Analysis On Genomic Organization Of Dendrolimus Punctus Densovirus And Study On Function Of Non-Structural Protein NS1

Dendrolimus punctatus larva is the most destructive defoliator of massopine forests. Recently, a non-enveloped icosahedral DNA virus has been isolated from dead larvae of Dendrolimus punctatus (pine caterpillar) in the Xinxian Forestry Center, Henan province, China. This virus is likely an important pathogen of Dendrolimus punctatus. The virus was named Dendrolimus punctatus densovirus (DpDNV).Virus particles were about 22 nm in diameter and contained a single-stranded DNA genome of approximately 5.0 kb. Four major capsid proteins were resoved by 12% SDS-PAGE, and the molecular mass of these were 79kDa, 65 kDa, 55 kDa and 51 kDa, respectively.The taxonomy of densovirus is based on the structure and organization of their genomes. To identify the taxonomy position of DpDNV, a serial overlap clones were constructed by digestion of genome and similar 5'RACE techniques. The complete genomic sequence was obtained by determining all these clones. The genomic DNA of DpDNV was 5039nt long (GeneBank No. AY665654). The base composition of the plus strand of genome was A/T rich (35.56% A, 18.49% C, 19.07% G and 26.87%T), similar to other densoviruses (for example, CeDNV 36.34% A, 25.80% T). The DpDNV genome sequence showed about 60% identity to both CeDNV and BmDNV-1.The DpDNV genome had ITRs of 200nt and its terminal 131nt could be folded into a J-shaped hairpin structure similar to those structures previously identified on the CeDNV and BmDNV genome within the genus Iteravirus. The plus strand of the DpDNV genome containned three large ORFs, whereas the minus strand did not contain any significant ORF. The ORFs within the 5'-half of the plus strand encoded the viral NS proteins, while ORF within the 3'-half encoded the viral proteins. The replication initiator motifs(â…  and â…¡) and the tripartite superfamily â…¢-type ATPase / helicase motifs (A, B and C) were present in N- and C-terminus of NS1 protein ofDpDNV, respectively, which are conserved among densovirus and vertebrate parvovirus. Meanwhile, the conserved motifs of pvPLA2 domain were present in VP1 unique region of DpDNV from aa2 to aa60. The amino acid sequence of the VP1 protein showed 76% and 72% identities with VPls of CeDNV and BmDNV, respectively. P7 and P54 promoters were likely functional for the putative NS and VP transcript of DpDNV. And 2 AATAAA sites at m.u.54 and m.u.95 were most likely polyadenylation sites. Sequence analysis revealed that a direct repeat of 18nt (2656-2691 nt) was located between the NS1 and VP sequences of DpDNV similar to that of BmDNV.Homologies in the genome organization, structural characteristics of the genome and sequence identities all suggest that DpDNV is a new member of the genus Iteravirus. Furthermore, Phylogenetic analysis based on DNA-dependent ATPase/helicase domain showed that DpDNV was most related to CeDNV and BmDNV-1 in the genus Iteravirus and formed another separate cluster. These results further support the classification of the Iteravirus, as a separate genus, within the subfamily Densovirinae.The gene of NS1 was cloned into the pFastBac-IEl vector, producing the combinant plasmid of pFastBac-IEl-NSl. Sf-9 cells were transfected with pFastBac-IEl-NSl, subsequently infected by AcMNPV lacking a functional P35. Cells were examined under light microscope. Apoptosis was evident, generating many apoptosis bodies, in contrast to the control cells. Western blot showed the expression of NS1 protein. Therefore, NS1 protein was predicted to likely function as an apoptosis activator.