Institut für Mikrobiologie und Molekularbiologie der Universität Giessen, Frankfurter Str. 107, 35392 Giessen, Germany1
Author for correspondence: Gerd Hobom. Fax +49 641 9935549.e-mail Gerd.Hobom{at}mikro.bio.uni-giessen.de
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Abstract |
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Introduction |
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The genome of BFDV is similar to that of other polyomaviruses and consists of an early region which encodes its large-T and small-t antigens (but no middle-t), and a late region, again in standard genetic arrangement, for structural proteins VP1, VP2 and VP3. However, in addition to the regular VP genes in the late downstream region, BFDV encodes another four proteins in the late upstream region, in two entirely overlapping reading frames located within the 5' segment of late mRNAs. Due to an alternative splice reaction either removing a shorter intron (2a) or a longer intron (2b) (Luo et al., 1995 ), both of the reading frames give rise to a pair of proteins that are structurally related, but differ in size; the pairs are not at all similar. In reference to the well-known simian virus 40, JC and BK agnoproteins (Jackson & Chalklay, 1981
; Rinaldo et al., 1998
), which are located in the same genetic position, the late upstream BFDV proteins have also been called agnoproteins. However, only the second pair of BFDV agnoproteins, agno-2a and agno-2b, which appears to assist in cell lysis bears some similarity at the protein level to other polyomavirus agnoproteins (Liu & Hobom, 1999
).
The first pair of agnoproteins (agno-1a and agno-1b), as translated from the group of promoter pL1-initiated viral late mRNAs, is unique to avian polyomaviruses. It has been observed to be essential during virus propagation, and the presence of either of these two proteins would allow the virus to complete its infection cycle, as determined by alternative intron deletion studies (see below). In contrast, the agno-2a and agno-2b pair that is translated in the second reading frame from late viral mRNAs initiated at promoter pL2, which itself is located within partially spliced intron 1, appears to be dispensable, at least in cell culture.
The dominant protein among these four, agno-1a, is observed in SDSPAGE separations to consist of multiple electrophoretic subspecies, which appear to be related to a primary subspecies (i.e. the lower-most band) of an apparent molecular mass of 31 kDa; its molecular mass as deduced from the cDNA sequence only accounts for 19·6 kDa. Both the multiplicity of electrophoretic subspecies, and the unexplained very large difference between its apparent molecular mass(es) under denaturing conditions in SDSPAGE and the size of the agno-1a polypeptide calls for an analysis of its post-translational modification(s). The same observations also apply to the smaller splice variant of agno-1a, protein agno-1b, which, because of an internal deletion of exon 3, only consists of exons 1, 2 and 4 of the agno-1 reading frame. It similarly displays an aberrant molecular mass in SDSPAGE, 26 kDa, relative to a calculated polypeptide mass of only 12·3 kDa, together again with a multiplicity of electrophoretic subspecies above that principal protein band. Besides leading to a full description of the BFDV agnoprotein 1 structure, analysis of its post-translational modifications will answer the question as to whether these modifications are important for either of the two functions determined for BFDV agno-1a and required during the virus life-cycle.
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Methods |
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Site-directed mutagenesis.
Single or multiple nucleotide substitutions at specific sites in the agno-1a gene were constructed either by primer oligonucleotide-directed PCR-fragment mutagenesis or via insertion of pairs of complementary oligonucleotides in between two restriction sites. Synthetic oligonucleotides were designed to cause mutations of either serine or threonine to alanine (or to leucine or glycine) and, in three cases, mutations of proline to alanine. Mutant clones were identified by sequencing across the respective regions and the mutated restriction fragments were subcloned into otherwise wild-type agno-1a influenza virus expression constructs. Multiple substitutions were obtained by combining the individual mutations using appropriately located restriction sites. As described above, in order to investigate the effects of the various substitutions in agno-1a with regard to distal VP1 translation and BFDV viability, the genetic variants were also transferred into BFDV genome-releasing plasmids, and again confirmed by DNA sequence analysis.
Cell culture, DNA transfection, and recombinant virus propagation.
Murine B82 cells (an L cell line), MadinDarby canine kidney (MDCK) cells and chicken embryo fibroblasts (CEF) were grown in Dulbeccos modified Eagle medium (DMEM; GIBCO/BRL) supplemented with 10% (v/v) foetal calf serum (FCS) and penicillin/streptomycin (100 U/ml and 100 µg/ml, respectively) for influenza virus propagation, and with 5% FCS and 2 mM L-glutamine for BFDV propagation.
Lipofectamine (GIBCO/BRL) was used for DNA transfection. Following transfection of 5 µg plasmid DNA into 107 cells of subconfluent B82, cells were infected with influenza helper-viruses (influenza A/FPV/Bratislava) at an m.o.i. of 3 at 6 h post-transfection. At 10 h post-infection, the supernatants containing recombinant influenza viruses (Zhou et al., 1998 ) were passaged for amplification once on MDCK cells and finally on CEF cells to facilitate agnoprotein production and isolation from the natural avian polyomavirus host cell.
For BFDV genome transfection, the prokaryotic vector segment was separated following MluI digestion from the BFDV DNA fragment (Stoll et al., 1994 ). The infectious DNA segment was isolated and approximately 1 µg was used for transfection into CEF cells. BFDV-containing supernatants were passaged twice on CEF. Propagation of BFDV and its variants was documented by Western blotting of cell lysates using anti-BFDV polyclonal antiserum.
Protein purification and desalting.
In order to obtain relatively large amounts of agno-1a protein for further analysis, CEF cells (10 cm Petri dishes, 60x) were infected by recombinant influenza viruses and harvested at 10 h post-infection. The cells were collected by centrifugation and lysed in a solution containing 8 M urea in 100 mM NaCl, 50 mM NaH2PO4, 10 mM TrisHCl, pH 8·0. His-tagged wild-type and mutant agno-1a proteins were isolated via metal chelate affinity chromatography under denaturing conditions using TALONspin columns and a pH gradient for elution according to the manufacturers instructions (Clontech). Protein chromatography was monitored via SDSPAGE and Western blotting as described below. Protein-containing fractions were desalted via reverse-phase (RP)-HPLC, using a Vydac 300 (5 µm) C4 column (100x2·1 mm) with a 2100% acetonitrile (ACN) gradient for elution in an H2O/ACN and trifluoroacetic acid system, within 20 min. As an alternative to RPHPLC, ultrafiltration was used for protein desalting and concentration. Before protein or peptide samples were subjected to mass spectrometry (MS), microdialysis was carried out (pore size, 0·025 µm; Millipore).
SDSPAGE and Western blotting.
Cell lysates or protein eluants were resolved by SDSPAGE using the Laemmli (1970) system with a 12·5% (v/v) separating gel, and then transferred to PVDF (Immobilon-P; Millipore) in a semi-dry system (Bio-Rad). The membrane was incubated with polyclonal anti-BFDV antibody (Stoll et al., 1993
) for 3 h. After incubation with biotinylated anti-rabbit immunoglobulin (Amersham), peroxidase-conjugated streptavidin (HRPStreptavidin; Zymed) was added; the membrane was then developed with 4-chloro-1-naphthol (3 mg/ml in methanol; Serva) and H2O2 (Merck) in PBS.
Alkaline phosphatase and protease treatments.
About 1 µg protein was incubated with alkaline phosphatase (from calf intestine; Boehringer Mannheim) at 30 °C, and the dephosphorylation reaction was terminated after 1 h by enzyme inactivation.
For proteinase digestions, sequencing grade Glu-C from Staphylococcus aureus V8 (Boehringer Mannheim) and Lys-C from Lysobacter enzymogenes (Boehringer Mannheim) were dissolved in 25 mM ammonium carbonate, pH 7·8 or 25 mM TrisHCl, pH 8·5/1 mM EDTA, respectively, giving rise to a final concentration of 1 µg/µl. Purified agno-1a (10 µg) was dissolved in 25 µl of either reaction buffer, then incubated overnight with 2 µl of the respective proteinase at 25 °C (Glu-C) or 37 °C (Lys-C).
MS.
After microdialysis, both individual protein fractions and mixtures of endoproteinase-digested peptides were used for determination of their molecular mass(es) by matrix-assisted laser-desorption/ionization-time of flight-MS (MALDITOFMS) following the technique described previously by Lochnit et al. (1998) , except that a change in the matrix material was introduced: 2,5-dihydroxybenzoic plus 2-hydroxy-5-methoxybenzoic acid (9:1) was used instead of only the former. In this method, full-size macromolecules become indirectly mobilized by way of energy absorption and evaporation directly only of the matrix molecules.
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Results |
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Expression of the agno-1a protein variant in recombinant influenza virus-infected CEF cells left the pattern of electrophoretic subspecies almost unchanged, except for a slight general shift-up, due to an increase in molecular masses resulting from the His-tag extension (see Fig. 1a). The variant agno-1 gene was also introduced by exchange into a plasmid carrying a 1·2-mer oversize BFDV genome, which, upon DNA transfection by homologous recombination in vivo, is known to give rise to circular BFDV DNA able to support replication and production of virus progeny (Stoll et al., 1994
). The GRH6 agno-1 variant BFDV could be propagated in spite of the C-terminal extension. It has been previously observed that either of the two genes, agno-1a or agno-1b, has to be present in the BFDV genome to support virus propagation (J. Li & G. Hobom, unpublished results).
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The C-terminal H6 sequence allowed a single-step purification procedure via standard metal chelate affinity chromatography, both for the group of wild-type modification subspecies and a series of mutant agnoproteins (see below). Separation techniques applied were also controlled to eliminate any shift in the position of electrophoretic subspecies during affinity column chromatography or consecutive RPHPLC purification and desalting steps (e.g. see Fig. 1b, lane 2).
Modification results from multiple, but fractional phosphorylation
Purified agno-1a-GRH6 consisting of a complex pattern of electrophoretically separable subspecies was treated with alkaline phosphatase, which converted the whole pattern into a single band co-migrating with the lower-most subspecies at 32 kDa (Fig. 1b). This demonstrated that the agno-1a modification pattern resulted from protein phosphorylation, apparently occurring at several residues simultaneously, but only to a moderate extent in each case. Alternatively, the overall intermediate level of phosphorylation might originate in part from secondary dephosphorylation reactions or an equilibrium between the two. In any case, a considerable fraction of agno-1a is observed in the position of the dephosphorylated protein. In the absence of any cysteine residue in the agno-1a protein sequence, and with negative results in several reactions to demonstrate the presence of any carbohydrate moiety, phosphorylation remained the only type of modification identified in agno-1a, except for a blocked N terminus as initially concluded from futile attempts of sequence analysis via Edman degradation, and later confirmed by MS (see below).
In a first approach to identify the position of phosphates in the agno-1a polypeptide chain, we used a MAb directed against phosphotyrosine residues. A negative result excluded its three tyrosine residues from further consideration as phosphate-accepting sites.
Determination of phosphorylated peptides via proteinase digestion and MS
Of the 17 serine and 23 threonine residues in total present in the agno-1a polypeptide chain, 14 of these are in favourable context with regard to the consensus sequences (Pinna, 1990 ; Roack, 1991
; Li & Garcea, 1994
) of either protein kinase C (1), casein kinase II (4) and proline-directed protein kinases (PDPK) (9) (see Fig. 2
). In a first analytical procedure, purified agno-1a was digested by two endo-proteinases, and the digestion mixtures were subjected to MALDITOFMS as described in Methods.
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Identification of agno-1a phosphorylation sites through substitution mutagenesis
Modification by only three phosphate groups as observed in MS measurements would not be able to explain the much more complex pattern of agno-1a electrophoretic subspecies, but at least provided a starting point for site-directed mutagenesis. In this approach, the majority of the fourteen predicted potential positions for serine or threonine phosphorylation identified by consensus sequence searches (Fig. 2) were individually converted into alanine, glycine or leucine residues. Since PDPK phosphorylation strictly requires a distal adjacent proline residue, other mutagenization reactions also converted this amino acid (and not the preceding serine or threonine) into alanine, thereby also analysing the specificity of that particular phosphorylation reaction. Finally, after having determined seven residues one by one to be involved in agno-1a phosphorylation as presented in Fig. 3
, and resulting in a single, unphosphorylated agno-1a species upon their combination in a heptamer mutant (lane 11), the remaining less likely phosphorylation sites have been mutagenized not individually, but in small groups. These mutagenization reactions regarding positions T3, S17, T21, S36, T39 and S51 did not change the wild-type agno-1a electrophoretic distribution pattern (data not shown). It was therefore concluded that, in the first round of mutagenization experiments, all of these sites remained unmodified in the agno-1a polypeptide chain.
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P147A, controlling adjacent S146, only caused a slight decrease in several of the low mobility bands in the electrophoretic pattern but, in contrast to P136A, left the general mobility unchanged (lane 9) and therefore, the involvement of S146 in phosphorylation remained in doubt. However, in a control experiment with all other six relevant substitutions (plus companion T141G) combined in a single variant polypeptide chain, a single phosphorylation band of low mobility was clearly observed above the basic unphosphorylated agno-1a species, both of which also demonstrated an expected increase in mobility (indicating a conformational alteration) apparently resulting from P136A plus T137A. By an additional substitution, P147A, the remaining site S146 could be knocked out (lanes 10, 11), resulting in a single, dephosphorylated type of agno-1a. Correlation of individual electrophoretic subspecies in the original series with (mono) phosphorylation in either of the six other positions except S146 appeared to be difficult at this point, since minor changes in conformation and electrophoretic mobility, and also in phosphorylation yields if hierarchically influenced by other sites (Roack, 1991 ), might occur in any single substitution reaction. The prominent monophosphorylation band (lane 10) resulting from modified S146 (in a polypeptide carrying seven substitutions) is a case in point, since it appeared to have increased in amount compared with the corresponding band in the original pattern.
While that first group of substitution experiments essentially constituted a minus one series, i.e. inactivating individual phosphorylation sites one by one, we also wanted to confirm the phosphorylation sites determined in this way by a plus one series, i.e. by restoring these individual phosphorylation sites one or two at a time, starting out with the fully dephosphorylated multiple variant as shown in lane 11 of Fig. 3. The results of these experiments are displayed in Fig. 4
, with individual substitutions at all relevant positions indicated above the lanes. All residues identified previously in the dephosphorylation series did accept phosphates under these conditions (for S146, see Fig. 3
), while several other potential sites including S51 did not result in (mono) phosphorylated agno-1a polypeptide chains (data not shown). Wide differences were observed for the shift in electrophoretic mobility resulting from single phosphate group additions at individual residues, e.g. see S135 and T137 (lanes 7 and 8) versus T142 and S53 (lanes 3 and 4).
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Fully phosphorylated agno-1a is required in BFDV propagation
The series of single or double substitution mutants in the minus one series that resulted in phosphorylation variants of protein agno-1a as presented in Fig. 3 was recloned into the above-mentioned plasmid vector known to produce infectious (wild-type) BFDV DNA upon DNA transfection into CEF cells (Stoll et al., 1994
). As a variation, however, the multiple expression potential of the viral agno-gene region was reduced down to agno-1a only by appropriately deleting all of the intron sequences in that region. Also, the H6 C-terminal variation of the influenza virus-carried agno-1a mutants was removed in the cloning step. As shown in lane 1 of Fig. 5
, wild-type agno-1a, in the absence of agno-1b (and agno-2a and -2b), is able to support expression of downstream structural proteins VP1, VP2 and also VP3 (hidden behind the unphosphorylated species of agno-1a; see Fig. 1a
) up to standard levels in infected CEF cells. Upon passage of the supernatant of these cells, BFDV can be further propagated, yielding the same pattern of late translation products as observed in the transfected cells, i.e. in lane 1.
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Unphosphorylated agno-1a is incorporated into BFDV virions
Protein analyses of purified BFDV virions demonstrated the presence of an additional structural protein of 31 kDa besides standard VP1, VP2 and VP3 (and histone proteins), which has been determined to be agno-1a (J. Li, H. Müller & G. Hobom, unpublished results). The dominant species of agno-1a at 31 kDa appeared to be accompanied only by traces of proteins with lower electrophoretic mobility. Upon phosphatase treatment of denatured virion proteins, all of these traces disappeared, whereas the 31 kDa protein remained unchanged, i.e. in the position of unmodified agno-1a (Fig. 6). In conclusion, it is almost exclusively the unphosphorylated subspecies of agno-1a that is sequestered into the virion as a fourth component of its shell, which differs from the situation with BFDV VP1 which is incorporated in various phosphorylated states (Haynes & Consigli, 1992
). Since agno-1a has to fulfil two functions in the virus life-cycle, acting in the infected cell in a phosphorylated state, and incorporated into the virion as an unmodified polypeptide, the series of agno-1a subspecies observed in the infected cell may reflect that situation in an overall fractional level of phosphorylation.
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Discussion |
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Since agno-1a has been shown, on the one hand, to be an essential element in the BFDV infection cycle, and is also present as an additional capsid protein in the virus shell (J. Li, H. Müller & G. Hobom, unpublished results), we were interested in investigating the functional significance of the various protein phosphorylation states. As shown here, virions and infected cells appear to use different forms of agno-1a, i.e. unphosphorylated and phosphorylated, both of which are provided through a pattern of fractional phosphorylation. In infected cells, it has been shown that agno-1a is required for sufficient expression of late distal structural proteins through interaction with ribosomes moving along viral late mRNAs (Li, 1997 ).
Since unphosphorylated agno-1a is observed in the virion almost exclusively (see Fig. 6), sequestration of this subspecies into the protein shell during morphogenesis should be due to specific interaction with other viral proteins. Alternatively, the phosphate groups might be removed upon VP binding of agno-1a by phosphatase action. Since all three VP proteins of BFDV, in particular the major component VP1, are very similar in size and sequence to standard polyomavirus VPs, and are maintained in a closely related genetic organization (Rott et al., 1988
), it is very surprising that an additional viral protein is required for the formation of infectious avian polyomavirus particles. Its function and even location within those virions, which by electron microscopy look very much like standard polyomavirus particles (Lehn & Müller, 1986
), are presently unknown and need to be analysed further. Not even a trace of agno-1b, the internally deleted splice variant of agno-1a is detected in BFDV virions.
Contrary to the state of agno-1a in virions, in its function during the infection cycle it is required in a phosphorylated state, and unphosphorylated agno-1a is not active in this capacity (Fig. 5). It has been observed that agno-1a binds to late viral mRNAs in a sequence-specific manner (Li, 1997
), which is supported by a standard RNA-binding element at positions 7183 of the agno-1a sequence, within the central segment that is missing in agno-1b. A similar requirement of phosphorylation for specific RNA binding has been observed for human T-lymphotropic virus rex (Green et al., 1992
) and human immunodeficiency virus rev (Fouts et al., 1997
).
The two functions known for agno-1a, i.e. mRNA binding/interaction with ribosomes and incorporation into the viral protein shell, are surprisingly divergent, but at least these differences in function are reflected in a requirement for the unphosphorylated and phosphorylated subspecies of the same protein, which is similar to the situation observed for phosphorylated and dephosphorylated calmodulin (Quadroni et al., 1998 ). This in turn is able to explain the presence of a series of subspecies of agno-1a in the cell, i.e. representing intermediate stages of phosphorylation. Whether the pattern that was routinely observed at 72 h post-infection is variable during the infection cycle remains to be determined in kinetic experiments, which may also answer the question as to whether the pattern results from partial phosphorylation, partial dephosphorylation or indeed reflects a balanced state between both reactions.
BFDV agno-1a is a special genetic element present only in avian polyomaviruses that is devoid of any similarity in structure or function to other polyomavirus agno proteins (Resnick & Shenk, 1986 ). A more closely related function instead appears to be maintained by BFDV agno-2a and agno-2b, which are expressed very late in infection (Luo et al., 1995
) and apparently assist in cell lysis (Liu & Hobom, 1999
).
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Acknowledgments |
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Footnotes |
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References |
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Received 3 August 1999;
accepted 30 September 1999.