Studies On The Genome Structure And Function Of Bombyx Mori Densovirus 3

Densoviruses(DNVs) are small no-enveloped particles particles of 18～26 nm in diameter and their genomes consist of 4～6.5 kb single-stranded linear DNA encapsidated as plus and minus strands in separate virions.DNVs tend to be highly host-specific and cause death of the host in most cases.The current interest in the molecular biology of these viruses is fostered by their potential as modified virus pesticide,and by their use as vectors for the expression of foreign proteins.Due to important of the silkworm industry,many isolates were obtained from silkworm,they were termed as BmDNV-1(Ina isolate),BmDNV-2(Saku isolate and Yamanashi isolate),BmDNV-5,BmDNV-3(China isolate) and BmDNV-4(Kenchu insolate). BmDNV-3 and BmDNV-2(Yamanashi isolate) both have bipartite genomes(VD1,VD2) and have the same host species,but their virulence and the symptoms of infection are different.In present studies,we reported the characterizations and potential function of Bombyx mori densonucleosis virus type 3.The results are shown as follows.1.The genome of BmDNV-3 contains two kinds different single-stranded linear DNA molecules(VD1,VD2),and each of them is encapsidated respectively in the form of single-stranded liner DNA(+VD1,-VD1,+VD2,-VD2) in equal percentage.Viral DNA was extracted from the purified viral particles under high salt concentrations,the viral DNA hybridized upon extraction was blunted using T4 DNA polymerase(10 U each with 300 ng of DNA),Restriction fragments digested with Hindâ…¢were cloned into Hindâ…¢/Smaâ… -digested pUC119,and the complete nucleotide sequence was determined.Sequence analysis showed VD1 genome consisted of 6543 nts including inverted terminal repeats(ITRs) of 224 nts,and VD2 genome consisted of 6022 nts including ITRs of 524 nts.The GenBank accession numbers are DQ017268 and DQ017269,respectively.2.Computer analysis showed that the 'plus' strand of the genome contained three large open reading frames(ORF1 to 3),ORF1 and ORF2 most likely encoded non-structural proteins(NS) since they contained conserved replication initiator motifs and NTP-binding and helicase domains shared by all parvoviruses,and ORF3 encoded viral capsid proteins(VP).The 'minus' strand in the right-half had one open reading frame(ORF4),it most likely encoded NS since it contains sequences conserved among a putative DNA polymerases.Analysis of the predicted DNA polymerase sequence using neighbor-joining and protein parsimony algorithms indicated the predicted 1115-residue polypeptide contain five motifs associated with DNA polymerases synthetic activities and three additional motifs associated with polymerases possessing 3′to 5′exonuclease activity.In VD2,one major open reading frame(ORF1) in the plus strand and one minor ORF2 in the complementary strand were identified. Computer analysis suggested the minus strand ORF2 most likely encode the major nonstructural protein,while the plus stand ORF1 most likely encodes the major structural protein.3.Comparison of the complete genome sequence between BmDNV-3 and BmDNV-2(Yamanashi isolate) showed an identity of 98.4%in VD1 and 97.7%in VD2, with a total number of 228 bp substitutions,11 bp deletions and 2 bp insertions found in BmDNV-3.Comparative polymorphisms of ORFs and ITRs regions of the two virus genomes showed that there were three highly variable regions(VD1 ORF3,VD2 ORF1, VD1ORF4 and ITRs).These results make us further understand the strain heterogeneity and the possible reasons for variation of the virulence in this virus,and provide clues for their evolution.4.The transcripts of the virus were mapped to examine the transcription strategy of VD1.Northern blotting revealed one NS genes of 1.1 kb and another VP transcript of 1.5 kb in the left-half 'plus' strand,and one transcript about 3.3 kb of 'minus' strand in the right-half.Sequencing of 3′and 5′ends products of the 1.1 kb NS transcript demonstrated that it started nt 290 and ended at nt 1437.ORF1 and ORF2 were contained completely within this NS cassette but translated from a different reading frame,allowing leaky scanning translation of NS1 and NS2.The 1.5 kb VP transcript was found to start nt 1423 and ended at nt 2931.The VP(ORF3) and NS(ORF4) transcripts overlapped for 10 nts at the 3′ends(antisense RNAs) at 50 m.u.These results indicated that this genus transcription and translation strategies are radically different from that of other reported densonucleosis viruses.5.The midguts was homogenized and isopycnic centrifuged in 40%(W/W) cesium chloride(CsCl),then each fraction of the gradient was submitted in parallel to quantitative PCR,the result showed the buoyant density of VD₁ and VD₂ were all approximately 1.29 g/cm³.The buoyant density of BmDNV-3 is smaller than that of other DNVs.Judging from copies of VD1 and VD2 in different gradients,we think they are probably in different capsids.In addition,the relative quantity of each type of viral protein was estimated after separation on SDS-polyacrylamide gel,four structural proteins with molecular weights of 48 kDa,55 kDa,72 kDa and 97 kDa were found, and 55 kDa was predominance.6.The prokaryotic expression plasmid pET28a(+)-VD1 ORF4 3′fragment was constructed,and then was transformed into BL21.The gene was expressed in the formation of inclusion bodies induced by IPTG.The expression products were with the weight of 62 kDa.In addition,the eukaryotic expression plasmid pF_ASTB_ACHTB-VD1 ORF4 3′fragment was constructed.