| Human parvovirus B19 belonging to the Parvoviridae family, Parvovirinae subfamily, Erythrovirus Genus, was first discovered and named by Cossart. in 1975. It is one of the two pathogenic human parvoviruses. B19 is widespread all over the world, different clinical syndromes may be displayed due to the different ages and physical condition of the host, and the infection usually occurs in the late autum, spring and early summer. Several diseases have been reported to be associated with B19 infection, including erythema infectiosum (fifth disease) in children, polyarthropathy syndromes in adults, transient aplastic crisis in patients with underlying chronic hemolytic anemia, and chronic anemia due to persistent infection in immunocompromised patients. Infection during pregnancy can lead to hydrops fetalis with possible fetal loss or congenital infection. It has reported recently that myocarditis, hepatitis and myelitis may be related to B19 infection.B19 is about 25nm in diameter, the particle comprises of icosahedral capsid containing a single-stranded linear DNA genome of approximately 5.6kb. The left part of the genome encodes the non-structural protein NS1, whereas the right part of the genome codes for two structural capsid proteins,VP1(84kDa) and VP2(58kDa), of which VP2 is a truncated form of VP1. The additional 227 amino acids at the amino-terminal end of the VP1-protein, so-called VP1-unique part (VPlu) have been demonstrated to exhibit a PLA2 activity that showed in most of other parvoviruses. The PLA2 activity of the VPl-up may play an important role in the process of B19 infection by an unknown mechanism. The VP1u has become a potential target for anti drug design.In this study, the key amino acids (130,132,134,154) of the VP1u which involves in the Ca2+binding were mutated by the site-directed mutagenesis. In the functional study of the VP1 unique part, PLA2 activity detection in vitro and the mutant infectious clones were carried out at the same time. Briefly, the PLA2 domain of B19 VP1u was cut out and inserted into the plasmid PUC-18a to generate the recombinant plasmid PUC-muVPl serving as a template in the site-directed mutagenesis. The mutated clones were confirmed by sequencing. Then the mutated VPlu was cut and inserted into the prokaryotic expression plasmid pMal-c2x to produce the pMal-VPu. The fusion protein was induced by EPTG in the E.coli DH5a cells and analyzed by SDS-PAGE and western-blot. Then the target proteins were purified by affinity column, sPLA2 activity for both the wild type and mutants were assayed and compared. Our data showed that no PLA2 activity was detected when the Asp in the position of 154 was mutated into Ala suggesting the importance of 154Asp in the PLA2. At the same time, the mutant infectious clones encompassing the full genome of B19 were constructed. The fragment from 2251 to 4291bp (2040 bp) in the B19-4244 (the infectious clone) was cut out and inserted into pBluescript‖KS (+) to generate the recombinant plasmid PB-2040 which was used as the template for site-directed mutagenesis by PCR. The mutated fragment was first confirmed by sequencing and then will be cloned back into B19-4244(-2040). the mutant clones PBG132R、PBG134R and PBD154A that were presently constructed and confirmed will be served as the intermediate for construction of the mutant infectious clones. These constructions provide us with useful tool for further study of effect of amino acid changes in the critical domain of B19 VP1u on virus replication. |
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