Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, PO Box 56, FIN-00014, Helsinki, Finland1
Author for correspondence: Petri Auvinen. Fax +358 9 19158952. e-mail Petri.Auvinen{at}helsinki.fi
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The processing of SIN virus P123 and P1234 has been studied extensively (Hardy & Strauss, 1988 , 1989
; Ding & Schlesinger, 1989
; Hardy et al., 1990
; Shirako & Strauss, 1990
; Strauss et al., 1992
; Strauss & Strauss, 1994
). According to these studies, P2 is responsible for all proteolytic cleavages of P1234 and P123. Experiments performed in vitro indicate that P12, P123, P23 and P2 are proteases with slightly different substrate specificities (Shirako & Strauss, 1990
). Protease activity has been mapped to the carboxy-terminal half of P2, which has a domain similar to papain-like proteinases with active site cysteine (C481) and histidine (H558) residues (Ding & Schlesinger, 1989
; Hardy & Strauss, 1989
; Strauss et al., 1992
). Mutation of these residues results in the loss of P2 protease activity and failure to produce infectious RNA when transcribed from the infectious SIN virus cDNA clone (Strauss et al., 1992
). Construction of cleavage site mutants of the SIN virus polyprotein, which express P123 and P4 or P1, P23 and P4, results in low yields of virus but the early minus-strand RNA synthesis is as efficient as that of the wild-type virus (Shirako & Strauss, 1994
). The specific role of P123 and possibly P23 in SIN virus minus-strand RNA synthesis has also been demonstrated by expressing SIN virus P123 and P4 using recombinant vaccinia viruses (Lemm & Rice, 1993a
, b
; Lemm et al., 1994
). Only a low level of plus-strand RNA synthesis can be carried out by P123 together with properly cleaved P4, with tyrosine as the amino-terminal residue (Lemm & Rice, 1993 b
; Lemm et al., 1994
). Thus, SIN virus-directed RNA synthesis is controlled by expression and cleavage of the non-structural polyprotein.
In SFV-infected cells, translation of the non-structural polyprotein should yield equimolar amounts of P1P4. However, only about 20% of the expected amount of P4 is found in pulse-labelling experiments (Takkinen et al., 1991 ). The appearance of P4 as the first mature protein, during in vitro translation and in SFV-infected cells, suggested that it may be cleaved immediately after completion of the translation of P1234. Hypertonic synchronization of translation initiation in cells infected with polyprotein cleavage mutants ts4 and ts6 of SFV supported immediate P4 cleavage (Takkinen et al., 1991
). There was also rapid processing of the P34 precursor, which yielded prevalent amounts of mature P3, while P4 seemed to be degraded. SFV P4 has at its amino terminus a series of amino acids with similarity to retroviral and cellular aspartic proteases with a conserved DTG sequence (Takkinen et al., 1990
). On the basis of in vitro translation experiments, it was proposed that the P3/4 site was cleaved by a protease domain localized within the first 102 amino acid residues of P4 (Takkinen et al., 1990
). A nascent autocatalytic cleavage of P4 from P1234 would produce the active catalytic polymerase subunit, whereafter P2 would cleave at sites P1/2 and P2/3. If the latter cleavage takes place first, the P4 sequences in P34 cannot be utilized for RNA polymerase production but would be degraded instead (Takkinen et al., 1991
).
We have produced SFV P1234 and its shorter derivatives by recombinant baculovirus expression in insect cells and by in vitro translation. In order to reinvestigate the processing of the non-structural polyprotein, we have introduced mutations into the putative active sites of the P2 and P4 proteases and followed their effects on the processing of P1234 and its derivatives. Although both putative active site mutations were lethal for the virus, processing of P1234 was carried out exclusively by the P2 protease.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vitro transcription/translation of full-length cDNA clones and transfection of cells.
DNA from icSFV4, icSFVMut2CA, icSFVMut4DA and icSFVMut2CA4DA was linearized by digestion with SpeI and purified on QIAquick PCR purification column (Qiagen). Capped RNA was transcribed from 5 µg of linearized plasmid in 100 µl reaction mix comprising 20 µl of 5x transcription buffer (Promega), 1 mM m7GpppG (Pharmacia), 5 mM DTT, 3 mM each of UTP, ATP and CTP, 0·3 mM GTP (Pharmacia), 100 U RNasin (Promega) and 30 U SP6 RNA polymerase (Promega). The mixture was incubated at 37 °C for 2·5 h, GTP was added to a final 3 mM concentration and incubation was continued for a further 1 h at 37 °C. RNA transcripts were used for in vitro translation with a coupled T7-transcription/translation kit (Promega), without the T7 polymerase. The standard 25 µl reaction mixture contained 1 µg of RNA and 10 µCi of [35S]methionine (Amersham). Reaction products were analysed by SDSPAGE in 10% gels and by autoradiography. The same transcripts were used to transfect HeLa and BHK cells, essentially as described (Peränen & Kääriäinen, 1991 ). Infection was monitored at 7 and 16 h post-transfection by immunofluorescence with polyclonal antisera against P1 and the envelope protein E2 (Laakkonen et al., 1998
).
In vitro translation was performed using the T7-transcription/translation kit (Promega), according to the manufacturers instructions. The translation mixture was incubated at 30 °C for 40 min. In some experiments, the in vitro translation reaction was stopped by the addition of cycloheximide to a final concentration of 0·6 mg/ml. For in trans cleavage experiments, substrate and protease proteins produced in vitro were incubated, mixed and incubated for a further 40 min at 30 °C to allow cleavages to occur.
Construction of recombinant baculoviruses expressing SFV polyproteins.
The BAC-to-BAC Recombinant Baculovirus system (Gibco BRL) was used according to the manufacturers protocols. The SFV polyprotein-coding fragments were released by digestion with BamHI/SphI from plasmids P1234, P12CA34, P1234DA, P12CA34DA, P123, P12CA3, P23, P2CA3, P34, P34DA, P34, P
34DA, P3
4 and P3
4DA and cloned into the plasmid vector pFASTBAC1 (Gibco BRL), which was digested with the same enzymes. Recombinant baculoviruses expressing these constructs were created and designated Bac1234, Bac12CA34, Bac1234DA, Bac12CA34DA, Bac123, Bac12CA3, Bac23, Bac2CA3, Bac34, Bac34DA, Bac
34, Bac
34DA, Bac3
4 and Bac3
4DA, respectively. The correct size of the expressed polyproteins was verified by SDSPAGE and Western blotting with SFV non-structural protein-specific antisera (Peränen et al., 1988
; Ahola et al., 1997
). Baculovirus stocks were amplified and titrated using an insect cell culture (Sf9, Gibco BRL) and Sf-900 II culture media (Gibco BRL) containing 10% foetal calf serum and 50 mg/l gentamycin. However, for polyprotein expression and pulsechase experiments, HighFive cells (BTI-TN-5B1-4) (Invitrogen) and HighFive medium (Invitrogen) were used. Optimal expression time was determined to be 4048 h post-infection (p.i.) at 27 °C with 10 p.f.u. per cell. These conditions were used in all experiments.
Pulsechase experiments.
Approximately 2x106 HighFive cells per 35 mm dish were infected with each recombinant baculovirus. The original medium was changed to a methionine-free Grace medium (Invitrogen) at 40 h p.i. for 60 min followed by a 15 min pulse (500 µCi/ml [35S]methionine, Amersham). Samples were collected after a short pulse (<1 min) or 90 min chase with HighFive medium containing a tenfold excess of unlabelled methionine. In some experiments, proteosome inhibitor MG132 (Calbiochem) was added to the medium at 50 µM. Samples were collected and immunoprecipitated with rabbit polyclonal antisera raised against SFV P1, P2, P3 or P4, essentially as described elsewhere (Kujala et al., 1997 ; Suopanki et al., 1998
). Samples were then run on SDSPAGE gels, which were dried and then exposed to phosphorimaging plates. Data were analysed using a BAS-1500 phosphorimager (FuJiFILM) and TINA program, version 2.09c.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Effects of P2CA and P4DA mutations on processing of polyprotein intermediates
To further analyse the processing of SFV non-structural polyprotein, we studied the in vitro cleavages of previously identified intermediates P123, P23 and P34 and the effects of active site mutations in P2 and P4 on them. No cleavage of P12CA3 or P2CA3 could be seen in the translation products (Fig. 2A, lanes 2, 4), whereas the wild-type P123 and P23 were processed (Fig. 2A
, lanes 1, 3). By precipitation with antisera against P1, P2 and P3, it was possible to identify the cleavage products of P123 and P23. Thus, P123 yielded P12, P2, P3/P1 and a truncated form of P1 (
P1) (P1 and P3 migrate similarly on the gels used). Translation of construct P23 yielded both P2 and P3 (Fig. 2A
, lane 3). However, the translation products of both P34 and P34DA constructs remained uncleaved (Fig. 2A
, lanes 5, 6). Thus, we concluded that in vitro processing of P123 requires active P2 protease. In contrast, processing of P34 did not take place, irrespective of the mutation in the putative active site of the proposed P4 protease. In addition, we used truncated versions of P34 containing the P3/4 cleavage site (Takkinen et al., 1990
): P
34 and P
34DA, with a complete P3 (482 residues) plus 102 residues from the N terminus of P4 (about 64 kDa), and P3
4 and P3
4DA, with only 78 residues from the C terminus of P3 joined to the complete P4 (about 76 kDa). Combined in vitro transcription/translation experiments were carried out with these constructs (Fig. 2B
, lanes 14). No cleavage of the putative precursors, which would yield either P3 (Fig. 2B
, lanes 1, 2) or P4 (Fig. 2B
, lanes 3, 4), was observed as compared with the in vitro translation of P3 and P4 (Fig. 2B
, lanes 5, 6).
|
The non-structural proteins produced by Bac1234 were identified in pulsechase experiments. A 15 min pulse was followed by a 90 min chase, as described in Methods. Anti-P1 antibodies precipitated P12 (only faintly seen in this exposure) and P1 after the pulse and only P1 after the chase (Fig. 3, lanes 1, 2). Similar analysis with antibodies against P2, P3 and P4 revealed the precursors P12 and P34 as well as the final products (Fig. 3
, lanes 38). Thus, all mature SFV non-structural proteins were detectable by immunoprecipitation (Fig. 3
) as well as by Western blotting (data not shown).
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Here we have studied the processing of SFV polyprotein P1234 and its previously identified processing intermediates P123, P12 and P34 (Lachmi & Kääriäinen, 1976 ; Glanville et al., 1978
; Keränen & Kääriäinen, 1979
; Kääriäinen et al., 1987
) as well as the putative intermediate P23 by in vitro translation and by expression through recombinant baculoviruses. To identify the responsible proteases for processing the polyprotein, we introduced a point mutation into the active site of the P2 protease (Strauss et al., 1992
; Strauss & Strauss, 1994
; ten Dam et al., 1999
) and transferred it into constructs encoding the non-structural polyprotein and its cleavage intermediates. To specifically investigate the putative autoprotease activity of P4 (Takkinen et al., 1990
, 1991
), we prepared constructs encoding P34, and truncated forms thereof (P
34, P3
4), representing major deletions of P3 or P4 but preserving the P3/4 junction sequence. Mutation in the putative active site aspartyl residue D6 in P4 was introduced into the above constructs as well as into P1234. In vitro translation and expression in insect cells through recombinant baculoviruses failed to support the previously proposed autoprotease activity of P4. In contrast, mutation in the active site of the P2 protease (cysteine residue C478) inhibited the processing of the non-structural polyprotein and its partial cleavage products. This confirms that P2 is the sole protease responsible for the complete processing of the polyprotein, as has been previously shown for SIN virus (Strauss & Strauss, 1994
).
The design of these experiments allowed us to make conclusions concerning polyprotein processing at the different cleavage sites. Co-infection with Bac23- and Bac12CA34- or Bac12CA3-encoding substrate polyproteins, which cannot self-cleave, did not yield P1, indicating that the P1/2 junction could not be cleaved in trans by P23 or its processing product P2. On the other hand, the ability of wild-type P123 and P12 to generate P1 showed that the P1/2 site could be cleaved but neither cis nor trans cleavage could be distinguished. In SIN virus P123, the P1/2 bond could be cleaved in a bimolecular reaction by P123, but it could not be cleaved in cis (Shirako & Strauss, 1990 ; de Groot et al., 1990
). Also in our in vitro translation experiments using P12CA3 as a substrate for proteolysis and P123 as a protease, we did not observe cleavage of the P1/2 bond in trans. However, the protease used is itself cleavable. Under similar conditions, the cleavage of SIN virus P1/2 is inefficient (Hardy & Strauss, 1989
). However, we have not succeeded in demonstrating in trans cleavage of the P1/2 bond in our in vitro system.
P2 and P3 appeared rapidly in Bac23-infected insect cells. P123 yielded P1, P2 and P3 in both insect cells and in vitro translation. In the SIN virus non-structural polyprotein, cleavage at the P1/2 site has been shown to be necessary before the second cleavage at the P2/3 site can occur (Shirako & Strauss, 1990 ). Thus, we assume that SFV P123 is also cleaved first at the P1/2 site followed by rapid cleavage of the P2/3 site. Co-infection with recombinant viruses Bac12CA3 and Bac23 resulted in the production of P12, P2 and P3, but not P1, showing that cleavage had taken place at the P2/3 site in trans.
Co-infection with Bac23 and Bac12CA34 or Bac34 yielded P4 as an indication of in trans cleavage at the P3/4 junction. Cleavage at the P3/4 site produces the early RNA polymerase responsible for the synthesis of 42S RNA minus strands (Lemm et al., 1994 ; Shirako & Strauss, 1994
). P123 is short-lived like minus-strand RNA synthesis in the presence of protein synthesis inhibitors (Sawicki & Sawicki, 1980
). Later in infection, when the concentration of P2 protease increases, the probability of cleavage in trans at the P2/3 site increases, leading to shut-off of minus-strand RNA synthesis. At the same time, P12 and P34 are seen as short-lived intermediates of the processing pathway (Lachmi & Kääriäinen, 1976
; Keränen & Kääriäinen, 1979
; Lehtovaara et al., 1980
; Keränen & Ruohonen, 1983
). Thus, it is possible to explain how the previously identified intermediates (P123, P12 and P34) are processed from SFV P1234 non-structural polyprotein. The complete cleavage products P1P4 serve as the late RNA polymerase, which synthesizes only 42 and 26S plus-strand RNAs (Suopanki et al., 1998
).
Although all SFV non-structural proteins are synthesized principally in equimolar amounts, there is clearly less mature P4 (about 20%) as compared with P1P3 (Takkinen et al., 1991 ). Addition of proteosomal inhibitor MG132 to the medium of insect cells infected with recombinant baculoviruses producing P4 resulted in an increased amount of P4, suggesting that this protein is normally degraded by proteosomes through the ubiquitination pathway, similar to SIN virus P4 (de Groot et al., 1991
).
In addition to the central role in the timing of minus- and plus-strand RNA synthesis, P2 has other important functions in alphavirus RNA replication. It is an NTPase (Rikkonen et al., 1994 ; Rikkonen, 1996
) and RNA helicase (Gomez de Cedron et al., 1999
). We found recently that P2 has RNA 5' triphosphatase activity needed in the capping of viral RNAs (Vasiljeva et al., 2000
). Triphosphatase, NTPase and RNA helicase activities are localized in the amino-terminal half of the molecule.
About 50% of P2 molecules are transported to the nucleus during virus infection (Peränen et al., 1990 ; Rikkonen et al., 1992
). The role of this nuclear transport in virus replication is not known, but elimination of the nuclear localization signal of P2 resulted in viable virus; however, the virus had lost its pathogenicity for mice (Rikkonen, 1996
). Finally, P2 regulates the synthesis of the subgenomic 26S mRNA of the structural proteins (Sawicki et al., 1978
; Suopanki et al., 1998
; Strauss & Strauss, 1994
). Protease activity, nuclear localization signals and probably the 26S RNA regulatory domain are located in the carboxy-terminal half of P2. As shown previously (reviewed by Strauss & Strauss, 1994
) and in this paper, P2 protease is also acting in the context of the non-structural polyprotein and its cleavage intermediates, which makes it a fascinating object for future studies. The availability of an in vitro system for alphavirus RNA replication (Lemm et al., 1998
) enables further investigation into the regulation of the RNA replication process.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
de Groot, R. J., Hardy, W. R., Shirako, Y. & Strauss, J. H. (1990). Cleavage-site preferences of Sindbis virus polyproteins containing the non-structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo. EMBO Journal 9, 2631-2638.[Abstract]
de Groot, R. J., Rümenapf, T., Kuhn, R. J., Strauss, E. G. & Strauss, J. H. (1991). Sindbis virus RNA polymerase is degraded by the N-end rule pathway. Proceedings of the National Academy of Sciences, USA 88, 8967-8971.[Abstract]
Ding, M. X. & Schlesinger, M. J. (1989). Evidence that Sindbis virus nsP2 is an autoproteinase which processes the virus nonstructural polyprotein. Virology 171, 280-284.[Medline]
Glanville, N., Lachmi, B.-E., Smith, A. E. & Kääriäinen, L. (1978). Tryptic peptide mapping of the nonstructural proteins of Semliki Forest virus and their precursors. Biochimica et Biophysica Acta 518, 497-506.[Medline]
Gomez de Cedron, M., Ehsani, N., Mikkola, M., Garcia, J. A. & Kääriäinen, L. (1999). RNA helicase activity of Semliki Forest virus replicase protein nsP2. FEBS Letters 448, 19-22.[Medline]
Hardy, W. R. & Strauss, J. H. (1988). Processing the nonstructural polyproteins of Sindbis virus: study of the kinetics in vivo by using monospecific antibodies. Journal of Virology 62, 998-1007.[Medline]
Hardy, W. R. & Strauss, J. H. (1989). Processing the nonstructural polyproteins of Sindbis virus: nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans. Journal of Virology 63, 4653-4664.[Medline]
Hardy, W. R., Hahn, Y. S., de Groot, R. J., Strauss, E. G. & Strauss, J. H. (1990). Synthesis and processing of the nonstructural polyproteins of several temperature-sensitive mutants of Sindbis virus. Virology 177, 199-208.[Medline]
Kääriäinen, L. & Söderlund, H. (1978). Structure and replication of alphaviruses. Current Topics in Microbiology and Immunology 82, 15-69.[Medline]
Kääriäinen, L., Takkinen, K., Keränen, S. & Söderlund, H. (1987). Replication of the genome of alphaviruses. Journal of Cell Science 7, 231-250.
Keränen, S. & Kääriäinen, L. (1979). Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses. J ournal of Virology 32, 19-29.[Medline]
Keränen, S. & Ruohonen, L. (1983). Nonstructural proteins of Semliki Forest virus: synthesis, processing, and stability in infected cells. Journal of Virology 47, 505-551.[Medline]
Kujala, P., Rikkonen, M., Ahola, T., Kelve, M., Saarma, M. & Kääriäinen, L. (1997). Monoclonal antibodies specific for Semliki Forest virus replicase protein nsP2. Journal of General Virology 78, 343-351.[Abstract]
Laakkonen, P., Auvinen, P., Kujala, P. & Kääriäinen, L. (1998). Alphavirus replicase protein nsP1 induces filopodia and rearrangement of actin filaments. Journal of Virology 72, 10265-10269.
Lachmi, B.-E. & Kääriäinen, L. (1976). Sequential translation of nonstructural proteins in cells infected with a Semliki Forest virus mutant. Proceedings of the National Academy of Sciences, USA 73, 1936-1940.[Abstract]
Lehtovaara, P., Ulmanen, I., Kääriäinen, L., Keränen, S. & Philipson, L. (1980). Synthesis and processing of Semliki Forest virus-specific nonstructural proteins in vivo and in vitro. European Journal of Biochemistry 112, 461-468.[Abstract]
Lemm, J. A. & Rice, C. M. (1993a). Assembly of functional Sindbis virus RNA replication complexes: requirement for coexpression of P123 and P34. Journal of Virology 67, 1905-1915.[Abstract]
Lemm, J. A. & Rice, C. M. (1993b). Roles of nonstructural polyproteins and cleavage products in regulating Sindbis virus RNA replication and transcription. Journal of Virology 67, 1916-1926.[Abstract]
Lemm, J. A., Rümenapf, T., Strauss, E. G., Strauss, J. H. & Rice, C. M. (1994). Polypeptide requirements for assembly of functional Sindbis virus replication complexes: a model for the temporal regulation of minus- and plus-strand RNA synthesis. EMBO Journal 13, 2925-2934.[Abstract]
Lemm, J. A., Bergquist, A., Read, C. M. & Rice, C. M. (1998). Template-dependent initiation of Sindbis virus RNA replication in vitro. Journal of Virology 72, 6546-6553.
Li, G. & Rice, C. M. (1989). Mutagenesis of the in-frame opal termination codon preceding nsP4 of Sindbis virus: studies of translational readthrough and its effect on virus replication. Journal of Virology 63, 1326-1337.[Medline]
Li, G. & Rice, C. M. (1993). The signal of translational readthrough of the UGA codon in Sindbis virus RNA involves a single cytidine residue immediately downstream of the termination codon. Journal of Virology 67, 5062-5067.[Abstract]
Liljeström, P., Lusa, S., Huylebroeck, D. & Garoff, H. (1991). In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release. Journal of Virology 65, 4107-4113.[Medline]
Peränen, J. & Kääriäinen, L. (1991). Biogenesis of type I cytopathic vacuoles in Semliki Forest virus-infected cells. Journal of Virology 65, 1623-1627.[Medline]
Peränen, J., Takkinen, K., Kalkkinen, N. & Kääriäinen, L. (1988). Semliki Forest virus-specific nonstructural protein nsP3 is a phosphoprotein. Journal of General Virology 69, 2165-2178.[Abstract]
Peränen, J., Rikkonen, M., Liljeström, P. & Kääriäinen, L. (1990). Nuclear localization of Semliki Forest virus-specific nonstructural protein nsP2. Journal of Virology 64, 1888-1896.[Medline]
Rikkonen, M. (1996). Functional significance of the nuclear-targeting and NTP-binding motifs of Semliki Forest virus nonstructural protein nsP2. Virology 218, 352-361.[Medline]
Rikkonen, M., Peränen, J. & Kääriäinen, L. (1992). Nuclear and nucleolar targeting signals of Semliki Forest virus nonstructural protein nsP2. Virology 189, 462-473.[Medline]
Rikkonen, M., Peränen, J. & Kääriäinen, L. (1994). ATPase and GTPase activities associated with Semliki Forest virus nonstructural protein nsP2. Journal of Virology 68, 5804-5810.[Abstract]
Sawicki, D. L. & Sawicki, S. G. (1980). Short-lived minus-strand polymerase for Semliki Forest virus. Journal of Virology 34, 108-118.[Medline]
Sawicki, D. L., Kääriäinen, L., Lambek, C. & Gomatos, P. J. (1978). Mechanism for the control of synthesis of Semliki Forest virus 26S and 42S RNA. Journal of Virology 25, 19-27.[Medline]
Sawicki, D. L., Sawicki, S. G., Keränen, S. & Kääriäinen, L. (1981a). Specific Sindbis virus-coded function for minus-strand RNA synthesis. Journal of Virology 39, 348-358.[Medline]
Sawicki, S. G., Sawicki, D. L., Kääriäinen, L. & Keränen, S. (1981b). A Sindbis virus mutant temperature-sensitive in the regulation of minus-strand RNA synthesis. Virology 115, 161-172.[Medline]
Shirako, Y. & Strauss, J. H. (1990). Cleavage between nsP1 and nsP2 initiates the processing pathway of Sindbis virus nonstructural polyprotein P123. Virology 177, 54-64.[Medline]
Shirako, Y. & Strauss, J. H. (1994). Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis. Journal of Virology 68, 1874-1885.[Abstract]
Strauss, J. H. & Strauss, E. G. (1994). The alphaviruses: gene expression, replication, and evolution. Microbiological Reviews 58, 491-562.[Abstract]
Strauss, E. G., Rice, C. M. & Strauss, J. H. (1983). Sequence encoding the alphavirus nonstructural proteins is interrupted by an opal termination codon. Proceedings of the National Academy of Sciences, USA 80, 5271-5275.[Abstract]
Strauss, E. G., de Groot, R. J., Levinson, R. & Strauss, J. H. (1992). Identification of the active site residues in the nsP2 proteinase of Sindbis virus. Virology 191, 932-940.[Medline]
Suopanki, J., Sawicki, D. L., Sawicki, S. G. & Kääriäinen, L. (1998). Regulation of alphavirus 26S mRNA transcription by replicase component nsP2. Journal of General Virology 79, 309-319.[Abstract]
Takkinen, K. (1986). Complete sequence of the nonstructural protein genes of Semliki Forest virus. Nucleic Acids Research 14, 5667-5682.[Abstract]
Takkinen, K., Peränen, J., Keränen, S., Söderlund, H. & Kääriäinen, L. (1990). The Semliki Forest virus-specific nonstructural protein nsP4 is an autoproteinase. European Journal of Biochemistry 189, 33-38.[Abstract]
Takkinen, K., Peränen, J. & Kääriäinen, L. (1991). Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein. ournal of General Virology 72, 1627-1633.
ten Dam, E., Flint, M. & Ryan, M. D. (1999). Virus-encoded proteinases of the Togaviridae. Journal of General Virology 80, 1879-1888.
Vasiljeva, L., Merits, A., Auvinen, P. & Kääriäinen, L. (2000). Identification of a novel function of the alphavirus capping apparatus. RNA 5'-triphosphatase activity of nsP2. Journal of Biological Chemistry 275, 17281-17287.
Received 28 July 2000;
accepted 24 November 2000.