Characterization Of Functional Sequences In BmNPV-encoded Bm65 And The Effect Of The Sequences On Viral Proliferation

The silkworm?Bombyx mori?is an important economic insect in China.The silk spited by Bombyx mori has high economic value and is widely used in clothing,medical treatment and cosmetics.However,silkworm is easily suffered from the infection of BmNPV,which results to the outbreak of virulent infectious diseases in silkworm population,leading to the death of a large number of silkworms and significantly reduces the production of silkworm cocoons.So,the infection of silkworm will bring about great losses to the silkworm economy.Our previous study showed that BmNPV-encoded Bm65 with molecular mass of about 12.2 kDa was expressed in the early stage of viral infection.Bm65 was distributed mainly in the nucleus,which was involved with the repair of DNA damage caused by ultraviolet radiation.However,the mechanism of nuclear import of Bm65 remains unclear.In this study,several functional sequences of Bm65 were characterized,and the effect of their mutations on Bm65 into the nucleus and viral proliferation were revealed.It will provide theoretical basis for further development and utilization of baculovirus.The main results in the study are as follows:1.³³RRIK and ⁷⁶KRKCSK of basic amino acid clusters and two hydrophobic amino acid clusters of ⁹²PLLL and ⁹⁸FLLA were conserved in the sequence of Bm65and its homologous sequence.The sequences may be involved with the dynamic shuttle of Bm65 protein from cytoplasm to nucleus.2.A series of Bm65 truncated mutants fused with EGFP under the control of ie1promoter were constructed.The fluorescence results indicated that the motif of⁷⁶KRKCSK was directly involved with the nuclear import of Bm65,while ³³RRIK was not involved in nuclear import of Bm65.To further confirm the results,a series of mutant transient expression vectors containing mutations in the motif of ³³RRIK and⁷⁶KRKCSK were constructed,which were also under the control of ie1 promoter and fused with EGFP.The results showed that the ³³RRIK mutant did not affect the nuclear import of Bm65.However,the Bm65 protein was blocked into the nucleus and accumulated mainly in the cytoplasm after the motif of ⁷⁶KRKCSK was mutated.It indicated that ⁷⁶KRKCSK played an important role in the nuclear import of Bm65.A single point mutation was made on the ⁷⁶KRKCSK sequence,and we found that the amino acids of 78K and 81K in the ⁷⁶KRKCSK sequence were very essential for the nuclear import of Bm65.3.To determine whether ⁹²PLLLHKFLL was the nuclear export signal of Bm65protein,mutations were made in the sequence of ⁹²PLLLHKFLL and the mutants of Bm65 fused with EGFP was used to study the localization of Bm65.The results showed that Bm65 protein was accumulated exclusively in the nucleus.No green fluorescence could be seen in the cytoplasm when the mutation was made in the motif of ⁹²PLLL and ⁹⁸FLLA,respectively.Therefore,we believed that the motif of ⁹²PLLL and ⁹⁸FLLA play a role in the nuclear export of Bm65.4.Recombinant virus for the expression of Bm65 fused with Flag tag was constructed in the study.Western blot analysis of the total protein of virus-infected BmN cells revealed that Bm65 existed as a tetramer in virus-infected cells.Three cysteine residues C12,C46 and C79 in Bm65 sequence were mutated into alanine.However,the mutation did not alter the formation of Bm65 tetramer,but affected the stability of Bm65 tetramer.Moreover,recombinant virus fused EGFP tag to the C-terminus or N-terminus of the Bm65 protein was constructed,respectively.Western blot results showed that the fusion protein existed as a monomer,indicating that the fusion of the EGFP tag could affect the formation of Bm65 tetramer.5.The mutations in the motif of ⁷⁶KRKCSK could block of Bm65 into nucleus.Western blot analysis revealed that the mutations did not affect the formation of Bm65tetramer.However,the mutations in the motif of ⁷⁶KRKCSK greatly reduced the production of progeny infectious virions.Compared with the wild-type virus,the mutant virus had significantly lower the production of progeny virions in the silkworm after subcutaneous injection of the silkworm,which delayed the pathogenicity or death of the silkworm.