Agnoprotein-1a of avian polyomavirus budgerigar fledgling disease virus: identification of phosphorylation sites and functional importance in the virus life-cycle

Qiang Liub,1 and Gerd Hobom1

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


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The avian polyomavirus budgerigar fledgling disease virus (BFDV) encodes an unusual set of four agnoproteins in its late upstream region. Of the two pairs of these proteins, which overlap each other in two different reading frames, the pL1-promoted agnoprotein-1a (agno-1a) is the dominant species and is able to support virus propagation in the absence of the other three polypeptides. Viral BFDV agno-1a, and also agno-1a expressed via an influenza virus vector, consists of a complex series of electrophoretically separable subspecies that can be reduced by phosphatase action down to a primary unphosphorylated protein with an apparent molecular mass of 31 kDa. Through peptide mass spectrometry and site-directed mutagenesis, the positions of four serine and three threonine residues have been determined as phosphate-accepting groups, which are partially modified by the combined action of three different cellular kinases. Since extensively phosphorylated agno-1a is required for its intracellular function, control over VP protein expression, and unphosphorylated agno-1a is observed as an additional component in the BFDV virion, both extreme subspecies appear to be drawn from that complex mixture, which also includes the intermediate stages of phosphorylation.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Avian polyomaviruses were initially detected as the causal agent of severe contagious diseases among fledgling budgerigars (Bozeman et al., 1981 ; Lehn & Müller, 1986 ), and hence were called budgerigar fledgling disease virus (BFDV, which later became BFDV-1). An initial characterization based on morphological and serological criteria has been confirmed and extended by DNA sequence analysis of its circular double-stranded genome of 4981 bp (Rott et al., 1988 ). Characterization of other, closely related avian viruses, isolated from a chicken (BFDV-2), parrot (BFDV-3) (Stoll et al., 1993 ) and other birds (BFDV-4, BFDV-5; Johne & Müller, 1998 ), has been reported more recently, indicating a rather frequent occurrence among these species, which is further supported by serological data.

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 SDS–PAGE 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 SDS–PAGE 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 SDS–PAGE, 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.


   Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Construction of agno-1a expression plasmids and viral variants.
The cDNAs of BFDV agno-1a (initially called agno-1d; Luo et al., 1995 ) were cloned into influenza virus/RNA polymerase I expression vector pHH1 (E. Hoffmann & G. Hobom, unpublished results), carrying promoter-up mutation pHL1104 (Neumann & Hobom, 1995 ). For increasing the translation efficiency, an optimal initiation sequence, 5' CCACCATGG 3' (Kozak, 1987 ), was introduced via incorporation into the forward PCR primer sequence, which consequently converted the N-terminal sequence from MS- into MA-. Also, for ease of protein purification, a poly-histidine tag, GRH6, was fused onto the C terminus via incorporation into the backward PCR primer sequence. In a second step, both modifications were also incorporated into an infectious plasmid DNA vector as described by Stoll et al. (1994) , which, upon DNA transfection, gives rise to circular BFDV viral genomes and progeny viruses.

{blacksquare} 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.

{blacksquare} Cell culture, DNA transfection, and recombinant virus propagation.
Murine B82 cells (an L cell line), Madin–Darby canine kidney (MDCK) cells and chicken embryo fibroblasts (CEF) were grown in Dulbecco’s 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.

{blacksquare} 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 Tris–HCl, 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 manufacturer’s instructions (Clontech). Protein chromatography was monitored via SDS–PAGE 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 2–100% acetonitrile (ACN) gradient for elution in an H2O/ACN and trifluoroacetic acid system, within 20 min. As an alternative to RP–HPLC, 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).

{blacksquare} SDS–PAGE and Western blotting.
Cell lysates or protein eluants were resolved by SDS–PAGE 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 (HRP–Streptavidin; Zymed) was added; the membrane was then developed with 4-chloro-1-naphthol (3 mg/ml in methanol; Serva) and H2O2 (Merck) in PBS.

{blacksquare} 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 Tris–HCl, pH 8·5/1 mM EDTA, respectively, giving rise to a final concentration of 1 µg/µl. Purified agno-1a ({approx}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).

{blacksquare} 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 (MALDI–TOF–MS) 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.


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
cDNA cloning and protein purification of BFDV agnoproteins
Only during the very late period of BFDV infection is structural protein VP1 the dominant protein product. VP2 and VP3 are also synthesized at higher rates than agno-1a, which in turn is synthesized four to fivefold in excess of agno-1b. At intermediate stages of the infection cycle, agno-1a and agno-1b are the major products. In SDS–PAGE analysis, proteins VP3 and agno-1a directly overlap due to similar apparent molecular masses (31 kDa), whereas smaller-sized agno-1b is well-separated, but in danger of contamination by proteolytic breakdown products of dominant and larger proteins. Therefore, the agno-1a and the agno-1b genes were separately cDNA-cloned via PCR into a foreign vector plasmid to produce recombinant influenza viruses able to infect CEF, the host cell of BFDV. The influenza virus/RNA polymerase I vRNA expression system for RNA molecules and proteins as developed in this laboratory makes use of a high-yielding promoter-up mutation (pHL1104; Neumann & Hobom, 1995 ) incorporated into vector pHH1 (E. Hoffmann & G. Hobom, unpublished results). The cDNA of agno-1a to be inserted into pHH1 was derivatized by flanking variations which were introduced via the forward primer sequence carrying an optimal ribosomal initiation sequence (Kozak, 1987 ), and via the backward primer sequence extending the agnoprotein reading frame with a tail sequence including six histidines, GRH6.

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).



View larger version (54K):
[in this window]
[in a new window]
 
Fig. 1. A series of electrophoretically separated agno-1a subspecies is caused by fractional phosphorylation reactions. (a) C-terminal His-tag addition (GRH6) causes a general shift-up of the agno-1a modification pattern, but no further variation in relative mobilities. Lanes: 1, SDS–PAGE separation followed by Western blot of wild-type BFDV late proteins in the cell lysate of virus-infected CEF cells; 2, analogous analysis of CEF cells infected by variant BFDV carrying a C-terminal His-tag elongation of the agno-1a reading frame. Normally hidden VP3 becomes visible because of the agno-1a shift-up. (b) Agno-1a modification is caused by phosphorylation. Lanes: 1, size marker; 2, agno-1a-H6 modification pattern present in CEF infected by recombinant influenza virus encoding BFDV agno-1a-GRH6; 3, the same CEF cell lysate after treatment with alkaline phosphatase.

 
The general shift-up of the entire electrophoretic agno-1a pattern caused by the addition of eight C-terminal amino acids (GRH6) did not change its distribution, except for a slight relative increase in the concentrations of several subspecies of lower electrophoretic mobility. Protein analysis upon cloning into high-level influenza virus expression vectors proved that all agno-1a subspecies originated from the same reading frame (Fig. 1a). The same result was obtained for the agno-1b subspecies (data not shown). Both control experiments demonstrated that proteins agno-1a and agno-1b, in spite of N- and C-terminal variations, represented an unchanged and certainly functional pattern of polypeptide modifications, at least in the standard BFDV host cell, CEF. Also, the higher level of expression in an influenza vector system did not cause aberrations.

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 RP–HPLC 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 MALDI–TOF–MS as described in Methods.



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 2. Amino acid sequence of BFDV agno-1a indicating the potential and the actual phosphorylation sites for various kinases by their recognition consensus sequences. The single letter amino acid code is used and the residues are shown in groups of ten. Roman letters represent the polypeptide chain as present in both agno-1a and its internal splice variant, agno-1b, while the central sequence region, present only in agno-1a, is italicized. Protein kinase C sites, {blacktriangleup}; casein kinase II sites, {square}, {blacksquare}; and PDPK sites, {circ}, • (potential and actual sites are indicated by open and closed symbols, respectively). Tyrosine residues are marked by an open diamond ({lozenge}). A dash above three of the sites refers to those residues that are phosphorylated in agno-1b (unpublished results).

 
Purified agno-1a was digested by endoproteinase Lys-C (five fragments expected) and by endoproteinase Glu-C, predicted to result in 14 peptides in complete cleavage. However, since the agnoprotein substrate consists of a mixture of several partially phosphorylated subspecies more than one molecular mass was expected for each of the phosphopeptides, a regular 80 Da apart from each other (i.e. phosphorylated and unmodified). Also, not all of the peptides expected were indeed observed in the two MALDI–TOF mass spectra obtained upon loading the two digestion mixtures. While two out of five fragments (the large N-terminal peptide and another highly acidic fragment) were not detectable in the Lys-C experiment, one of the fragments was observed in duplicate, and the C-terminal one was even detected in triplicate (apparently containing zero, one or two phosphate groups), suggesting the determination of two phosphopeptides. Major losses also occurred in the Glu-C experiment, with exemption of the three N-terminal fragments; again, one of these peptides was detected in duplicate. This incomplete, but complementary information obtained from the two peptide series allowed us to combine the individual data into an initial picture regarding the position of the more frequently used phosphorylation sites (see Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Masses of peptides present in the Glu-C (nos 1–3) and Lys-C (nos 4–7) digestion mixtures of BFDV agno-1a-H6 as measured by MALDI–TOF–MS

 
While any positive evidence of phosphorylation obtained with this technique could be used further and was indeed confirmed in consecutive experiments, a negative observation by MALDI–TOF–MS, i.e. a specific fragment detected only in the unphosphorylated state, did not allow its (partial) phosphorylation to be excluded, e.g. fragment 5 (aa 93–103) later turned out to contain up to two phosphates (see below). The respective phosphorylated, and therefore more acidic, molecular species may have been lost in that technique, similarly to several constitutionally acidic peptides, and only the unphosphorylated molecular species were observed. Another type of modification was clearly demonstrated in the MALDI–TOF–MS study: an N-terminal deletion of methionine, and its substitution by an acetyl group, as expected from the ‘blocked’ N terminus observed in amino acid sequencing.

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.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3. Site-directed mutagenesis of (potential) phosphorylation sites in agno-1a. Western blot patterns of purified mutant agno-1a proteins carrying the respective substitutions, in comparison to the wild-type construct (lane 1) are presented. Individual amino acid substitutions at relevant positions are outlined above each lane for the respective variant, in comparison to the wild-type construct in lane 1. In principle, only the amino acids exchanged are indicated, such as for mutant S53A in lane 2. Residues functionally inactivated due to adjacent changes such as serine-135 in mutant P136A (lane 5) are indicated in parentheses.

 
Fig. 3 shows several single substitution reactions each of which did result in a partial reduction of the agno-1a subspecies pattern: S53A, S93A, T97A, T137A and also P136A, which indirectly indicated that S135 was phosphorylated by PDPK. However, the latter substitution not only abolished phosphorylation at S135, but apparently also caused a conformational change in the agno-1a polypeptide chain resulting in a slightly higher electrophoretic mobility of the remaining subspecies pattern (lane 5). While this might not be unexpected for a change involving proline, a similar conformational change was also observed for substitution T137A in the adjacent position (lane 6), suggesting in turn a conformational change resulting from phosphorylation by PDPK at T137 (or both at S135 and T137). While the double substitution T141G plus T142A indicated that one of these two residues was heavily involved in phosphorylation, T141G alone retained a full multi-subspecies pattern (lanes 7 and 8), which leaves T142A as the mutation responsible for the observed effect.

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).



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4. Restoration of individual phosphorylation sites in a fully and an almost fully dephosphorylated agno-1a variant. Individual amino acid substitutions executed at relevant positions within the influenza virus vector-encoded gene are indicated above each lane, in comparison to a fully dephosphorylated variant (lane 2, compare with lane 11 in Fig. 3), and the wild-type construct (lane 1). Only amino acid substitutions relative to lane 2, effectively reversions with respect to the wild-type sequence, have been indicated. (S) and (T) refer to non-accepting residues, either constitutively (T141) or resulting from indirect inactivation via adjacent P->A substitutions (S135, T137 and S146). SDS–PAGE followed by Western blotting of purified mutant agno-1a proteins showed the increased agno-1a modification patterns as obtained after restoration of individual accepting residues in a fully dephosphorylated variant of agno-1a.

 
The phosphorylation sites determined twice for agno-1a in both of these experimental approaches are summarized in Fig. 2, as a subset of the potential sites indicated previously by consensus sequence searches for the various known protein kinases. According to the substitution results, the single protein kinase C site is indeed used for phosphorylation (T97), two out of four potential casein kinase II sites are found to be phosphorylated (S53 and S93), while a group of four out of nine PDPK-specific sequences was converted by that enzyme into phosphorylated residues (S135, T137, T142 and S146).

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.



View larger version (54K):
[in this window]
[in a new window]
 
Fig. 5. Full-level phosphorylated agno-1a is required in infectious DNA transfection for VP synthesis and BFDV propagation. Relevant sequence information for individual agno-1a variants is indicated above each lane, in comparison to the series of wild-type sequence residues involved in phosphorylation as shown above lane 1. The principal construct (lane 1) has its BFDV agno gene region reduced to expression of only agno-1a, and single (lanes 2–8) or multiple (lanes 9 and 10) variants are agno-1a-internal substitution derivatives of that construct, inserting DNA fragments as derived from the series in Fig. 3. SDS–PAGE followed by Western blotting was done using CEF lysates prepared at 72 hours post DNA transfection. For electrophoretic mobility increases of agno-1a mutants in lanes 5–7, 9–10, compare with Fig. 3.

 
Fig. 5 demonstrates that any single phosphorylation site that is missing in the agno-1a polypeptide chain will either fully (lanes 4–7) or at least to a large extent (lanes 2–3, 8–10) inhibit expression of the viral structural genes and, in accordance with this result, no virus passage was achieved for any of the variants. Somewhat surprisingly, no partial modification was observed for any of the agno-1a variants under these conditions, which differs from the results shown in Fig. 3, whereas the unmodified protein is present at almost normal levels after 72 h post-transfection. The BFDV transfection system is known to go through a bottleneck before the recircularized viral genome appears and becomes amplified, and, even in BFDV wild-type infection, expression is clearly below the levels achieved in recombinant influenza virus production of foreign proteins using both a promoter-up mutation and an optimal ribosomal initiation sequence. We also have evidence that oligomerization of agno-1a is required for its function in amplifying the expression of late distal structural genes (Li, 1997 ), which possibly might depend on and in turn stimulate multiple phosphorylation. In conclusion, extensive phosphorylation of agno-1a appears to be a prerequisite for at least one vital function in infected cells, and only after this modification will significant amplification of viral structural proteins be observed. Unphosphorylated agno-1a, which is always present in the series of protein subspecies, may not be active in this regard.

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.



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 6. BFDV virions contain agno-1a (31 kDa) as a structural component almost exclusively in the unphosphorylated state. Lanes: 1, phosphatase treatment of denatured BFDV virion proteins, analysed by SDS–PAGE and Western blotting; 2, control lane without phosphatase treatment. A size marker is indicated on the left.

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
In order to study the role of BFDV agno-1a protein and its prominent series of subspecies as observed by SDS–PAGE during the BFDV replication cycle, we have expressed this protein in CEF cells using an influenza virus/RNA polymerase I vector system developed in our laboratory. The series of multiple forms of this protein originates from fractional phosphorylation as indicated by phosphatase treatment of purified agno-1a. Some of the phosphorylation sites were regionally localized by phosphopeptide MS, but the full series of seven phosphate-accepting residues in total (Fig. 2) has only been identified by two series of site-directed mutagenesis reactions, forward and backward. Only one of these residues, threonine-97, is located within a protein kinase C motif; two residues, serine-53 and serine-93, are located in casein kinase II consensus sequences; and the remaining four residues, serine-135, serine-146, threonine-137 and threonine-142, are adjacent to downstream proline residues, indicating that PDPK are also involved in agno-1a phosphorylation. Since fractional phosphorylation turned out to be the only modification responsible for the multiplicity of agno-1a protein bands in SDS–PAGE, the discrepancy between the apparent molecular mass (unphosphorylated, 31 kDa) and the calculated mass of the polypeptide chain (19·6 kDa) has to result from an unusual molecular shape effectively determined by its primary sequence. The same is true for its smaller variant, agno-1b, which, in its unphosphorylated state, migrates to position 26 kDa in SDS–PAGE as opposed to a theoretical mass of 12·3 kDa. Besides such overall similarity indicating an elongated molecular shape for either of these proteins, the conformation of agno-1b should be different in detail from that of agno-1a, since the phosphorylation sites identified in agno-1b were not identical to those in agno-1a even in the segments that are common to both proteins (Q. Liu, unpublished results).

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 71–83 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 ).


   Acknowledgments
 
We thank J. Li for valuable cooperation and discussions, H. Müller for various BFDV antisera, and M. Hintz and M. Linder for help in protein fragmentation and mass spectrometry analyses. Expert technical assistance by U. Ruppert and N. Pohl is gratefully recognized. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 272).


   Footnotes
 
b Present address: Virology Group, Veterinary Infectious Disease Organization, Saskatoon, Saskatchewan, Canada S7N 5E3.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Bozeman, L. H., Davis, R. B., Gaudry, D., Lukert, P. D., Fletcher, O. J. & Dykstra, M. J. (1981). Characterization of a papovavirus isolated from fledgling budgerigar. Avian Disease 25, 972-980.[Medline]

Fouts, D. E., True, H. L., Cengel, K. A. & Celander, D. W. (1997). Site-specific phosphorylation of the human immunodeficiency virus type-1 rev protein accelerates formation of an efficient RNA-binding conformation. Biochemistry 36, 13256-13262.[Medline]

Green, P. L., Yip, M. T., Xie, Y. & Chen, I. S. Y. (1992). Phosphorylation regulates RNA binding by the human T-cell leukemia virus rex protein. Journal of Virology 66, 4325-4330.[Abstract]

Haynes, J. I.II & Consigli, R. A. (1992). Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1. Journal of Virology 66, 4551-4555.[Abstract]

Jackson, V. & Chalklay, R. (1981). Use of whole-cell fixation to visualize replicating and maturing simian virus 40: identification of new viral gene product. Proceedings of the National Academy of Sciences, USA 78, 6081-6085.[Abstract]

Johne, R. & Müller, H. (1998). Avian polyomavirus in wild birds: genome analysis of isolates from Falconiformes and Psittaciformes. Archives of Virology 143, 1501-1512.[Medline]

Kozak, M. (1987). An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Research 15, 8125-8148.[Abstract]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]

Lehn, H. & Müller, H. (1986). Cloning and characterization of budgerigar fledgling disease virus, an avian polyomavirus. Virology 151, 362-370.[Medline]

Li, J. (1997). Molecular analysis of late gene expression in BFDV. PhD thesis, University of Giessen.

Li, M. & Garcea, R. L. (1994). Identification of the threonine phosphorylation sites on the polyomavirus major capsid protein VP1: relationship to the activity of middle T antigen.Journal of Virology 68, 320-327.[Abstract]

Liu, Q. & Hobom, G. (1999). Recombinant expression of late genes agno-2a and agno-2b of avian polyomavirus BFDV. Virus Genes 19, 183–187.[Medline]

Lochnit, G., Nispel, S., Dennis, R. D. & Geyer, R. (1998). Structural analysis and immunohistochemical localization of two acidic glycosphingolipids from the porcine parasitic nematode, Ascaris suum.Glycobiology 8, 891-899.[Abstract/Free Full Text]

Luo, D., Müller, H., Tang, X.-B. & Hobom, G. (1995). Early and late pre-mRNA processing of budgerigar fledgling disease virus 1: identification of viral RNA 5' and 3' ends and internal splice junctions. Journal of General Virology 76, 161-166.[Abstract]

Neumann, G. & Hobom, G. (1995). Mutational analysis of influenza virus promoter elements in vivo. Journal of General Virology 76, 1709-1717.[Abstract]

Pinna, L. A. (1990). Casein kinase 2: an ‘eminence grise’ in cellular regulation? Biochimica et Biophysica Acta 1054, 267-284.[Medline]

Quadroni, M., L’Hostis, E. L., Corti, C., Myagkikh, I., Durussel, I., Cox, J., James, P. & Carafoli, E. (1998). Phosphorylation of calmodulin alters its potency as an activator of target enzymes. Biochemistry 37, 6523-6532.[Medline]

Resnick, J. & Shenk, T. (1986). Simian virus 40 agnoprotein facilitates normal nuclear location of the major capsid polypeptide and cell-to-cell spread of virus. Journal of Virology 60, 1098-1106.[Medline]

Rinaldo, C. H., Traavik, T. & Hey, A. (1998). The agnogene of the human polyomavirus BK is expressed. Journal of Virology 72, 6233-6236.[Abstract/Free Full Text]

Roack, P. J. (1991). Multisite and hierarchical protein phosphorylation. Journal of Biological Chemistry 266, 14139-14142.[Abstract/Free Full Text]

Rott, O., Kroeger, M., Mueller, H. & Hobom, G. (1988). The genome of budgerigar fledgling disease virus, an avian polyomavirus. Virology 165, 74-86.[Medline]

Stoll, R., Luo, D., Kouwenhoven, B., Hobom, G. & Mueller, H. (1993). Molecular and biological characteristics of avian polyomaviruses: isolates from different species of birds indicate that avian polyomaviruses form a distinct subgenus within the polyomavirus genus. Journal of General Virology 74, 229-237.[Abstract]

Stoll, R., Hobom, G. & Müller, H. (1994). Host restriction in the productive cycle of avian polyomavirus budgerigar fledgling disease virus type 3 depends on a single amino acid change in the common region of structural proteins VP2/VP3. Journal of General Virology 75, 2261-2269.[Abstract]

Zhou, Y., König, M., Hobom, G. & Neumeier, E. (1998). Membrane-anchored incorporation of a foreign protein in recombinant influenza virions. Virology 246, 83-94.[Medline]

Received 3 August 1999; accepted 30 September 1999.