| Hypoviruses are a group of unencapsidated,cytoplasmically replicating RNA viruses of the chestnut blight fungus Cryphonectria parasitica that can attenuate virulence (hypovirulence).Four virus species have been identified as belonging to the genus Hypovirus in the Family Hypoviridae:CHV1,CHV2,CHV3 and CHV4.Six members finished genome sequencing,and three members CHV1-EP713,CHV1-Euro7 and CHV1-EP721 belong to CHV1.Although Cryphonectria parasitica strains infected with CHV1-EP713,CHV1-Euro7 and CHV1-EP721 all exhibit typical hypovirulence-associated traits,compared with CHV1-EP713,CHV1-Euro7 and CHV1-EP721 are milde viruses.Comparison with other two hypoviruses,the accumulation of CHV1-EP721 in the cells of fungus is significantly lower, although it showes extensive sequence identities with CHV1-Euro7 and CHV1-EP713,with an average of 99%and 90%identities at nucleotide level and 99%and 92%identities at amino acids level respectively.To further examine whether different fungal hosts would have an impact on the accumulation of viral dsRNA,hypoviruses CHV1-EP713,CHV1-Euro7 and CHV1-EP721 were introduced via transfection into virus-free and genetically different fungal strains EP155, Euro7(-v)and EP721(-v),respectively.A change of colony pigmentation from orange to white indicated that a productive infection for all of these viruses had been established.An examination of viral dsRNA in the infected cells revealed that the viral dsRNA accumulation level was solely determined by the input virus.The high level of sequence identity shared by CHV1-EP713,CHV1-Euro7 and CHV1-EP721 suggested the possibility that viral coding domains responsible for differences in viral dsRNA accumulation in a given host fungus could be mapped through the construction of chimeric viruses by genome domain swapping.Four chimeric viruses, 721A713B,713A721B,721AE7B and E7A721B with ORF A and ORF B from different hypovirus strains were obtained through swapping of the polypeptide coding domains between CHV1-EP721 and CHV1-EP713,and between CHV1-EP721 and CHV1-Euro7.All these chimeric viruses were able to establish a productive infection in the host fungus EP721(-v)by transfection.An examination of the viral dsRNA by argarose gel electrophoresis revealed that both 713A721B and E7A721B dsRNA accumulated to the lowest level and comparable to that of CHV1-EP721 and 721A713B dsRNA accumulated to the highest level and comparable to that of CHV1-EP713.The results showed a correlation between the origin of ORF B and viral dsRNA accumulation level.Comparing fungal strains infected with wild-type virus strains and chimeric viruses for their virus transmission efficiency into the asexual conidial spores,the results showed that the transmission efficiency correlates with virus accumulation level in the host.In order to map the determinant for low viral dsRNA accumulation in CHV1-EP721 to a more defined domain within the ORF B,a series of additional chimeric viruse:721/E7 Narâ… and E7/721 Narâ… ,721/E7 PH and E7/721 PH,721/E7(5.3-7.8K)and E7/721(5.3-7.8K)were constructed.All resulting chimeras were infectious upon transfection of the fungal host EP721(-v)and all transfectants showed a white phenotype.An examination of the dsRNA from the transfectants revealed that the chimera E7/721(5.3-7.8K)accumulated to a level similar to that observed for CHV1-EP721,while the reciprocal chimera 721/E7(5.3-7.8K) accumulated to a level about 70%of the wild-type CHV1-Euro7 dsRNA level.Combined, these results indicate that the determinant for low viral dsRNA accumulation in CHV1-EP721 is located within the Narâ… /Mluâ… (5.3-7.8 kb)domain of ORF B.In order to determine whether the domain between 5.3 to 7.8 kb of CHV1-Euro7 would function in trans to enhance the viral dsRNA accumulation of CHV1-EP721,three plasmids, pTr5.4,pTr4.1 and pTr2.5,were constructed to transform the spheroplasts of EP721.The examination of dsRNA from transformants showed that the viral accumaltion of CHV1-EP721 was restored to near the level of CHV1-Euro7,which suggested that the determined domain from 5.3 to 7.8 kb function in trans to increase the viral dsRNA accumulation.And we also found the fusion of EGFP gene egfp to 3' end of the domain from 5.3 to 7.8 kb not affect its functions to enhance the accumulation of CHV1-EP721 in trans. Other three plasmids containing different domains from 5.3 to 7.8 kb of CHV1-Euro7 were further constructed to transform the spheroplasts of EP721.The plasmid pTr57G contained the domain from 5.3 to 7.0 kb,pTr68G contained the domain from 6.4 to 7.7 kb and pTr67G contained the domain from 6.4 to 7.0 kb,and all these three fragments were tagged with egfp in 3' end.The detection of dsRNA accumulation in the transformants showed that the accumulation of CHV1-EP721 was nearly increased to the level of CHV1-Euro7,which suggested that the viral gene determining the hypoviral dsRNA accumulation maybe locate in the 600 bp domain from 6.4 to 7.0 kb.And the same domain of CHV1-EP721 was found increasing the dsRNA accumulation of itself to the level similar to that of CHV1-Euro7 in trans.Point mutations were performed on four different amino acid residues in the domain from 6.4 to 7.0 kb between CHV1-EP721 and CHV1-Euor7.And the residue Val 1426 was substituted with Ala,Glu substituting for Lys 1439,Gly for Glu 1495 and Arg for His 1496 in the domain from 6.4-7.0 kb of CHV1-EP721.The dsRNA examination results showed that the dsRNA accumulation of CHV1-EP721 could not be restored to the level of CHV1-Euro7 in the fungal cells transformed with the 6.4-7.0 kb fragment of CHV1-EP721 containing these point mutations.The fragment containing the domain from 6.1 to 6.8 kb of CHV1-EUro7 was expressed in E.coli BL21(DE3)system and obtained the product produced in the form of inclusion body, designated in P4.Recombinant purified proteins were injected into rabbits for polyclone antibodies preparation.And the interaction between P4 and the other proteins encoded with the hypovirus was examined with the Biacore system.The study on the determinant for hypoviral dsRNA accumulation should provide some help to understand the mechanism of hypoviral dsRNA replication.
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