP2 and its carboxyl-terminal fragment Pro39 as proteolytic enzymes. As substrates we used in vitro synthesized polyproteins P1234, P123, P23 and P34, as well as recombinant thioredoxin fusion proteins, which contain short SFV-specific fragments, spanning the polyprotein processing sites 1/2, 2/3, and 3/4. We show for the first time that purified nsP2 has proteolytic activity, which cleaves readily the 3/4 site of P1234 and P34. Deletion series of nsP2 resulted in a soluble, active carboxyl-terminal fragment consisting of amino acid residues 459–799, which was designated as Pro39. It was purified to near homogeneity by metal-affinity chromatography. The specificities of Pro39 and nsP2 were identical, indicating that the amino-terminal half of nsP2 does not affect the fidelity of the protease. According to sequence alignments with the thiol protease superfamily, Pro39 contains a conserved protease domain (459), but also almost 200 carboxyl-terminal extra amino acids (9ten Dam E. Flint M. Ryan M.D. J. Gen. Virol. 1999; 80: 1879-1888Crossref PubMed Scopus (23) Google Scholar,13Gorbalenya A.E. Koonin E.V. Lai M.-C. FEBS Lett. 1991; 288: 201-205Crossref PubMed Scopus (270) Google Scholar). Our attempts to delete 40–120 amino acids from the carboxyl terminus of nsP2 resulted in insoluble or inactive proteins (Fig. 1). Experiments with temperature-sensitive mutants of SIN and SFV nsP2 have shown that amino acid replacements N700K in SIN ts133, K736S in SIN ts24, and M781T in SFV ts4 result in inhibition of protease activity at 39 °C, suggesting that the extreme carboxyl terminus of nsP2 participates somehow in the protease function (24Suopanki J. Sawicki D.L. Sawicki S.G. Kääriäinen L. J. Gen. Virol. 1998; 79: 309-319Crossref PubMed Scopus (51) Google Scholar). Establishment of a biochemical assay system consisting of isolated Pro39 and thioredoxin attached cleavage regions of the SFV nonstructural polyprotein allowed characterization of the viral protease under defined experimental conditions. Pro39 was inactivated by N-ethylmaleimide but not with pepstatin, EDTA, or PMSF. These are properties, which are in accordance with its classification as a thiol proteinase of the papain superfamily. However, Pro39 is not inhibited by E-64, which is a typical inhibitor of cysteine proteases (37Beynon R.J. Bond J.S. Beynon R.J. Bond J.S. Proteolytic Enzymes: A Practical Approach. Oxford University Press, Oxford, United Kingdom1989: 83-104Google Scholar). Sensitivity for NEM and resistance for E-64 have been previously reported for poliovirus 3C thiol proteinase (38Lawson M.A. Semler B.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9919-9923Crossref PubMed Scopus (53) Google Scholar, 39Yu F.Sh. Lloyd R.E. Virology. 1991; 182: 615-625Crossref PubMed Scopus (64) Google Scholar). Another interesting feature of Pro39 is the inhibition by zinc ions (Fig.8). When Pro39 (or nsP2) was added to the reaction mixture, after in vitro translation of P12 or P123, almost a quantitative release of nsP1 was observed. Similarly, when P34 or P1234 were used as substrates, quantitative release of nsP4 was seen, whereas only a small amount of nsP3 was released from P23, P123, or P1234 (Fig. 3). These results suggested that sites 1/2 and 3/4 were exposed to the added protease, whereas site 2/3 was not. To study this phenomenon under controlled conditions, in which the large protein domains would not interfere sterically with the proteolysis, we constructed fusion proteins with a different number of amino acid residues around the cleavage sites. Constructs with less than 10 amino acids on both sides of the cleavage site were not digested by Pro39 or nsP2 (data not shown). We ended up using thioredoxin fusion proteins with about 40 residues of each cleavage region (Trx12, Trx23, and Trx34). Isolation of the cleavage products and their mass spectrometric analysis, as well as amino-terminal sequencing showed that Pro39 cleavage products were derived exactly from the predicted cleavage sites, determined previously by radiosequence analysis (29Kalkkinen N. Laaksonen M. Söderlund H. Jörnvall H. Virology. 1981; 113: 188-195Crossref PubMed Scopus (10) Google Scholar, 30Kalkkinen N. Wittmann-Liebold B. Salnikow J. Erdmann V.A. Advanced Methods in Protein Microsequence Analysis. Springer-Verlag, Heidelberg1986: 194-206Crossref Google Scholar) (Table II). As controls we used thioredoxin fusion proteins with mutations close to the cleavage site, which were not digested by Pro39 or nsP2 (Fig. 4). Thus, we conclude that both proteases recognize specifically the three cleavage sites of the SFV nonstructural polyprotein. However, there were large differences in the sensitivity of the different cleavage sites, the 3/4 junction being most sensitive. Roughly 5000-fold more Pro39 was needed for complete cleavage of site 1/2 (Fig. 5). Under the same conditions, only a small amount of Trx23 was cleaved. The different sensitivities of the three cleavage sites may well reflect the different specificities of the protease associated with the polyproteins, in which the cleavage at site 2/3 of P123 or P1234 does not take place unless preceded by cleavage of nsP1. The fact that Pro39 can catalyze cleavage at 1/2 site of in vitro translated SFV P12, P123, and P1234, which normally undergoes cleavage incis (14Merits A. Vasiljeva L. Ahola T. Kääriäinen L. Auvinen P. J. Gen. Virol. 2001; 82: 765-773Crossref PubMed Scopus (60) Google Scholar), is better understood when realized that the estimated molar enzyme to substrate ratio represents an excess of 50 to 1, which is difficult to imagine to take place during virus infection. Thus, we cannot exclude the possibility that cleavages at sites 1/2 and 2/3 require cofactor(s), which might be derived from the other nsPs. Such a situation has been characterized thoroughly for the NS3 protease of hepatitis C virus. The site-specific proteolytic activity of NS3 protease was greatly increased by a short amino acid sequence of NS4 protein adjacently located in the polyprotein (40Failla C. Tomei L. De Francesco R. J. Virol. 1994; 68: 3753-3760Crossref PubMed Google Scholar, 41Lin C. Rice C.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7622-7626Crossref PubMed Scopus (84) Google Scholar, 43Love R.A. Parge H.E. Wickersham J.A. Hostomsky Z. Habuka N. Moomaw E.W. Adachi T. Hostomska Z. Cell. 1996; 87: 331-342Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar,44Shimizu Y. Yamaji K. Masuho Y. Yokota T. Inoue H. Sudo K. Satoh S. Shimotohno K. J. Virol. 1996; 70: 127-132Crossref PubMed Google Scholar). The processing intermediate P123, together with nsP4, enables the synthesis of the complementary RNA for a short time period, whereafter P123 is autocatalytically cleaved to yield the components of the stable RNA polymerase among them nsP2. The released nsP2 exercises its role in two different forms. As a part of the RNA polymerase complex (45Kujala P. Ikäheimonen A. Ehsani N. Vihinen H. Auvinen P. Kääriäinen L. J. Virol. 2001; 75: 3873-3884Crossref PubMed Scopus (177) Google Scholar), the amino-terminal domain provides RNA triphosphatase and RNA helicase activities (15Vasiljeva L. Merits A. Auvinen P. Kääriäinen L. J. Biol. Chem. 2000; 275: 17281-17287Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 17Gomez de Cedrón M. Ehsani N. Mikkola M.L. Garcı́a J.A. Kääriäinen L. FEBS Lett. 1999; 448: 19-22Crossref PubMed Scopus (132) Google Scholar). As "soluble nsP2," the carboxyl-terminal domain acts as a regulator of 26 S RNA synthesis (24Suopanki J. Sawicki D.L. Sawicki S.G. Kääriäinen L. J. Gen. Virol. 1998; 79: 309-319Crossref PubMed Scopus (51) Google Scholar) and as atrans-acting protease, which catalyzes the rapid cleavage of P1234 and P123, thus preventing the negative strand RNA synthesis late in infection. We thank Airi Sinkko for excellent technical assistance. We are grateful to Dr. Nisse Kalkkinen for valuable advice and discussions. We thank Dr. Marja Makarow and Dr. Tero Ahola for critical reading of the manuscript.
Referência(s)