Intracellular Localization of Blattella germanica Densovirus (BgDV1) Capsid Proteins.

Densovirus genome replication and capsid assembly take place in the nucleus of the infected cells. However, the mechanisms underlying such processes as the delivery of virus proteins to the nucleus and the export of progeny virus from the nucleus remain elusive. It is evident that nuclear transport signals should be involved in these processes. We performed an in silico search for the putative nuclear localization signal (NLS) and nuclear export signal (NES) motifs in the capsid proteins of the Blattella germanica Densovirus 1 (BgDV1) densovirus. A high probability NLS motif was found in the common C-terminal of capsid proteins together with a NES motif in the unique N-terminal of VP2. We also performed a global search for the nuclear traffic signals in the densoviruses belonging to five Densovirinae genera, which revealed high diversity in the patterns of NLSs and NESs. Using a heterologous system, the HeLa mammalian cell line expressing GFP-fused BgDV1 capsid proteins, we demonstrated that both signals are functionally active. We suggest that the NLS shared by all three BgDV1 capsid proteins drives the trafficking of the newly-synthesized proteins into the nucleus, while the NES may play a role in the export of the newly-assembled BgDV1 particles into the cytoplasm through nuclear pore complexes.

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.

Four amino acids of an insect densovirus capsid determine midgut tropism and virulence.

Densoviruses are insect parvoviruses that are orally infectious for Lepidoptera. To assess the mechanisms underlying their specificity and their virulence, we investigated the role of eight candidate residues in the densovirus capsid. We showed that the substitutions of four amino acids were associated with decreased virulence due to a decreased ability to cross the host midgut epithelium, without an effect on viral replication in other tissues.

Structure of Penaeus stylirostris densovirus, a shrimp pathogen.

Penaeus stylirostris densovirus (PstDNV), a pathogen of penaeid shrimp, causes significant damage to farmed and wild shrimp populations. In contrast to other parvoviruses, PstDNV probably has only one type of capsid protein that lacks the phospholipase A2 activity that has been implicated as a requirement during parvoviral host cell infection. The structure of recombinant virus-like particles, composed of 60 copies of the 37.5-kDa coat protein, the smallest parvoviral capsid protein reported thus far, was determined to 2.5-Å resolution by X-ray crystallography. The structure represents the first near-atomic resolution structure within the genus Brevidensovirus. The capsid protein has a ß-barrel "jelly roll" motif similar to that found in many icosahedral viruses, including other parvoviruses. The N-terminal portion of the PstDNV coat protein adopts a "domain-swapped" conformation relative to its twofold-related neighbor similar to the insect parvovirus Galleria mellonella densovirus (GmDNV) but in stark contrast to vertebrate parvoviruses. However, most of the surface loops have little structural resemblance to any of the known parvoviral capsid proteins.

The nuclear localization signal of the NS1 protein is essential for Periplaneta fuliginosa densovirus infection.

The regulatory protein NS1 is a key molecule in life cycle of Periplaneta fuliginosa densovirus (PfDNV). When we ectopically expressed the PfDNV NS1 protein in non-P. fuliginosa insect cells, the NS1 protein could not enter the nucleus and remained in the cytosol. However, the NS1 was localized to both the cytosol and nucleus of cockroach hemocyte cells. So we investigated the abilities of the potential nuclear localization signal (NLS) of P. fuliginosa Densovirus non-structural protein 1 (NS1) to translocate NS1 and a carrier protein to the nucleus following transfection into insect cells. Possible nuclear localization sequences were chosen from the NS1 on the basis of the presence of basic residues, which is a common theme in most of the previously identified targeting peptides. Nuclear localization activity was found within the residues 252-257 (RRRRRR) of the NS1, while replacement of a single arginine in this region with glycine abolished it. The targeting activity was enhanced with the arginine residues added.

[Bioinformatics analysis of mosquito densovirus nostructure protein NS1].

OBJECTIVE: To analyze and predict the structure and function of mosquito densovirus (MDV) nostructual protein1 (NS1). METHODS: Using different bioinformatics software, the EXPASY pmtparam tool, ClustalX1.83, Bioedit, MEGA3.1, ScanProsite, and Motifscan, respectively to comparatively analyze and predict the physic-chemical parameters, homology, evolutionary relation, secondary structure and main functional motifs of NS1. RESULTS: MDV NS1 protein was a unstable hydrophilic protein and the amino acid sequence was highly conserved which had a relatively closer evolutionary distance with infectious hypodermal and hematopoietic necrosis virus (IHHNV). MDV NS1 has a specific domain of superfamily 3 helicase of small DNA viruses. This domain contains the NTP-binding region with a metal ion-dependent ATPase activity. A virus replication roller rolling-circle replication(RCR) initiation domain was found near the N terminal of this protein. This protien has the biological function of single stranded incision enzyme. CONCLUSION: The bioinformatics prediction results suggest that MDV NS1 protein plays a key role in viral replication, packaging, and the other stages of viral life.

Structure comparisons of Aedes albopictus densovirus with other parvoviruses.

Parvoviridae is a family of the smallest viruses known with a wide variety of hosts. The capsid structure of the Aedes albopictus C6/36 cell densovirus (C6/36 DNV) at 1.2-nm resolution was obtained by electron cryomicroscopy (cryoEM) and three-dimensional (3D) image reconstruction. Structure comparisons between the C6/36 DNV and other parvoviruses reveal that the degree of structural similarity between C6/36 DNV and the human parvovirus B19 is higher than that between C6/36 DNV and other insect parvoviruses. The amino acid sequence comparisons of structural and non-structural proteins also reveal higher levels of similarity between C6/36 DNV and parvovirus B19 than those between C6/36 DNV and other parvoviruses. These findings indicate that C6/36 DNV is closely related to the human virus B19, and the former might evolve from the human species other than from other insect viruses.

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.

Three-dimensional structure determination of capsid of Aedes albopictus C6/36 cell densovirus.

The three-dimensional structure of capsid of Aedes albopictus C6/36 densovirus was determined to 14-A resolution by electron cryomicroscopy and computer reconstruction. The triangulation number of the capsid is 1. There are 12 holes in each triangular face and a spike on each 5-fold vertex. The validity of the capsid and nucleic acid densities in the reconstructions was discussed.

A new virus infecting Myzus persicae has a genome organization similar to the species of the genus Densovirus.

The genomic sequence of a new icosahedral DNA virus infecting Myzus persicae has been determined. Analysis of 5499 nt of the viral genome revealed five open reading frames (ORFs) evenly distributed in the 5' half of both DNA strands. Three ORFs (ORF1-3) share the same strand, while two other ORFs (ORF4 and ORF5) are detected in the complementary sequence. The overall genomic organization is similar to that of species from the genus DENSOVIRUS: ORFs 1-3 most likely encode the non-structural proteins, since their putative products contain conserved replication motifs, NTP-binding domains and helicase domains similar to those found in the NS-1 protein of parvoviruses. The deduced amino acid sequences from ORFs 4 and 5 show sequence similarities with the structural proteins of the members of the genus DENSOVIRUS: These data indicate that this virus is a new species of the genus Densovirus in the family PARVOVIRIDAE: The virus was tentatively named Myzus persicae densovirus.

Biochemical characterization of Junonia coenia densovirus nonstructural protein NS-1.

Junonia coenia densovirus (JcDNV) is an autonomous parvovirus that infects the larvae of the common buckeye butterfly, Junonia coenia. Unlike vertebrate parvoviruses, the genes encoding the structural protein and nonstructural (NS) proteins of JcDNV are in opposite orientations; thus, each strand contains a sense and antisense open reading frame (ORF). The promoter at map position 93 controls expression of NS ORFs 2, 3, and 4, which encode three NS proteins, NS-1, NS-2, and NS-3. These proteins are likely to be involved in viral DNA replication, among other functions. In contrast to the nonstructural proteins of the vertebrate parvoviruses, the NS proteins of the Densovirinae have not been characterized. Here, we describe biochemical properties of the NS-1 protein of JcDNV. The NS-1 ORF was cloned in frame with the Escherichia coli malE gene, which encodes the bacterial maltose binding protein (MBP). Using electrophoretic mobility shift and DNase I protection assays, we identified the region of the JcDNV terminal sequence that is recognized specifically by the MBP-NS-1 fusion protein. The site consists of (GAC)4 and is located on the A-A' region of the terminal palindrome. In addition, the MBP-NS-1 fusion protein catalyzes the cleavage of single-stranded DNA (ssDNA) substrates derived from the JcDNV putative origin of replication, primarily at two sites in the motif 5'-G*TAT*TG-3'. One cleavage site is between the thymidine dinucleotide at positions 92 and 93 and the other site corresponds to thymidine at nucleotide 95; both sites are on the complementary strand of the sequence assigned GenBank accession number A12984. Cleavage of ssDNA is dependent on the presence of a divalent metal cofactor but does not require nucleoside triphosphate hydrolysis. Parvovirus NS proteins contain the phylogenically conserved Walker A- and B-site ATPase motifs. These sites in JcDNV NS-1 diverge from the consensus, yet despite these atypical motifs our analyses support that MBP-NS-1 has ATP-dependent helicase activity. These results indicate that JcDNV NS-1 possesses activities common to the superfamily of rolling-circle replication initiator proteins in general and the parvovirus replication proteins in particular, and they provide a basis for comparative analyses of the structure and function relationships among the parvovirus NS-1 equivalents.

A new densovirus isolated from the mosquito Culex pipiens (Diptera: culicidae).

We have isolated from a laboratory strain of Culex pipiens in which abnormal larval mortality occurred a small icosahedral nonenveloped DNA virus sharing the main biological and biophysical properties of densoviruses (DNVs). Unlike DNVs isolated previously from Aedes species, i.e. the AaeDNV and the AalDNV (Afanasiev, B.N., Galyov, E. E., Buchatsky, L.P., Kozlov, Y.V., 1991. Nucleotide sequence and genomic organization of Aedes densonucleosis virus. Virology 185, 323-336; Boublik, Y., Jousset, F.-X., Bergoin, M., 1994. Complete nucleotide sequence and genomic organization of the Aedes albopictus Parvovirus (AaPV) pathogenic for Aedes aegypti larvae. Virology 200, 752-763), this mosquito DNV named CpDNV possesses a genome of 6 kb separately encapsidated in stoichiometric proportion as 'plus' and 'minus' strands. The lack of sequence homology between the CpDNV and the AalDNV genome and of antigenic cross-reactivity between their capsid polypeptides indicates that these two mosquito DNVs are phylogenetically distant. In contrast, the CpDNV appears to be related to the Junonia coenia densovirus (JcDNV) at both serological and genomic levels.

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.

The structure of an insect parvovirus (Galleria mellonella densovirus) at 3.7 A resolution.

BACKGROUND: Parvoviruses infect vertebrates, insects and crustaceans. Many arthropod parvoviruses (densoviruses) are highly pathogenic and kill approximately 90% of the host larvae within days, making them potentially effective as selective pesticides. Improved understanding of densoviral structure and function is therefore desirable. There are four different initiation sites for translation of the densovirus capsid protein mRNA, giving rise to the viral proteins VP1 to VP4. Sixty copies of the common, C-terminal domain make up the ordered part of the icosahedral capsid. RESULTS: The Galleria mellonella densovirus (GMDNV) capsid protein consists of a core beta-barrel motif, similar to that found in many other viral capsid proteins. The structure most closely resembles that of the vertebrate parvoviruses, but it has diverged beyond recognition in many of the long loop regions that constitute the surface features and intersubunit contacts. The N termini of twofold-related subunits have swapped their positions relative to those of the vertebrate parvoviruses. Unlike in the vertebrate parvoviruses, in GmDNV there is no continuous electron density in the channels running along the fivefold axes of the virus. Electron density corresponding to some of the single-stranded DNA genome is visible in the crystal structure, but it is not as well defined as in the vertebrate parvoviruses. CONCLUSIONS: The sequence of the glycine-rich motif, which occupies each of the channels along the fivefold axes in vertebrate viruses, is conserved between mammalian and insect parvoviruses. This motif may serve to externalize the N-terminal region of the single VP1 subunit per particle. The domain swapping of the N termini between insect and vertebrate parvoviruses may have the effect of increasing capsid stability in GmDNV.

Evidence for two small viruses persistently infecting established cell lines of Phthorimaea operculella, deriving from embryos of the potato tuber moth.

Two small viruses were isolated from established cell lines of P. operculella deriving from embryos. The first one probably related to the Nodaviridae family, is a 30 nm in diameter icosahedral virus, with a bisegmented RNA genome and a single polypeptide of 39 kilodaltons. The second one related to the Parvoviridae family, is a 25 nm in diameter icosahedral virus with a DNA genome and a capsid constituted of 4 polypeptides of respectively, 90,000; 64,000; 56,000 and 43,500 daltons. The two viruses probably chronically infect the cell lines and may be consider latent viruses.

A densovirus newly isolated from the smoky-brown cockroach Periplaneta fuliginosa.

We purified a causing agent of fetal disease for smoky-brown cockroach Periplaneta fuliginosa, which was designated as "cockroach small spherical virus (CSSV)". Purified virus particles had a diameter of 22 +/- 0.6 nm and contained DNA as a single-stranded form. However, the extraction of DNA under condition of appropriate high salt and elevated temperature yielded a double-stranded DNA with a size of 5,500 nucleotides. These results were quite similar to those of other densoviruses (DNVs). The CSSV had five structural proteins (VP1: 52 KDa, VP2: 56 KDa, VP3: 79 KDa, VP4: 82 KDa, and VP5: 105 KDa). The SDS-PAGE profile of these proteins was quite different from that of the cockroach DNV previously reported and was rather similar to that of Bombyx mori (Bm) DNV-1. An immunochemical study, however, demonstrated that there was no immunological relationship between the CSSV and the Bm DNV-1. These data suggest that the CSSV is a new member of DNV.