1 Unité de Virologie et Immunologie Moléculaires, INRA, F-78350 Jouy-en-Josas, France
2 Unité de Biochimie et Structure des Protéines, INRA, F-78350 Jouy-en-Josas, France
Correspondence
Bernard Delmas
delmas{at}jouy.inra.fr
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ABSTRACT |
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MAIN TEXT |
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The genomic segment B encodes VP1, the putative viral RNA-dependent RNA polymerase (Gorbalenya et al., 2002). Translation of genomic segment A yields a polyprotein, pVP2VP4VP3 (Kibenge et al., 1988
; Muller et al., 2003
) and a small protein (VP5) dispensable for replication (Mundt et al., 1997
; Yao et al., 1998
). The polyprotein processing is controlled by VP4, the viral protease (Birghan et al., 2000
; Lejal et al., 2000
; Petit et al., 2000
).
The complete proteolytic cascades involved in processing the IBDV and BSNV polyproteins and generating VP2, the structural peptides and VP3 have been characterized. The polyproteins are cotranslationally cleaved to generate pVP2 (the precursor of VP2), VP4 and VP3. Cleavage sites have been identified at the pVP2VP4 and VP4VP3 junctions (Sanchez & Rodriguez, 1999; Lejal et al., 2000
; Da Costa et al., 2003
). For both viruses, processing of pVP2 generates VP2 and four small peptides derived from the C terminus of pVP2 (Da Costa et al., 2002
, 2003
). These peptides are associated with the virus particles. For IPNV, only the VP4 target sites generating pVP2, VP4 and VP3 have been defined, being between aa 508509 and 734735 of the polyprotein (Petit et al., 2000
). In this study we characterized the final processing of the IPNV polyprotein. By using mass spectrometry and N-terminal sequencing, we identified peptides derived from the C-terminal domain of pVP2 that were present in the virus particles.
Fig. 1(a) shows a multi-alignment of the C-terminal domains of the birnavirus pVP2s. The sequence comparison is anchored to the multiple cleavage sites identified in the C terminus of pVP2 of BSNV and IBDV and to the cleavage site mapped at the pVP2VP4 junction of IPNV and DXV (Chung et al., 1996
; Sanchez & Rodriguez, 1999
; Petit et al., 2000
; Da Costa et al., 2002
, 2003
). We predicted from this alignment that pVP2 processing of IPNV might result in the generation of mature VP2 (aa 1442 of the polyprotein) and three peptides (aa 443486, 487495 and 496508 of the polyprotein). The [M+H]+ theoretical monoisotopic masses of the three peptides were expected to be 4796·59, 949·43 and 1451·73 Da, respectively. As most IBDV and BSNV pVP2-derived peptides have been identified on virus particles, we postulated that their IPNV homologues should be identifiable in purified IPNV virions.
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Mass spectrometry analysis was carried out on the virus particles present in band 1 using a Voyager-DE STR time-of-flight mass spectrometer (Applied Biosystems). One main peptide with a monoisotopic [M+H]+ mass of 1451·72 was identified (Fig. 2a, top panel). This [M+H]+ mass fitted well with the theoretical mass of the predicted 13 aa long peptide extending from Ala-496 to Ala-508, which is 1451·73 Da. As cleavage at the pVP2VP4 junction occurs between aa 508 and 509, this peptide is indeed the C terminus of pVP2. An additional peptide with an [M+H]+ mass of 4796·14 was detected (Fig. 2a
, lower panel). No other peptide with the same range of mass (35005500 Da) was detected. This [M+H]+ mass fitted well with the mass of a peptide extending from Trp-443 to Ala-486, which is 4796·59 Da and represents the N-terminal part of this pVP2 domain. The presence of these two peptides from residues [443486] and [496508] in the virus particles prompted us to locate the putative 9 aa peptide derived from residues 487495. To identify this peptide, which had a theoretical [M+H]+ monoisotopic mass of 949·43 Da, we analysed the magnified signal in this mass range. As shown in Fig. 2(a)
(top panel), a peptide with an [M+H]+ monoisotopic mass of 949·61 was detectable. Fourteen additional peptides were also identified by mass spectrometry in the mass range 5902300 Da (Fig. 2a
, top panel). Thirteen of these appeared to be cleaved products derived from peptide [443486] (Fig. 2b, c
). All possessed the same N terminus as peptide [443486]. No complementary peptide harbouring the corresponding C-terminal domains of peptide [443486] was identified. These observations suggest that a fraction of peptide [443486] was cleaved by a carboxypeptidase with low specificity. A cellular carboxypeptidase, or possibly VP4, might be associated with these cleavage events. Finally, a peptide, with an [M+H]+ mass of 1380·70, deriving from the amino- or carboxy-cleavage of peptide [496508], was also detected.
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The existence of peptides derived from pVP2 suggests that aa [443508] represent the domain that is processed during pVP2 maturation. To determine whether the pVP2-processed domain involved residues upstream of residue 442, we engineered a baculovirus driving expression of the IPNV polyprotein with a stop codon at position 443, and compared the electrophoretic mobility of the mature VP2 (present on virus particles) with this truncated form. To this end, plasmid SK-IPNA (Petit et al., 2000
) was used as a template to amplify the VP2 reading frame (the first 442 codons of the polyprotein) by PCR. The PCR product was cloned into pFastBac1 (Gibco-BRL) using suitable restriction sites. The resulting plasmid was used to generate the recombinant baculovirus BacVP2IPNV by standard procedures. Production and purification by density centrifugation of the baculovirus-expressed IPNV VP2 were carried out as described previously (Chevalier et al., 2002
). As shown in Fig. 1(b)
, the virion-associated protein VP2, the VP2 released from the virus particles and the VP2-stop443 co-migrated. As determined by mass spectrometry analysis, the experimental masses of viral VP2 and VP2-stop443 were 48 578·3 and 48 531·6 Da, respectively. These masses fit well with the theoretical [M+H]+ mean mass of a VP2 extending from residues 1 to 442 with an initial acetylated methionine (48 569·9 Da), suggesting that the pVP2 characteristic domain does not extend upstream of residue 442.
In this study we showed that processing of pVP2 of IPNV is similar to the maturation of the IBDV and BSNV pVP2s. Three peptides (not four, as for IBDV and BSNV) define the C-terminal domain of pVP2. For IPNV, peptide [443486] is processed further to generate a large number of additional peptides. For BSNV, processing of the peptide [443486] homologue was also observed (Da Costa et al., 2003). Although processing of the IPNV peptide [443486] appeared to involve a carboxypeptidase, an endoprotease appeared to control the cleavages of its BSNV homologue. Similar additional peptides were not identified in IBDV virions (Da Costa et al., 2002
).
Three target cleavage sites were identified in the pVP2 maturation process. We previously proposed two of these (486487, 495496) as targets for the IPNV VP4 protease (Petit et al., 2000). These two sites, and the primary cleavage site at the pVP2VP4 junction, were defined by the motif [S/T]XA
A. This consensus sequence shared some similarity with the sequence SKA
W surrounding the maturation cleavage site at position 442443, suggesting that VP4 could be involved in the cleavage generating the mature VP2.
When the virus particles were altered by ultracentrifugation, the released VP2 was able to self-assemble into particles with a diameter of about 25 nm. These observations suggest that the VP2 of IPNV has assembly properties similar to the VP2/pVP2 of IBDV (Martinez-Torrecuadrada et al., 2000; Caston et al., 2001
; Chevalier et al., 2002
).
The role of the birnavirus structural peptides remains to be elucidated. We favour the hypothesis that they may be involved in virus entry into the target cells, but it cannot be ruled out that they play a role in capsid assembly or genome encapsidation.
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ACKNOWLEDGEMENTS |
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Received 3 February 2004;
accepted 27 April 2004.