The Nuclear Localization Signal Of The NS1 Protein Is Essential For Periplaneta Fuliginosa Densovirus Infection

Periplaneta fuliginosa Densovirus (PfDNV) isolated from cockroaches in Wuhan, PRC, has been systematically studied for about 20 years. In 2004, International Committee on Taxonomy of Viruses (ICTV) created a new genus (Pefudensovirus) in the subfamily Densovirinae with Periplaneta fuliginosa densovirus as type species owing to its divergences in the genome structure and the gene expression strategy between PfDNV and other DNVs.The regulatory protein NS1 is a key molecule in viral replication.It is a multi-functional protein that has several catalytic activities, including ATP binding and hydrolysis, oligomerization, sitespecific DNA binding to a cognate recognition motif, site-and strand-specific nicking, helicase activity, and promoter trans-regulation. These activities allow this pleiotropic protein to execute the different functions necessary for progeny virion production, including regulation of viral DNA amplification and gene expression. Additionally, some of the latter functions may also act upon the host cell genome and may contribute to the cytotoxic activities of NS1. Moreover, the ability of NS1 physically interacts with a variety of host cell proteins, including components of the DNA replication machinery and transcriptional regulators may represent another "scavenging"mechanism by which NS1 negatively interferes with essential cellular processes.The host ranges of different densoviruses are variable. JcDNV can infect butterfly and some nymph of nocturnal moth; while PfDNV just infects Periplaneta fuliginosa cochroch. Lack of a suitable cell line that permits virus replication to high titers has been a long-standing problem in densovirus research. In this study, infected primary hemocyte cells produced a typical cytopathic effect characterized by cell clumping and syncytia. So the hemocyte cells of P. fuliginosa nymphs, which were permissive for both infection with the purified PfDNV virions and transfection by plasmids, were used to study the nuclear localization signal of the NS1 protein and PfDNV infection.Firstly, the cDNA clone encoding NS1 of PfDNV was prepared from the total mRNA of PfDNV-infected crockroach by reverse transcription-PCR. For expression in E. coli, the DNA fragment was cloned in pET28a plasmid and the protein with his-tagged was expressed and purified by Ni-NTA affinity chromatography for generating polyclonal rabbit antibodies. Affinity and specificity of the anti-NS1 serum were tested by Western blot analysis.The infectious plasmid pUC119-PfDNV was mixed with DEAE-dextran and injected in 3 star nymph of Periplaneta fuliginosa. The PfDNV virion was purified from the infected cochroch. To understand clearly the sensitivity of insect cells, we infected the insect cells of hemocyte, S2 and C6/36 by PfDNV strains. The characteristic cytopathic effect typical for PfDNV, in the form of clumping and ballooning of cells and syncytia formation, was observed in hemocyte cells at around 48 h post-infection. Hemocyte showed an extensive CPE (60-70%) at 72 h post-infection. And the cells were split seriously after 72 h post-infection, and no difference was found between the mock-cells and the insect cell lines, S2 and C6/36.To understand clearly the localization of NS1, we constructed a plasmid, pAcNS1, by PCR the nsl sequence from pUC-PfDNV. Immunofluorescence was then used to determine the cellular distribution of NS1 in the transfected cells. After the pAcNS1 was transfected into the insect cells (hemocytes, S2 and C6/36), NS1 protein was detected at 48 h post-transfection.We found that NS1 was localized to both cytoplasm and nucleus of hemocytes, while it was localized to only the cytoplasm of S2 and C6/36. Transfection efficiency of the plasmid pAcNS1 of hemocyte, S2 and C6/36 cells were 32%,41%and 38%, respectively. In order to confirm these results, cytoplasmic and nuclear extracts were isolated from NS1-transfected cells and analyzed by Western blot. NS1 protein (approximately 71 kDa, with tag) was present in the cytoplasm of the insect cell lines, while NS1 was detected in the nuclear extracts of hemocytes.We next asked whether the NS1 protein was expressed and localized to the nucleus in these insect cells following PfDNV infection. Infection of insect cells using virions of PfDNVwas carried out. Because of the cytopathic effect, immunofluorescence diagnosis directed against NS1 using polyclonal antibodies were performed at 36 h post-infection. NS1 could be observed in insect cells, and was localized to the nucleus exclusively in the hemocyte cells. Infection efficiencies of hemocyte, S2 and C6/36 cells, which accorded to the number of cells expressing NS1, were 16%,31%and 25%, respectively. Western blotting of the fractionated lysates confirmed that NS1 was in the nucleus of hemocyte.