Endogenous Viral Element-Derived Piwi-Interacting RNAs (piRNAs) Are Not Required for Production of Ping-Pong-Dependent piRNAs from Diaphorina citri Densovirus.

Piwi-interacting RNAs (piRNAs) are a class of small RNAs primarily responsible for silencing transposons in the animal germ line. The ping-pong cycle, the posttranscriptional silencing branch of the piRNA pathway, relies on piRNAs produced from endogenous transposon remnants to direct cleavage of transposon RNA via association with Piwi-family Argonaute proteins. In some mosquito species and mosquito-derived cell lines expressing a functionally expanded group of Piwi-family Argonaute proteins, both RNA and DNA viruses are targeted by piRNAs in a manner thought to involve direct processing of exogenous viral RNA into piRNAs. Whether viruses are targeted by piRNAs in nonmosquito species is unknown. Partial integrations of DNA and nonretroviral RNA virus genomes, termed endogenous viral elements (EVEs), are abundant in arthropod genomes and often produce piRNAs that are speculated to target cognate viruses through the ping-pong cycle. Here, we describe a Diaphorina citri densovirus (DcDV)-derived EVE in the genome of Diaphorina citri We found that this EVE gives rise to DcDV-specific primary piRNAs and is unevenly distributed among D. citri populations. Unexpectedly, we found that DcDV is targeted by ping-pong-dependent virus-derived piRNAs (vpiRNAs) in D. citri lacking the DcDV-derived EVE, while four naturally infecting RNA viruses of D. citri are not targeted by vpiRNAs. Furthermore, a recombinant Cricket paralysis virus containing a portion of the DcDV genome corresponding to the DcDV-derived EVE was not targeted by vpiRNAs during infection in D. citri harboring the EVE. These results demonstrate that viruses can be targeted by piRNAs in a nonmosquito species independently of endogenous piRNAs.IMPORTANCE Small RNAs serve as specificity determinants of antiviral responses in insects. Piwi-interacting RNAs (piRNAs) are a class of small RNAs found in animals, and their primary role is to direct antitransposon responses. These responses require endogenous piRNAs complementary to transposon RNA. Additionally, piRNAs have been shown to target RNA and DNA viruses in some mosquito species. In contrast to transposons, targeting of viruses by the piRNA pathway in these mosquito species does not require endogenous piRNAs. Here, we show that piRNAs target a DNA virus, but not RNA viruses, in an agricultural insect pest. We found that targeting of this DNA virus did not require endogenous piRNAs and that endogenous piRNAs did not mediate targeting of an RNA virus with which they shared complementary sequence. Our results highlight differences between mosquitoes and our experimental system and raise the possibility that DNA viruses may be targeted by piRNAs in other species.

Structural proteins of Helicoverpa armigera densovirus 2 enhance transcription of viral genes through transactivation.

Herein, we report the identification of putative promoters for the non-structural proteins (NS) and capsid structural proteins (VP) of Helicoverpa armigera densovirus (HaDV2) as well as a potential mechanism for how these promoters might be regulated. For the first time, we report that VP is able to transactivate the VP promoter and, to a lesser degree, the NS promoter in densoviruses. In addition to this, another promoter-like sequence designated P2, when co-transfected with the VP gene, enhanced luciferase activity by approximately 35 times compared to a control. This suggests that there are two promoters for VP in HaDV2 and that the VP of parvoviruses might play a more important role in viral transcription than previously appreciated.

[Construction of AalDV-3 Mediated mirRNA Sponge Delivery System and Its Effect on Cell and Larva of Aedes aegypti].

microRNAs (miRNAs) not only play the key roles in regulation of the growth and development of mosquito, but also has an important function in interaction between the pathogen and vector. So, miRNAs can be used as the molecular target for development of an alternative method for mosquito and mosquito-borne disease control. However, an effective delivery system is still need. Mosquito densovirus have the potential for vector control as transducing agents to express foreign toxins or small interfering RNAs molecules in vitro and in vivo. In this study, we report the development of a recombinant Aedes albopictus densovirus-3(AalDV-3) miRNA sponge expression system, using an intronic miRNA sponge expression strategy. To test the inhibition effect of recombinant virus medicated miRNA sponge on endogenous miR-210, Aedes aegypti Aag2 cell and 1st-2nd larvae were infected, respectively. The splicing of the intronic miRNA sponge expression constructs in vitro and in vivo were tested by RT-PCR with intron span primers, and the relatively expression level of miR-210 was confirmed by qPCR. As results,AalDV-3can be used to decrease endogenous miRNAs by generating an antisense sponge in vitro and in vivo, which represents a tool for the functional analysis of mosquito genes and lays the foundation for the application of densovirus for vector control.

In vitro and in vivo host range of Anopheles gambiae densovirus (AgDNV).

AgDNV is a powerful gene transduction tool and potential biological control agent for Anopheles mosquitoes. Using a GFP reporter virus system, we investigated AgDNV host range specificity in four arthropod cell lines (derived from An. gambiae, Aedes albopictus and Drosophila melanogaster) and six mosquito species from 3 genera (An. gambiae, An. arabiensis, An. stephensi, Ae. albopictus, Ae. aegypti and Culex tarsalis). In vitro, efficient viral invasion, replication and GFP expression was only observed in MOS55 An. gambiae cells. In vivo, high levels of GFP were observed in An. gambiae mosquitoes. Intermediate levels of GFP were observed in the closely related species An. arabiensis. Low levels of GFP were observed in An. stephensi, Ae. albopictus, Ae. aegypti and Cx. tarsalis. These results suggest that AgDNV is a specific gene transduction tool for members of the An. gambiae species complex, and could be potentially developed into a biocontrol agent with minimal off-target effects.

Periplaneta fuliginosa densovirus nonstructural protein NS1 contains an endonuclease activity that is regulated by its phosphorylation.

Periplaneta fuliginosa densovirus (PfDNV) is a single-stranded DNA virus, belonging to Densovirinae subfamily, Parvoviridae family. Parvovirus nonstructural protein 1 (NS1) contains various activities required for parvoviral DNA replication, like endonuclease, helicase and ATPase, which are regulated by serine/threonine phosphorylation. However, for PfDNV, NS1 endonuclease activity has not been determined. Moreover, for densoviruses, whether NS1 is phosphorylated, and if so, phosphorylation pattern and impact on NS1 activities have not been investigated. Here, we demonstrated that PfDNV NS1 possesses endonuclease activity, covalently attaches to 5'-end of nicking site, and includes an active-site tyrosine (Y178). Moreover, using different phosphatases, we uncovered that both serine/threonine and tyrosine phosphorylations are critical for NS1 endonuclease and helicase activities. Further mass-spec and mutational analyses revealed that Y345 is phosphorylated and functions as a critical regulatory site for NS1 activities. This study should foster our understanding of NS1 activities and regulations in PfDNV and other densoviruses.

Densovirus infectious pathway requires clathrin-mediated endocytosis followed by trafficking to the nucleus.

Junonia coenia densovirus (JcDNV) is an ambisense insect parvovirus highly pathogenic for lepidopteran pests at larval stages. The potential use of DNVs as biological control agents prompted us to reinvestigate the host range and cellular mechanisms of infection. In order to understand the early events of infection, we set up a functional infection assay in a cell line of the pest Lymantria dispar to determine the intracellular pathway undertaken by JcDNV to infect a permissive lepidopteran cell line. Our results show that JcDNV particles are rapidly internalized into clathrin-coated vesicles and slowly traffic within early and late endocytic compartments. Blocking late-endocytic trafficking or neutralizing the pH with drugs inhibited infection. During internalization, disruption of the cytoskeleton, and inhibition of phosphatidylinositol 3-kinase blocked the movement of vesicles containing the virus to the nucleus and impaired infection. In summary, our results define for the first time the early endocytic steps required for a productive DNV infection.

[Expression of non-structural protein 1 of Bombyx mori densovirus-3].

We cloned the NS1 gene of BmDNV-3 into the prokaryotic expression vector pET-30a after we amplified the NS1 gene by PCR, and then we transformed the pET-30a-NS1 plasmid into BL21 star to express BmDNV-3 NS1. After we induced NS1 expression by IPTG, we used Western blot analysis to indentify the recombinant protein, the result indicated that the recombinant protein was BmDNV-3 NS1. After purification, we used NS1 to immunize New Zealand white rabbits following standard protocol to harvest anti-NS1 anti serum. On the other hand, we cloned the BmDNV-3 NS1 into pFastBacHTb-eGFP vector, and then transformed the pFastBacHTb-NS1-eGfp into BmDH10BAC to harvest recombinant bacmid genome. We obtained the recombinant virus from the cells, which was transfected by the recombinant bacmid genome using liposomes. We used the virus genome to infect Bombyx mori larvae. We observed the fluorescence in the cells and silkworm larvae at 2 days post infection, and then we used SDS-PAGE and fluorescence image analysis to identify the fusion protein. The result showed that the size of the fusion protein was not consistent with the expected size of NS1-eGFP, indicating the fusion protein was degraded randomly by the intrinsic digestive protease. To further confirm the fusion protein, we used Western blot with an anti-NS1 antibody.

Characterization of the promoter elements and transcription profile of Periplaneta fuliginosa densovirus nonstructural genes.

Periplaneta fuliginosa Densovirus (PfDNV), an autonomous invertebrate parvovirus that infects the cockroach, is unusual in that alternative splicing is involved in the structural gene expression. The expression strategy for nonstructural (NS) genes has yet not been reported. Northern blot analysis of cockroach larvae infected with PfDNV revealed two transcripts for the NS genes, one of 2.6 kb, and the other of 1.9 kb. The two transcripts were shown to begin at a common initiator consensus sequence, CAGT, located in the terminus of ITR. The 1.9 kb transcript was produced by splicing out the ns3 gene from the 2.6 kb transcript. To understand the mechanism of transcriptional regulation of NS genes, the 5'-flanking sequence of ns3 gene (325 bp), which encompasses the region from the 5'-terminus of the viral genome to the initiator ATG codon of the ns3 gene, was cloned and fused to a luciferase reporter gene. The luciferase reporter assay showed that this sequence possessed promoter activity in Sf9, Ld652, Tn368, and S2 cell lines. Subsequent promoter deletion analysis showed that the promoter exhibited TATA-dependent and TATA-independent transcriptional activities. Moreover, we found that the promoter activity of the 325-bp fragment in S2 cells could be enhanced significantly by co-transfection of the nonstructural protein NS1 and that the NS1 binding element, (CAC)(4) repeat, mediated the promoter activity activated by NS1 protein.

Transmission of viruses to mosquito larvae mediated by divalent cations.

The two major groups of pathogenic viruses in mosquitoes are the occluded viruses, represented by baculoviruses and cypoviruses, and the non-occluded viruses, represented by the densoviruses and the iridoviruses. Baculoviruses, densoviruses, and iridoviruses are DNA viruses, while cypoviruses are the major group of RNA viruses reported from mosquitoes. Research on mosquito pathogenic viruses has been limited, in part, due to the inability to effectively transmit them to the larval mosquito host. Recently, there have been tremendous advancements in the ability to transmit mosquito baculoviruses and cypoviruses with the finding that transmission is mediated by divalent cations. Oral transmission of both baculoviruses and cypoviruses to mosquito larvae is enhanced by magnesium and inhibited by calcium ions. The current status of transmission for each of the major groups is reviewed with emphasis on the common role of divalent cations in transmission of the distantly related baculoviruses and cypoviruses.

Biochemical characterization of Periplaneta fuliginosa densovirus non-structural protein NS1.

The non-structural (NS) proteins of parvoviruses are involved in essential steps of the viral life cycle. Various biochemical functions, such as ATP binding, ATPase, site-specific DNA binding and nicking, and helicase activities, have been assigned to the protein NS1. Compared with the non-structural proteins of the vertebrate parvoviruses, the NS proteins of the Densovirinae have not been well characterized. Here, we describe the biochemical properties of NS1 of Periplaneta fuliginosa densovirus (PfDNV). We have expressed and purified NS1 using a baculovirus system and analyzed its enzymatic activity. The purified recombinant NS1 protein possesses ATPase- and ATP- or dATP-dependent helicase activity requiring either Mg(2+) or Mn(2+) as a cofactor. The ATPase activity of NS1 can be efficiently stimulated by single-stranded DNA. The ATPase coupled helicase activity was detected on blunt-ended double-stranded oligonucleotide substrate. Using South-Western and Dot-spot assays, we identified a DNA fragment that is recognized specifically by the recombinant NS1 protein. The fragment consists of (CAC)(4) and is located on the hairpin region of the terminal palindrome. The domain for DNA binding was defined to the amino-terminal region (amino acids 1-250). In addition, we found that NS1 can form oligomeric complexes in vivo and in vitro. Mutagenesis analysis showed that ATP binding is necessary for oligomerization. Based on these results, it seems that PfDNV NS1, a multifunctional protein, plays an important role in viral DNA replication comparable to those of vertebrate parvovirus initiator proteins.

Structure of an insect parvovirus (Junonia coenia Densovirus) determined by cryo-electron microscopy.

Junonia coenia densovirus (JcDNV) belongs to the densovirus genus of the Parvoviridae family and infects the larvae of the Common Buckeye butterfly. Its capsid is icosahedral and consists of viral proteins VP1 (88 kDa), VP2 (58 kDa), VP3 (52 kDa) and VP4 (47 kDa). Each viral protein has the same C terminus but differs in the length of its N-terminal extension. Virus-like-particles (VLPs) assemble spontaneously when the individual viral proteins are expressed by a recombinant baculovirus. We present here the structure of native JcDNV at 8.7A resolution and of the two VLPs formed essentially from VP2 and VP4 at 17 A resolution, as determined by cryo-electron microscopy. The capsid displays a remarkably smooth surface, with only two very small spikes that define a pentagonal plateau on the 5-fold axes. JcDNV is very closely related to Galleria mellonella densovirus (GmDNV), whose structure is known (94% sequence identity with VP4 and 96% similarity). We compare these structures in order to locate the structural changes and mutations that may be involved in the species shift of these densoviruses. A single mutation at the tip of one of the two small spikes is a strong candidate as a species shift determinant. Difference imaging reveals that the 21 disordered amino acid residues at the N terminus of the capsid protein VP4 are located inside the capsid at the 5-fold axis, but the additional 94 amino acid residue extension of VP2 is not visible, suggesting that it is highly disordered. There is strong evidence of DNA ordering associated with the 3-fold axes of the capsid.

Analysis of proteins encoded in the bipartite genome of a new type of parvo-like virus isolated from silkworm - structural protein with DNA polymerase motif.

Bombyx mori densonucleosis virus type 2 (BmDNV-2) is a small, spherical virus containing two complementary single-stranded linear DNA molecules (VD1, VD2). BmDNV-2 is a new type of virus with a unique, yet unspecified replication mechanism which is different from that of parvoviruses (Bando, H., Choi, H., Ito, Y., Nakagaki, M. , Kawase, S., 1992. Structural analysis on the single-stranded genomic DNAs of the virus newly isolated from silkworm: the DNA molecules share a common terminal sequence, Arch. Virol. 124, 187-193; Bando, H., Hayakawa, T., Asano, S., Sahara, K., Nakagaki, M. , Iizuka, T., 1995. Analysis of the genetic information of a DNA segment of a new virus from silkworm, Arch. Virol., 140, 1147-1155; Hayakawa, T., Asano, S., Sahara, K., Iizuka, T., Bando, H., 1997. Detection of replicative intermediate with closed terminus of Bombyx densonucleosis virus. Arch. Virol. 142, 1-7). Recent analyses on the genomic information of BmDNV-2 identified open reading frames which code for three tentative nonstructural proteins and four (VP1 to 4) of the six known structural proteins (Bando, H., Hayakawa, T., Asano, S., Sahara, K., Nakagaki, M., Iizuka, T., 1995. Analysis of the genetic information of a DNA segment of a new virus from silkworm, Arch. Virol., 140, 1147-1155; Nakagaki et al., in preparation). In this report we demonstrate that the two largest ORFs, VD1-ORF1 and VD2-ORF1, code for the two remaining structural proteins. In addition, computer-assisted analysis revealed that the structural protein encoded in VD1-ORF1 contains sequences conserved among various DNA polymerases, and showed an evolutionary relationship with the DNA polymerases involved in protein-primed replication.

Packaging of AeDNV-GFP transducing virus by expression of densovirus structural proteins from a sindbis virus expression system.

Genetic recombination resulting in the production of wild-type infectious virus is an obstacle in the current system for producing densovirus transducing particles. In order to eliminate this problem, a double subgenomic Sindbis virus (TE/3'2J/VP) was engineered that expresses the structural proteins (VPs) of Aedes densonucleosis virus (AeDNV) from the second subgenomic promoter. Expression of AeDNV VPs from TE/3'2J/VP was confirmed by Northern analysis of RNA from infected C6/36 (Aedes albopictus) cells and by indirect immunofluorescence in infected C6/36 cells and BHK-21 cells. TE/3'2J/VP was used to infect C6/36 cells transfected with p7NS1-GFP, a plasmid expressing the nonstructural genes of AeDNV and green fluorescent protein (GFP) as a reporter gene. This infection resulted in the production of AeDNV-GFP transducing virus, which is infectious to C6/36 cells and Aedes aegypti larvae, as determined by GFP expression. The TE/3'2J/VP packaging system produced titers of transducing virus comparable to those produced by the standard two-plasmid method. The possibility of recombination resulting in wild-type infectious virus in transducing densovirus stocks was eliminated by employing an RNA virus expression system to supply AeDNV structural proteins.

A titration procedure of the Junonia coenia densovirus and quantitation of transfection by its cloned genomic DNA in four lepidopteran cell lines.

A sensitive and reproducible tissue culture biossay method was developed based on indirect immunofluorescence to titrate virus suspensions of the Junonia coenia densovirus (JcDNV) and to quantify transfections by its cloned genomic DNA. Four lepidopteran cell lines, the SPC-SL 52 from Spodoptera littoralis, the SPC-PL 40 and the SPC-PL 65 cells derived from Spodoptera litura ovaries and hemocytes, respectively, and the SC-LD 135 from Lymantria dispar were compared for their efficiency to support viral replication. The viral titres expressed as TCID50/ml averaged 10(5) for SPC-SL 52, SPC-PL 40 and SC-LD 135 cells, but were above 10(7) for SPC-PL 65 cells. Even with this most sensitive cell line, the rate of infected cells did not exceed 75% and decreased progressively by serial subcultures. Two transfection protocols were used to compare the sensitivity of the same four cell lines to a recombinant plasmid encompassing an infectious sequence of JcDNV genome. SPC-SL 52 cells were found to be the most sensitive, and the lipofection method resulted in about a 5-fold increase compared to the calcium phosphate precipitation protocol. The rescued virions proved to be infectious and the restriction profiles of their DNA were identical to that of wild type virions.