The nucleotide sequence and genome organization of Plasmopara halstedii virus

P. halstedii pathotypes were cultured, characterized, harvested and intermediately stored as described earlier [1]. Isolates of P. halstedii used in this study are listed in Table 1. Sporangium samples are deposited in the Oomycetes collection of O. Spring, University of Hohenheim and stored at -70°C. Vouchers of field collections of downy mildew infected sunflower are deposited in the Hohenheim herbarium (HUH) under the collection numbers #405 (Waldenbuch, Germany, 2001), #406 (Wurmlingen, Germany, 2001), #407 (Steinenbronn, Germany, 2001), #530 (Balcarce, Argentina, 2003) and #1057 (Plieningen, Germany, 2008).

Virus extraction was based on former experiments [10] and was empirically adjusted to precipitate adequate amounts of PhV.

At least 180 g of frozen sunflower tissue (primary foliage leaves, cotyledones and stems) infected with different isolates of P. halstedii was used to purify virions. Per 1 g infected sunflower tissue, 2 ml 0.05 M sodium phosphate buffer, pH 7.0 were added. Additionally, 0.3% (w/v) of disodium sulfite was added and dissolved by stirring before the sunflower tissue was finely homogenized with an electrical blender. The homogenate was squeezed through four layers of cheesecloth and then centrifuged (5000 g, 4°C, 15 minutes). To the supernatant, 0.5 M sodium chloride was added and dissolved by stirring at room temperature. PEG 6000 was gradually added to a final ratio of 12% (w/v) and the suspension was stirred for an hour before the precipitation was conducted at 4°C for 60 hours. The suspension was centrifuged (7000 g, 4°C, 30 min) and the precipitate was re-suspended in ¹/₁₀₀ of the original volume of 0.05 M sodium phosphate buffer, pH 7.0 with additional 0.3% (w/v) disodium sulfite. To remove contaminants like ribosomes [11], 20% (v/v) chloroform was added and mixed with the re-suspension. A low-speed centrifugation step (1000 g, 4°C, 5 min) was performed to separate virus-containing aqueous from the organic phase. The chloroform extraction step was repeated once.

Success of the virus extraction and purification procedure was controlled by negative staining for transmission electron microscopy as described earlier [1].

The virus suspension was divided into aliquots and stored at -70°C. Under these conditions, virus suspensions were suitable for RNA extraction, purification and reverse transcription PCR (RT-PCR) experiments for more than three years.

Extraction of CP of PhV was carried out as described earlier [1]. Samples for nano-LC-ESI-MS analysis were further purified on 10% polyacryl amide gels [12] on a Mini-Protean System (Bio-Rad Laboratories, München, Germany) and stained with coomassie brilliant-blue (Roti-Blue, Carl Roth, Karlsruhe, Germany).

Gel bands of the CP were in-gel-digested using trypsin (Roche, Penzberg, Germany) [13]. After tryptic digestion, the supernatant was recovered and the gel pieces were extracted with 50% acetonitrile (ACN)/50% 0.1% formic acid (FA) (v/v). The pooled supernatants were then dried in a vacuum centrifuge and stored intermediately at -20°C.

Nano-LC-ESI-MS/MS experiments were performed on an ACQUITY nano-UPLC system (Waters, Milford, USA) directly coupled to a LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Fisher, Bremen, Germany). Tryptic digests of the PhV CP were concentrated and desalted on a precolumn (2 cm × 180 μm, Symmetry C18, 5 μm particle size; Waters, Milford, CT) and separated on a 20 cm × 75 μm BEH 130 C18 reversed phase column (1.7 μm particle size; Waters, Milford, CT) using a linear gradient of 1 to 50% ACN in 0.1% FA within 1 h. The LTQ-Orbitrap was operated under the control of XCalibur 2.0.7 software. Survey spectra (m/z = 250-1800) at a resolution of 60.000 at m/z = 400 were detected in the Orbitrap using lock-mass ions from ambient air for internal calibration [14]. Data-dependent tandem mass spectra were generated for the five most abundant peptide precursors in the linear ion trap.

Mascot 2.2 software (Matrix Science, London, UK) was used for protein identification. Spectra were searched against the NCBI protein sequence database downloaded as FASTA-formatted sequences from ftp://​ftp.​ncbi.​nih.​gov/​blast/​db/​FASTA/​nr.​gz and supplemented with the deduced ORF2 amino acid sequence of the PhV CP. Search parameters specified trypsin or "no enzyme" as cleaving enzyme allowing two missed cleavages, a 3 ppm mass tolerance for peptide precursors and 0.6 Da tolerance for fragment ions.

Mass spectra of purified PhV CP, in source decay (ISD)- and MS/MS-spectra were acquired on a AutoflexIII MALDI-TOF-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). The instrument was operated in the positive ion mode and externally calibrated using protein mass or peptide calibration standards (Bruker Daltonics, Bremen, Germany), respectively. PhV CP samples were desalted and concentrated on C4 ZipTips (Millipore, Schwalbach, Germany) following the manufacturer's protocols. Proteins were eluted directly onto a stainless steel target using 1 μl of a 2,5-Dihydroxybenzoic acid matrix solution (30 mg mL^-1 in 50% ACN/50% 0.1% TFA, v/v). Molecular masses of intact CP was obtained in the linear mode using an accelerating voltage of 20 kV with 1000 laser shots per sample to ensure good S/N ratio.

ISD-spectra and MS/MS-spectra (T3-Sequencing = terminus-specific pseudo-MS3 TOF-TOF analysis) of PhV CP were acquired in the reflector mode using an accelerating voltage of 20 kV. In order to achieve a good S/N ratio, 3000 and 2000-3000 laser shots were recorded for reflector-ISD and MS/MS spectra, respectively. Data were analyzed using Flex Analysis 3.0 and Bio-Tools 3.0 software (Bruker Daltonics, Bremen, Germany) taking into account the deduced amino acid sequence from ORF2 of PhV.

Viral ssRNA was extracted from purified virions, P. halstedii sporangia or herbarium specimens of P. halstedii infected sunflower using the Aurum Total RNA Mini Kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's protocols. RNA sample purity was checked spectroscopically (A₂₆₀ /A₂₈₀ ratio).

Viral dsRNA was extracted and purified from P. halstedii infected sunflower tissue [15].

cDNA synthesis was performed using either the First Strand cDNA Synthesis Kit or RevertAid First Strand cDNA Synthesis Kit (both kits: Fermentas, Glen Burnie, MD). A first fragment of RNA2 was obtained as stated earlier [1].

PhV specific primers used for sequence comparison were summarized in Table 2.

The PCR standard protocol was 94°C for 2 min; then 35 cycles of 94°C for 45 s, 54°C for 45 s, 72°C for 1 min 15 s; and a finale elongation step at 72°C for 10 min. Occasionally, a gradient thermal cycler set at different annealing temperatures was used to optimize the yield of PCR products.

Using a gradient thermal cylcer, a primer set for RNA2 (2-r7: 5'-AAG CGC GGC GTT TGT -3' and 2-f9: 5'-CAA AGC GTC TCC CAT TGG - 3') resulted in two additional DNA fragments (0.8 kb and 0.7 kb, respectively) at an annealing temperature of 48°C. These two additional amplicons were directly sequenced. Their deduced amino acid sequences showed similarity to the deduced amino acid sequence of the RdRp of SmV A. Based upon this similarity, the sequence data of the putative PhV RNA1 were aligned and virus-specific primers were designed to close the gap between these two partial nucleotide sequences.

The 3' ends of RNA1 and RNA2 were determined when viral RNA was transcribed into cDNA with an adaptor (5'-ATG ACT CGA GTC GAC ATC GAT TTT TTT TTT TTT TTT TTT TTT TTT TTV N -3') which included a poly(T) tract (adaptor and adaptor-specific primer modified after Sambrook and Russell, 2001). After its synthesis, cDNA was amplified in PCR with virus-specific forward primers and an adaptor-specific reverse primer (5'-ATG ACT CGA GTC GAC ATC GA -3'). PCR products were cloned in E. coli and sequenced with virus-specific forward primers. Control experiments with E. coli poly(A) polymerase (NewEngland Biolabs, Ipswich, MA) indicated the lack of internal annealing sites for poly(T), thus confirming that both viral RNA segments featured a poly(A) tract at their 3' ends.

The 5' ends were determined using the SMART RACE cDNA Amplification Kit (Clontech Laboratories, Carlsbad, CA).

A second 5' RACE technique based on purified viral dsRNA was carried out [16] in order to ascertain the 5' ends of RNA1 and RNA2. The results of the second 5' RACE experiments confirmed the 5' sequence of the RNA1 segment. For the RNA2 segment, the 5' end determined with the SMART RACE-kit was extended by additional 44 nt.

Sequence data were subjected to BLAST analysis and aligned using the software BioEdit (version 7.0.5.3; Hall, 1999). The GenBank accession numbers for the different PhV isolates are given in Table 1. The GenBank accession numbers for SmV A are AB083060 (RNA1), AB083061 (RNA2) and AB083062 (RNA3).

Based on the recent comparison of a partial nucleotide sequence of PhV in various samples of P. halstedii[1], the viral isolate Ph8-99 was selected for complete sequencing of the two ssRNA segments and genome analysis.

RNA1 consisted of 2793 nt excluding the 3' poly(A) tract. Analysis of the determined nucleotide sequence revealed the existence of one large ORF (ORF1) of 2745 nt (914 amino acid residues). The estimated molecular mass of the protein encoded by ORF1 was 104 kDa. RNA1 had a 5'-untranslated region (5' UTR) of 18 nt and a 3'-untranslated region (3' UTR) of 30 nt. Analysis of the deduced amino acid sequence of ORF1 revealed the presence of the putative RdRp domain containing the GDD motif.

RNA2 consisted of 1526 nt excluding the 3' poly(A) tract. Analysis of the determined nucleotide sequence revealed the existence of one ORF (ORF2) of 1128 nt. The estimated molecular mass of the protein encoded by ORF2 was 40 kDa (375 amino acid residues). RNA2 had a 5' UTR of 164 nt and a 3' UTR of 234 nt. A sequence comparison with data of SmV A [3] and other sequences indicated that ORF 2 encoded for the viral CP of PhV.

A molecular mass of the CP of ca. 37.5 kDa [8] to 36 kDa [1] was estimated previously by SDS-PAGE analysis. This was in discrepancy with the molecular mass of 40 kDa calculated for the 375 amino acids deduced from the nucleotide sequence of ORF2. Therefore, the CP band was cut off from an SDS-PAGE-gel, digested with trypsin and analyzed by mass spectrometry. Nano-LC-ESI-MS/MS analysis showed sequence coverage of 62% according to the deduced amino acid sequence from ORF2. The first 23 amino acids of the N-terminal sequence of CP were not covered by tryptic peptides and the peptide most proximal to the N-terminus of CP comprising the amino acids 24-35 (DYTVQSNSIVQR), had a non-tryptic cleavage site at its N-terminus (data not shown). This suggested that the N-terminus of the CP might be proteolytically processed in vivo leading to the lower molecular mass as observed in SDS-PAGE experiments. In order to verify this, the N-terminal sequence of the CP was analyzed by Top-Down MALDI-TOF analysis [17]. Two major in source decay (ISD) fragments of 1479.74 Da and 1765.93 Da were observed by Top-Down MALDI-TOF analysis (Figure 1A). Fragmentation of the smaller ISD fragment revealed the peptide sequence DYTVQSNSIVQR (Figure 1B), whereas the larger ISD fragment covered the sequence DYTVQSNSIVQRSLR (data not shown). Therefore, the Top-Down MALDI-TOF sequence analysis determined the start of the N-terminal sequence of CP at amino acid 24 and confirmed the result from the nano-LC-ESI-MS/MS experiment.

These results suggest that the CP of PhV is processed in vivo at its N-Terminus by a yet unknown protease to a final size of 352 amino acids and a mass of 38.0 kDa which is in consistency with the results obtained by SDS-PAGE analysis. Similar processing of the N-terminus was assumed for CP2 of SmV A [3].

Seven representative P. halstedii virus isolates (Ph4-93, Ph1-97, Ph9-98, Ph1-00, Ph10-00, Ph19-01 and Ph5-05) were chosen to be partially sequenced (Table 1). A 1.4 kb-sequence of RNA2 and a 1.9 kb-sequence of RNA1 were compared to the complete sequence of P. halstedii virus isolate Ph8-99 and compared among themselves. Accounting for more than three quarters of the total genome of P. halstedii virus, sequence variation was extremely low.

In terms of RNA1, the two French isolates (Ph8-99 and Ph1-00) were identical. The samples from Germany (Ph9-98), Hungary (Ph19-01), and the USA (Ph4-93) each differed from isolate Ph8-99 in single nucleotides at different positions. Only the sequence variation in the German sample led to an amino acid exchange in the deduced amino acid sequence.

In terms of RNA2, Ph8-99 differed in a single nucleotide from all other samples causing an amino acid exchange from alanine to valine. The Hungarian isolate Ph19-01 differed from isolate Ph8-99 in one nucleotide which led to an amino acid exchange from isoleucine to methionine. The French isolate Ph1-00 differed from Ph8-99 in two nucleotides causing a single amino acid exchange from glutamine to serine. The German isolate Ph5-05 showed one nucleotide insertion in the 3'-UTR and additionally one nucleotide exchange. The US isolate Ph4-93 showed only the nucleotide insertion like the German isolate Ph5-05 but not the additional nucleotide exchange. Since the insertion took place in the 3'-UTR, the frameshift was without consequences for the structure or function of the CP.

It appeared that alterations in these highly conserved sequences of RNA1 and RNA2 are detrimental for the self-assembly process during virus propagation.

Additionally five infected herbarium specimens of sunflower were tested to fathom the applicability of PhV sequence population studies on a broader set of samples. The herbarium specimens were stored in the herbarium of the University of Hohenheim for up to nine years. Small fragments between 410 and 650 nt of RNA2 were amplified and sequenced. Again, the sequence variation of the virus in these herbarium specimens was insignificant (data not shown).

A preliminary study revealed that PhV and SmV A shared several morphological, biochemical and molecular characteristics. Both viruses were isometric and showed a granular surface. PhV measured ca. 37 nm in diameter, whereas SmV A was slightly larger (ca. 40 nm in diameter). Both viral genomes are encoded in ssRNA. In SmV A, three RNA segments were detected whereas PhV only contained two segments [1, 3‐5]. The genome organization was now studied and compared to other viruses.

SmV A RNA1 with 2928 nt coded for the RdRp on ORF1a and another protein of unknown function in ORF1b. In PhV, RNA1 with its 2793 nt (excluding the 3' poly(A) tract) coded analogously for the RdRp. ORF1b as it was found in SmV A was not determined in PhV RNA1 (Table 3).

RNA2 of SmV A with a length of 1981 nt coded for the two CP. In PhV, RNA2 of 1526 nt (excluding the 3' poly(A) tract) was found to code for the single CP (Table 3). Between ORF2 of PhV and SmV A ORF2, amino acid sequence similarity of ca. 56% was observed. Within the first ten amino acids at the N-terminal sequences, PhV CP (DYTVQSNSIV) and CP2 of SmV A (DYKVSQNSLV) featured six identical amino acid residues. Two other amino acid QS (PhV) and SQ (SmV A), respectively, were interchanged. Amino acid sequence similarity of ca. 37% was observed between the CP of PhV and the CP of viruses within the Tombusviridae (e.g. Pelargonium leaf curl virus, Tomato bushy stunt virus).

In PhV, RNA1 and RNA2 both had poly(A) tracts at their 3' termini whereas in SmV A poly(A) tracts were lacking at the 3'-termini of all three viral RNA (Table 3).