Background
The subfamily of
Densovirinae within the family
Parvoviridae is a group of small (18–26 nm diameter), non-enveloped, icosahedral viruses containing a linear single-stranded DNA genome ranging between 4 and 6 kb with characteristic terminal hairpins [
1‐
3]. Members of this subfamily typically produce “cellular dense nucleosis” pathogenesis in their hosts, hence, they are commonly termed densoviruses (DVs) [
4‐
8]. Since the first identification of a densovirus in the greater wax moth
Galleria mellonella [
9], DVs have been isolated from many arthropods, including species from six insect orders (Lepidoptera, Diptera, Orthoptera, Dictyoptera, Odonata and Hemiptera) and decapod crustaceans (shrimps and crabs) [
10‐
12].
To date, many DVs have been identified and sequenced. Unlike vertebrate parvoviruses, which all exhibit a monosense organization of their genome with nonstructural protein (NS) and structural protein (VP) open reading frames (ORFs) located on the same strand, arthropod DVs possess two types of genomes: monosense and ambisense [
13‐
18]. Previously, the taxonomy of DVs was ambiguous, which was based on the organization of coding sequences, as well as genome size, terminal hairpin structure, gene expression strategy and host range [
19]. Under the proposal of the International Committee on Taxonomy of Viruses (ICTV), Cotmore et al. [
2] reconstructed the taxonomy of the family
Parvoviridae in which DVs were classified into five distinct genera:
Ambidensovirus,
Brevidensovirus, Iteradensovirus, Hepandensovirus and
Penstyldensovirus according to phylogenetic analysis and sequence homology.
DVs are highly pathogenic viruses to their hosts, and have been documented as being transmitted both horizontally and vertically [
7,
9,
16]. Traditionally, these properties have captured the interest of many researchers investigating the potential application of DVs as biopesticides for biological control of insect pests or vectors for transgenic insects [
20‐
26]. However, we previously reported a novel DV displaying a mutualistic interaction with its host (
Helicoverpa armigera), and named this virus HaDV2 (previously named HaDNV-1) to distinguish it from the HaDV1 reported by El-Far et al. [
27‐
29]. In this current study, we report the genome organization, transcription and expression strategies of the virus HaDV2.
Discussion
DVs are a group of viruses usually associated with causing high pathogenicity to their hosts [
7,
9,
12]. However, we previously reported a novel DV (HaDV2) which was found to be beneficial to its host by increasing larval and pupal developmental rate, fertility, adult female lifespan and enhancing host resistance to both a baculovirus and low doses of the Bt toxin [
28,
29]. This suggested a virus with quite different characteristics to the other previously described members within subfamily Densovirinae. In this current study, we determined the molecular biology of the HaDV2 virus, namely through examining its genome structure and ORF transcription and expression strategy. Based on our results, HaDV2 was a novel member of genus
Iteradensovirus, with new features differing from other members from this genus, such as an ITR of 101 nt at both termini, a single 90 nts hairpin structure at the 3′ end and the first ORF encoding NS2 protein [
17,
28,
35‐
40].
Phylogenetic analysis using both nucleotide and amino acid sequences showed that HaDV2 was clustered within the genus
Iteradensovirus. The sequence identities of the viral DNA and the amino acid identities for VP, NS1 and NS2 ORFs among members of the genus
Iteradensovirus exceed 58, 71, 35 and 28%, respectively. However, the sequence identities between HaDV2 and the current members of the genus
Iteradensovirus are no more than 44, 36, 28 and 19%, respectively. Thus, although the HaDV2 was clustered with
Iteradensovirus, it differs considerably from the other iteradensoviruses and appears to have a different function as described previously [
29].
Although phylogenetic analysis indicated that HaDV2 was clustered with members of the genus
Iteradensovirus, the NS1 and NS2 proteins of the HaDV2 are smaller than those of other
Iteradensovirus (more than 753 and 451 amino acids, respectively) [
17,
28,
35‐
40]. Are they functionally expressed as predicted? We used Western Blot analysis of transfected LD652 cells using anti-NS1 and anti-NS2 to show that the NS1 and NS2 proteins were 78 kDa and 48 kDa, respectively, consistent with the predicted size of the protein. NS proteins are a pivotal factor for viral transcription and replication as well as pathogenicity. The replication of DVs occured in the nucleus of their hosts [
19]. Therefore, the NS proteins of DVs should be located in the nucleus by nuclear localization signal (NLS) as reported by Yu et al. [
40]. To further investigate whether the NS proteins of the HaDV2 localized within the nucleus of their hosts (as those of other DVs), NS1 and NS2 proteins were expressed in LD652 cells using the recombinant plasmid NS1-GFP and NS2-GFP. The result indicated that the NS1 and NS2 proteins were completely located in the nucleus, suggesting that they possess a common function and could possibly play a role in the novel interactions between HaDV2 and its host. The experiments with the NS proteins were carried out by transient expression in LD652 cells, which were not the virus’s original host. It is acknowledged that this expression may not reflect the real role of HaDV2-NS promoter and how it works in the natural host.
Transcriptional patterns are diverse among the DVs. For example, JcDV,
Galleria mellonella densovirus (GmDV) and
Mythimna loreyi densovirus (MlDV) all have one transcript for the VP gene and two transcripts for the NS genes (the larger one for NS1 and the smaller one for NS2), in which the ORFs of NS1 and NS2 share a common TTS [
15,
30,
41]. Meanwhile, the transcripts of CpDV,
Periplaneta fuliginosa densovirus (PfDV) and
Myzus persicae nicotianae densovirus (MpnDV) arise from alternative splicing [
13,
42,
43]. The first ORFs of all known iteradensoviruses encode NS1 protein and the ORFs of NS2 are completely included in the ORFs of NS1 [
40]. However, the first ORF of HaDV2 encodes NS2, which may impact gene expression of NS2 compared to NS1. In addition, the NS1 and NS2 of other iteradensoviruses were translated from different transcripts and the TIS of NS1 was found to start 2–26 nt upstream of the start codon [
40]. Unexpectedly, our results suggested that the NS1 and NS2 of HaDV2 translated from the same transcript which started 63 nt upstream of the start codon of NS2. Surprisingly, although we provide evidence of the activity of the NS promoter, we failed to find the TATA-box upstream of the TIS of NS. Two TATA-box like sequences were located at nts 313 and 335 upstream of the start codon of NS1 and NS2; suggesting HaDV2, maybe like brevidensoviruses, has overlapping NS gene promoters responsible for different transcript starts and dictating the relative transcription rates of these transcripts. However, one of the two transcripts was in great excess, making it difficult to detect both transcripts by RACE. Like other DVs, the VP transcripts of HaDV2 had short-untranslated regions, located at 5 nts upstream of the start codon of VP.
Acknowledgements
We would like to thank Dr. Jie Wang (Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao, P.R. China) and Dr. Peng Xu (Central China Normal University, Wuhan, P.R. China) for providing suggestions and repairing figures.