Effect Of Amino Acids Outside The Conserved Domain Of The VP1u On The Phospholipase A2Activity Of Human Parvovirus B19

Human parvovirus B19(B19) that was discovered in1975by Yvonne Cossart is one of the parvoviruses to infect humans and causes many diseases and can potentially inhibit the formation of red blood cells. B19infection is specific,because of the receptor globoside on the surface of erythroid progenitor cells, B19displays a remarkable tropism for human hematopoietic cell. But expression of globoside on the cell surface is not sufficient for viral cell entry, therefore, the α5β1integrin as a cellular co-receptor and functional activity of KU80auto-antigen are also needed for viral replication. Infection of B19causes different clinical characteristics, including arthritis, joint pain and fever symptoms which occur in adults in relatively high frequency and anaemia which occur in children in relatively high frequency. B19's capsid is formed of only two proteins, VP1and VP2which are identical in their sequence except for an extra sequence of227amino acids at the VP1N-terminus. The study found that the227amino acids of VP1unique part (UP) have an activity of phospholipase A2(PLA2) in which the130-195amino acid is the conserved regions of enzyme activity. The anmino acids outside this conserved domain may also be important for the formation of the intact protein structure to keep the stable activity of the enzyme as well as for approaching the subtracts e.B19-VP1U has very important function in the life cycle of the virus and the activity of the PLA2is necessary for the infection of the virus, lack of the intact VP1will causes the loss of the infectivity of the virus and the mutant virus can not be transported into the cytoplasm from the nucleus,so the activity of the B19-VP1U is very important for the pathogenicity of the virus, autoimmune and the inflammation response. Mutation of critical amino acids in the phospholipase domain of B19-VP1U causes a partial or complete loss in enzymatic activity and viral infectivity. The present study mainly focuses on the impact of amino acids outside the conserved regions of the VP1u on the PLA2enzyme activity. The main results are summarized as follows:(1) Based on relevant literatures and our previous results, we, constructed a series of truncated mutants by deleting every10or30amino acids outside the VPlu conserved amino acids from the N-terminal and cloned these constructs into the pMAL-c2x plasmid to generate the corresponding recombinant vectors. The target proteins are expressed by a pilot experiment and SDS-PAGE and Western-blot were performed to confirm expression of the target proteins. Finally a large scale of the target proteins are produced and purified by the resin Amylose purification system.(2) Based on relevant literatures and our previous results, we constructed a series of truncated mutants by deleting every7to8amino acids outside the VPlu conserved amino acids from the C-terminal and cloned these constructs into the pMAL-c2x plasmid to generate the corresponding recombinant vectors. The target proteins are expressed by a pilot experiment and SDS-PAGE and Western-blot were performed to confirm expression of the target proteins. Finally a large scale of the target proteins are produced and purified by the resin Amylose purification system.(3) After production of sufficient targeted proteins, the concentration of the proteins was measured by the Broadford kit to assure the minimal concentration of the proteins was above the5mg/ml, then the PLA2enzyme activity was measured according to the instructions of the kit and the results were analyzed,Our results showed that the truncation of12A A from the N terminal the enzyme activity has been reduced by35percent compared to the entire VP1u. However, when the67AAs and96AAs from N-terminal were truncated the PLA2activity was significantly decreased and completely abolished, respectively.Therefore we hypothesized that the1to96AAs from N terminal have significant effect on enzyme activity. When the7AAs from the C terminal were truncated the enzyme activity was almost the same as that of the whole VP1u, but when16or24AAs were truncated, the enzyme activity was significantly decreased or completely lost, respectively.These results indicated that7to24AAs from the C terminal were also critical for enzyme activity. Outer the conserved region of amino acid deletion mutants of phospholipase A2activity may be changes B19-VP1U complete protein3D conformation and seams has a great relationship, because the protein to maintain activity are required to have a complete three-dimensional structure of protein to remain active. Our present study provides some useful information for further understanding the mechanism of B19infection.