Expression And Subcellular Localization Of The Periplaneta Fuliginosa Densovirus Capsid Proteins

Periplaneta fuliginosa Densovirus (PfDNV) isolated from cockroaches in Wuhan, PRC, has been studied for nearly20years. In2004, 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.PfDNV is a small non-enveloped single-stranded DNA virus. PfDNV is highly pathogenic and fatal to its hosts, and thus has the great potential to be used as a biopesticide to control cockroaches. PfDNV has an ambisense genome organization, with inverted terminal repeats (ITRs)of202nt at both ends. The minus strand of PfDNV encodes viral capsid proteins (VP).Previous analysis of PfDNV capsids by SDS-PAGE revealed that PfDNV virion is composed of five capsid proteins VPl-5(52kDa,56kDa,79kDa,82kDa and105kDa). However, previous transcription analysis predicted the existence of three capsid proteins as VP52(52kDa), VP65(65kDa) and VP85(85kDa). Therefore, the molecular masses of the predicted proteins do not match well with that of the reported VP1-5.To solve this discrepancy, VP mRNAs sequences were obtained from cockroaches, transfected with the infectious clone, pUC119-PfDNV. Comparing to previous studies, we found two extra splice donor sites (sd4and sd5), one extra splice acceptor site (sa4) and three new VP mRNAs (sd4sal&sd2sa2, sd4sal&sd3sa3, sdlsal&sd5sa4). Interestingly, although three new VP mRNAs were found, these new VP mRNAs did not encode new VP.After that, the opening reading frame (ORF) of VP52, VP65and VP85were cloned into pFastBacl vectors, and the relationship between the predicted VP52, VP65, VP85and the reported VP1-5was examined by ectopically expressing VP52, VP65and VP85in Sf9insect cells using baculoviral expression system, followed by comparing the expression patterns between VP52/VP65/VP85and the capsid proteins in PfDNV particles. Our findings determined that VP52, VP65and VP85are PfDNV capsid proteins, and that VP52corresponds to VP2, VP65corresponds to VP3&VP4, and VP85corresponds to VP5. We proposed that VP1was the N-terminal proteolytic product of VP2(VP52). Besides, we found that VP3&VP4were the different post-translation modification isoforms of VP65, and that the expressed VP65was composed of four protein bands in6%SDS-PAGE, which had the same N/C-terminals. Furthermore, we proved that PfDNV VPs were midified by phospholated tyrosine after translation. VP1was highly phospholated while VP2/VP3&4were poorly phospholated. We did not found phospholated tyrosine in VP5and the expressed VP65. However, the four different protein bands of VP65were not products of VP65phospholation as dephospholation of VP65did not change the four protein bands, indicating that some other unknown post-translation modifications may exist in VP65.Densoviruses replicate and assemble in nucleus of host cells. There are always basic amino acid dependent nuclear location sequenes (NLS) in parvovirus VPs. Moreover, we investigated the roles of VP52, VP65and VP85in the subcellular localization of capsid proteins and the process of viral assembly. Our findings revealed that VP52, VP65and VP85separately located in nucleus of Sf9cells, and that VP52or VP65alone can assemble into virus like particles (VLPs), while VP85alone can not. Analyses of VP sequences with PSORT II revealed the same potential NLS in all PfDNV VPs (PPNKKAKT). In order to test whether this potential NLS was functional, deficient VP65s were constructed and locations of these deficient VP65s were tested. It was surprising that both deleting the N-terminal242aa, which contained the potential NLS, and deleting the C-terminal100aa did not affect VP65nuclear transportation. We thought that the potential NLS was not function and that the nuclear transportation of PfDNV VPs may depend on VP protein structure or host cellular factors.All together, we determined VP52, VP65and VP85as the PfDNV capsid proteins and revealed relationships between VP52, VP65, VP85and VP1-5for the first time. Indeed, this relationship confused virologists, who are interested in PfDNV, and prevented our understandings to this important virus for a long time. This work is a primary study about PfDNV capsid proteins and it is also the first study about the roles of PfDNV capsid proteins in nuclear localization and viral particle assembly. Since the genomic organization of PfDNV resembles that of densovirus, while the expression profile of the genome, especially that for capsid gene, is similar to that of vertebrate parvovirus, it suggests that insect densovirus and vertebrate parvovirus have the same origin and that PfDNV might be an intermediate form of them.Wuhan nodavirus (WhNV) is a newly identified member of the Nodaviridae family. WhNV contains a bipartite genome of positive-sense RNAs, RNA1and RNA2. RNA1encodes the RNA dependent RNA polymerase (RdRp), while RNA2encodes the viral capsid protein. Subgenomic RNA3is produced by internal initiation of the negative RNA1. The nonstructural protein B2is encoded by subgenomic RNA3of nodaviruses. It is a viral suppressor of RNA silencing (VSRs) by sequestering RNA duplexes. WhNV B2blocks RNA silencing in cultured Drosophila cells. WhNV B2protein inhibited Dicer-mediated double-stranded RNA (dsRNA) cleavage and the incorporation of small interfering RNA (siRNA) into the RNA-induced silencing complex (RISC) by sequestering dsRNA and siRNA. B2protein dimerization is required for its binding to RNA, whereas RNA binding to B2protein in turn promotes its dimerization. The N-terminal20aa is essential for B2protein dimerization. Deletion of the N-terminal20aa hindered the RNA interference (RNAi) suppression of B2protein.RNAi is a gene silencing mechanism. It functions as antiviral immunity in diverse organisms. Viruses encode VSRs to combat RNAi-mediated immunity. These VSRs target protein components in the RNAi machinery and RNA to suppress RNAi. The endonuclease Dicer plays important roles in RNAi immunity, however little is known about how VSRs interact with Dicer. It has been reported that the B2protein from Flock House virus (FHV) interact with Dicer-2(Dcr-2) in vitro, reveals that the nodavirus B2protein may interact with Dicer and suppress the host RNAi immunity. Here, we showed that the WhNV B2protein suppressed Drosophila RNAi by directly interacting with Dcr-2but not Dcr-1and sequestering dsRNA and siRNA, and that RNA binding promoted the interaction of WhNV B2and Dcr-2. Furthermore, we found that WhNV B2binds to the PAZ and RNase III domains of Dcr-2via its C-terminal21aa region, thereby blocking the activities of Dcr-2in processing dsRNA and incorporating siRNA into the RISC. We also found that the interaction between WhNV B2and Dcr-2required the homodimeration of B2protein itself. Taken together, these results suggest that B2functions as a homodimer, and RNA binding to B2promotes the homodimerization of B2, thereby enhancing the B2-Dcr-2interaction.