Functional Studies And Specific Transduction Of B Lymphocytes Of A Baculovirus Acmnpv Orf38 Recombinant Baculovirus Construct

Partâ… Baculoviridae is a group of viruses that only infects arthropod in nature.The type species of Baculoviridae,Autographa californica nucleopolyhedrovirus (AcMNPV),has been extensively used in research and application.More and more knowledge about the Baculovirus,such as its life cycle,genome structure and replication,the regulation of gene expression,protein structure and function,and the interaction between virus and host,have been accumulated during last few decades.After the publication of the whole genome sequence of AcMNPV in 1994,many essential viral genes were identified.However,apart from these essential genes,there are still many other genes that play important roles in the life cycle of the virus, although not being essential for virus replication.Without them,virus replication would be impaired.AcORF38 of AcMNPV is one of such genes.Bioinformatics analysis revealed that it contains a conserved motif of Nudix superfamily.In the current study,the recombinant AcORF38 protein was prepared and shown to have the activity to hydrolyze ADP-Ribose in vitro,with a K_m of 204μM,and K_catof 6.96 s^-1at pH 8.0 and 5mM MgCl₂ when the concentration of AcORF38 was 28μM.This result implied that AcORF38 is truly a member of Nudix superfamily.The results of RT-PCR showed that orf38 gene is transcribed in the early phase of AcMNPV infection.An orf38 gene-deleted mutant virus,vAcGFP-â–³38,was constructed.The virus produced progeny virus,which meant that orf38 was non-essential for virus replication.However,the extracellular virus produced by the mutant virus was only 1/300 of that of the wild-type virus.This result indicated that orf38 played an important role in the infection cycle of virus.To further study the function of AcORF38,infected Sf9 cells were examined with electron microscopy.In the showed much more virus particles were observed in cells infected with vAcGFP-â–³38 than that infected with vAcGFP.In consistence with electronic microscopy,quantitation of viral DNA and mRNAs by real-time PCR found that the level of viral DNA,as well as four viral early mRNAs,including lef-2, lef-3,dnapol and p143,were significantly high in vAcGFP-â–³38-infected cells than vAcGFP-infected cells.The results suggested that AcORF38 might reduce the viral DNA synthesis and viral gene expression in the early phase of virus infection. Transient-expression assays further confirmed that AcORF38 reduced the expression of reporter gene egfp,as well as the mRNA level of cellular gene actin. Similar results were observed in transient assay in other 5 mammalian cells using AcORF38-expression vector driven by CMV promoter,indicating that AcORF38 might non-specifically reduce mRNA level in various cells.Taking the properties of Nudix hydrolase into account,it was postulated that AcORF38 might be an enzyme involved in degrading the cap structure at the 5' end of eukaryotic mRNA.To test it,both the analog of the cap,m7GpppG,and ADP-ribose were used as substrate for AcORF38.The K_m value was lower when m7GpppG was used as substrate,than ADP-Ribose,indicating that m7GpppG mignt be a more appropriate substrate than ADP-Ribose.Taking together,the current study suggested that AcORF38 was a negative regulator of viral and cellular gene expression.It did that in a non-specific way, possibly through hydrolyzing the cap structure of mRNAs.Such a negative regulator could prevent the over expression of viral early proteins and ensure the normal replication of the virus.The detailed mechanism of AcORF38 is still to be understoodPartâ…¡As a mammalian gene transfer vector,baculovirus has several unique advantages.For example,it is highly safe to use since it can not replicate in mammalian cells,it has a large capacity to hold foreign genes,since its rod-shaped nucleocapsid can accommodate an additional 100kb or more of DNA,and it has little cytotoxicity even at high multiplicity of infection.However,baculovirus also has its disadvantages such as the low transduction efficiency in some cells,including B-lymphocytes.In order to enhance baculovirus transduction of B cells,baculovirus surface display technology was applied to modify the virus surface protein of baculovirus. A short peptide motif from GP350/220 of Epstein-Barr virus,EDPGFFNVEI,which was known to bind to CD21,a surface protein on B-lymphocyte,was inserted into GP64,the main surface glycoprotein of AcMNPV.A recombinant virus, vAc-gp350EGFP,was constructed with the "avidity display" strategy,in which all copies of GP64 molecules on the virion had the motif.The recombinant virus, compared with a control virus with wild type GP64,showed increased transduction efficiency in B cell lines Raji,HR1,B95-8,BJAB,and DG75,regardless of their being EBV-positive or EBV-negative.The rate of EGFP positive Raji cell increased from 0.05%when transduced with the control virus,to 12.6%when transduced with vAc-gp350EGFP.The transduction of Raji cells by vAc-gp350EGFP was dose-dependently inhibited by pre-treatment of cells with anti-CD21 antibody, indicating that the improvement of the transduction was due to the interaction between the GP350/220 peptide and the CD21 molecules on the surface of B lymphocytes.The work significantly improved the transduction efficiency of baculovirus in B-lymphocytes.The approach used in the work might be applied to explore the known interactions of ligands and receptors,so as to expand the range of susceptible cells of baculovirus-mammalian cells vectors,or to increase the specificity of those vectors.