Improved method of purification of plum pox virus and serological analysis of the coat protein

Molecular weight heterogeneity of the coat protein (CP) is commonly observed in the purified potyvirus preparations due to partial proteolysis of the N-terminal domain exposed on the surface of viral particles. The cellular proteases released from disrupted plant tissues are believed to attribute to the partial CP proteolysis during purification and storage of the virus. The N-terminal domain is the most variable part of the CP. It displays virus- and strain-specific epitopes and is highly immunogenic. Degradation of the N-terminal domain creates a potential problem in the production of virus-specific antisera and strain-specific monoclonal antibodies and complicates the serological analysis of virus isolates. Plum pox virus (PPV) is considered the most important viral pathogen of stone fruit crops. Serological methods are of great importance for the diagnosis of PPV, strain identification, elaboration of PPV-resistant cultivars, epidemiological surveys and prevention of the uncontrolled spread of the virus in stone fruit plantings. The CP of PPV was studied by polyacrylamide gel electrophoresis, Western blotting and immunoelectron microscopy using monoclonal antibodies 5B and 4DG5 specific to the PPV universal and strain D epitopes, respectively. The purified virus particles have been shown to contain three types of subunits: full-size CP as well as the 28 and 31 kDa products of its partial proteolysis. Three bands of the similar molecular weight were also detected in the analysis of fresh extracts from PPV- infected Nicotiana benthamiana tobacco leaves and a number of stone fruits (peach, plum and cherry plum), homogenized directly in the sample buffer, by Western blotting with the antibody 5B suggesting that the CP can be proteolytically processed already in infected plant tissues. The improved procedure of PPV purification, based on the combination of two original methods (H.J. van Oosten, 1972; S. Lain et al., 1988) has been developed. The procedure included accumulation of the virus in N. benthamiana plants, its extraction from the infected leaves using a neutral HEPES buffer, the incubation of the clarified extract with 5 % Triton X-100, ultracentrifugation on a 20 % sucrose cushion and purification of the virus using the ultracentrifugation in sucrose concentration gradient (10-40 %) in a 0.1 М sodium borate buffer, рН 8.2. The method provided the yield up to 10 mg of the purified virus from 100 g of infected leaves. The virus particles contained mainly the full-size CP. The high yield of the virus seems to be due to an effective extraction of the virus using HEPES buffer as well as its negligible losses during clarification of the extract, treatment with Triton X-100 and ultracentrifugation on a sucrose cushion. The use of Triton X-100 in the specified concentration leads to a more complete separation of the virus from membrane complexes and solubilization of aggregates of viral particles, that increases the yield of the virus. Application of such the virus preparations for animal immunization can facilitate the high specific antisera and monoclonal antibodies production for reliable detection and serological analysis of PPV.