The Structures and Functions of Parvovirus Capsids and Missing Pieces: the Viral DNA and Its Packaging, Asymmetrical Features, Nonprotein Components, and Receptor or Antibody Binding and Interactions.

Parvoviruses are among the smallest and superficially simplest animal viruses, infecting a broad range of hosts, including humans, and causing some deadly infections. In 1990, the first atomic structure of the canine parvovirus (CPV) capsid revealed a 26-nm-diameter T=1 particle made up of two or three versions of a single protein, and packaging about 5,100 nucleotides of single-stranded DNA. Our structural and functional understanding of parvovirus capsids and their ligands has increased as imaging and molecular techniques have advanced, and capsid structures for most groups within the Parvoviridae family have now been determined. Despite those advances, significant questions remain unanswered about the functioning of those viral capsids and their roles in release, transmission, or cellular infection. In addition, the interactions of capsids with host receptors, antibodies, or other biological components are also still incompletely understood. The parvovirus capsid's apparent simplicity likely conceals important functions carried out by small, transient, or asymmetric structures. Here, we highlight some remaining open questions that may need to be answered to provide a more thorough understanding of how these viruses carry out their various functions. The many different members of the family Parvoviridae share a capsid architecture, and while many functions are likely similar, others may differ in detail. Many of those parvoviruses have not been experimentally examined in detail (or at all in some cases), so we, therefore, focus this minireview on the widely studied protoparvoviruses, as well as the most thoroughly investigated examples of adeno-associated viruses.

Viral Phrenology.

We introduce Viral Phrenology, a new scheme for understanding the genomic composition of spherical viruses based on the locations of their structural protrusions. We used icosahedral point arrays to classify 135 distinct viral capsids collected from over 600 capsids available in the VIPERdb. Using gauge points of point arrays, we found 149 unique structural protrusions. We then show how to use the locations of these protrusions to determine the genetic composition of the virus. We then show that ssDNA, dsDNA, dsRNA and ssRNA viruses use different arrangements for distributing their protrusions. We also found that Triangulation number is also partially dependent on the structural protrusions. This analysis begins to tie together Baltimore Classification and Triangulation number using point arrays.

ICTV Virus Taxonomy Profile: Parvoviridae.

Members of the family Parvoviridae are small, resilient, non-enveloped viruses with linear, single-stranded DNA genomes of 4-6 kb. Viruses in two subfamilies, the Parvovirinae and Densovirinae, are distinguished primarily by their respective ability to infect vertebrates (including humans) versus invertebrates. Being genetically limited, most parvoviruses require actively dividing host cells and are host and/or tissue specific. Some cause diseases, which range from subclinical to lethal. A few require co-infection with helper viruses from other families. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Parvoviridae, which is available at www.ictv.global/report/parvoviridae.

Atomic Resolution Structures of Human Bufaviruses Determined by Cryo-Electron Microscopy.

Bufavirus strain 1 (BuV1), a member of the Protoparvovirus genus of the Parvoviridae, was first isolated from fecal samples of children with acute diarrhea in Burkina Faso. Since this initial discovery, BuVs have been isolated in several countries, including Finland, the Netherlands, and Bhutan, in pediatric patients exhibiting similar symptoms. Towards their characterization, the structures of virus-like particles of BuV1, BuV2, and BuV3, the current known genotypes, have been determined by cryo-electron microscopy and image reconstruction to 2.84, 3.79, and 3.25 Å, respectively. The BuVs, 65-73% identical in amino acid sequence, conserve the major viral protein, VP2, structure and general capsid surface features of parvoviruses. These include a core ß-barrel (ßB-ßI), α-helix A, and large surface loops inserted between these elements in VP2. The capsid contains depressions at the icosahedral 2-fold and around the 5-fold axes, and has three separated protrusions surrounding the 3-fold axes. Structure comparison among the BuVs and to available parvovirus structures revealed capsid surface variations and capsid 3-fold protrusions that depart from the single pinwheel arrangement of the animal protoparvoviruses. These structures provide a platform to begin the molecular characterization of these potentially pathogenic viruses.

Human bocavirus capsid structure: insights into the structural repertoire of the parvoviridae.

Human bocavirus (HBoV) was recently discovered and classified in the Bocavirus genus (family Parvoviridae, subfamily Parvovirinae) on the basis of genomic similarity to bovine parvovirus and canine minute virus. HBoV has been implicated in respiratory tract infections and gastroenteric disease in children worldwide, yet despite numerous epidemiological reports, there has been limited biochemical and molecular characterization of the virus. Reported here is the three-dimensional structure of recombinant HBoV capsids, assembled from viral protein 2 (VP2), at 7.9-A resolution as determined by cryo-electron microscopy and image reconstruction. A pseudo-atomic model of HBoV VP2 was derived from sequence alignment analysis and knowledge of the crystal structure of human parvovirus B19 (genus Erythrovirus). Comparison of the HBoV capsid structure to that of parvoviruses from five separate genera demonstrates strong conservation of a beta-barrel core domain and an alpha-helix, from which emanate several loops of various lengths and conformations, yielding a unique surface topology that differs from the three already described for this family. The highly conserved core is consistent with observations for other single-stranded DNA viruses, and variable surface loops have been shown to confer the host-specific tropism and the diverse antigenic properties of this family.

Structure of adeno-associated virus type 4.

Adeno-associated virus (AAV) is a member of the Parvoviridae, belonging to the Dependovirus genus. Currently, several distinct isolates of AAV are in development for use in human gene therapy applications due to their ability to transduce different target cells. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. The structure of AAV type 4 (AAV4), one of the most antigenically distinct serotypes, was determined to 13-A resolution by cryo-electron microscopy and image reconstruction. A pseudoatomic model was built for the AAV4 capsid by use of a structure-based sequence alignment of its major capsid protein, VP3, with that of AAV2, to which AAV4 is 58% identical and constrained by its reconstructed density envelope. The model showed variations in the surface loops that may account for the differences in receptor binding and antigenicity between AAV2 and AAV4. The AAV4 capsid surface topology also shows an unpredicted structural similarity to that of Aleutian mink disease virus and human parvovirus B19, autonomous members of the genus, despite limited sequence homology.

Putative spawner-isolated mortality virus associated with mid-crop mortality syndrome in farmed Penaeus monodon from northern Australia.

Beginning in 1994, farms in northern Australia experienced a higher than normal mortality rate in 12 to 15 g prawns from growout ponds. The farmers named this problem mid-crop mortality syndrome (MCMS). Intramuscular injection of filtered (450 nm), cell-free extracts of moribund prawns from these ponds killed healthy prawns between 5 to 30 d post-injection. A 20 nm virus was visualized by electron microscopy from a 1.4 g ml-1 band recovered from caesium chloride gradients of extracts from the moribund prawns. DNA was extracted from this band, restriction enzyme digested and ligated into pGEM7zf(+) vector. A digoxigenin-labelled polymerase chain reaction (PCR)-generated, gene probe was subsequently prepared by amplifying an inserted sequence (approximately 2 kb) of one selected clone specific for the virus. Specimens of the moribund prawns stained positively by in situ DNA hybridization in endodermal tissues, including the apical ends of hepatopancreatic tubules, the midgut and hindgut caecae, the midgut, and the hindgut folds. In prawns that showed haemocytic enteritis, some haemocytes in the affected midgut showed limited staining. The positively-staining cells showed no cytolysis. In prawns injected with cell-free viral extracts, additional tissues were positive by probe analysis, including strong staining in the male reproductive tract, specifically in the terminal ampoule and the medial vas deferens. Limited staining also occurred in the ovary and in both the stromal matrix and spheroid cells of the lymphoid organ. It was evident that the infection was enteric by natural pathways and systemic by injection. Historical specimens of Penaeus monodon experimentally infected with spawner-isolated mortality virus (SMV) were probe-positive in exactly the same pattern as the naturally and experimental MCMS prawns. Altogether, the evidence suggested that the MCMS agent was a parvo-like virus very similar or identical to SMV.

Characterization of hepatopancreatic parvo-like virus, a second unusual parvovirus pathogenic for penaeid shrimps.

The hepatopancreatic parvo-like virus (HPV) of penaeid shrimp was extracted from infected shrimp tissues, purified and subsequently characterized. The viral particles, icosahedral in shape, are 22 nm in diameter and possess a buoyant density of 1.41 g/ml. They contain ssDNA, of approximately 5 kb in size which encodes a single polypeptide of 54 kDa. On the basis of its general characteristics this pathogenic agent belongs to the Parvoviridae family, but because of two unusual characteristics (capsid protein formed with a single polypeptide and genome structure more closely related to the autonomous parvoviruses rather than the densoviruses), it seems to constitute a novel group in the Parvoviridae family.

Topographical analysis of canine parvovirus virions and recombinant VP2 capsids.

The distribution of epitopes defined by monoclonal antibodies (MAbs) on the surface of canine parvovirus (CPV) virions and recombinant VP2-capsids was established using immunoelectron microscopy. A correlation appeared to exist between the linear position, neutralizing activity and immunogold staining. Both viral capsids and recombinant capsids gave similar patterns of immunostaining. The neutralizing MAbs that recognized epitopes not previously identified by Pepscan or immunoblotting gave a clear staining. However, MAbs 3C9 and 3C10, identified by Pepscan and immunoblotting as recognizing linear epitopes, did not show any labelling (3C9) or only scattered labelling (3C10). MAb 3C9 recognizes an N-terminal domain of VP2. MAb 4AG6, which recognizes the same linear epitope as 3C10, did not bind to the capsids, indicating a different orientation. An immunofluorescence assay was performed to supplement the B cell epitope characterization. In contrast to other MAbs that gave nuclear and cytoplasmic staining, MAb 3C9 gave a preferential nuclear staining. Based on these results, it is hypothesized that the N terminus of VP2 is barely, or not at all, exposed on the surface of the native virions, but becomes accessible after some virion steric change (e.g. after attachment to the cell receptor).

A parvo-like virus persistently infecting a C6/36 clone of Aedes albopictus mosquito cell line and pathogenic for Aedes aegypti larvae.

We have isolated and partially characterized from an apparently healthy C6/36 subclone of Aedes albopictus cell line a small icosahedral non-enveloped DNA virus, designated AaPV. This virus proved to be highly pathogenic for Aedes aegypti neonate larvae. Viral infection persisted for over 4 years in the cell culture without any cytopathic effect. Attempts to infect suckling mice, Drosophila melanogaster adults and Spodoptera littoralis larvae with AaPV were unsuccessful. Similarly, the AaPV failed to replicate in vertebrate and Drosophila cell lines. Virions, about 22 nm in diameter, had a buoyant density of 1.43 g/cm3 and contained three capsid polypeptides with molecular weights of 53, 41 and 40 kDa. A preliminary study of the viral genome indicated the presence of single-stranded DNA. By its biophysical and biochemical properties, this virus appears to be related to the genus Densovirus within the family Parvoviridae, but lacks serological relationships with the other members of this genus.

Structure determination of feline panleukopenia virus empty particles.

Various crystal forms of the single-stranded DNA, feline panleukopenia virus (FPV), a parvovirus, have been grown of both full virions and empty particles. The structure of empty particles crystallized in an orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 380.1 A, b = 379.3 A, and c = 350.9 A, has been determined to 3.3 A resolution. The data were collected using oscillation photography with synchrotron radiation. The orientations of the empty capsids in the unit cell were determined using a self-rotation function and their positions were obtained with an R-factor search using canine parvovirus (CPV) as a model. Phases were then calculated, based on the CPV model, to 6.0 A resolution and gradually extended to 3.3 A resolution by molecular replacement electron density averaging. The resultant electron density was readily interpreted in terms of the known amino acid sequence. The structure is contrasted to that of CPV in terms of host range, neutralization by antibodies, hemagglutination properties, and binding of genomic DNA.

Infectious entry pathway for canine parvovirus.

We have investigated whether canine parvovirus (CPV) infection involves a low pH-dependent entry pathway. The effects of two lysosomotropic bases, NH4Cl and chloroquine, on CPV entry were studied by immunofluorescence and ultrastructural and biochemical methods. In the presence of these reagents, input virions appear to accumulate in large vacuoles. Ultrastructural studies indicated that uptake of virions takes place predominantly in small uncoated vesicles that appear to fuse with larger vesicles. In the presence of NH4Cl, virions accumulate in the latter structures and their uncoating appears to be prevented. Viral DNA as well as antigen synthesis were found to be significantly inhibited in the presence of these reagents. In addition, inhibition of viral DNA and antigen synthesis appeared to be most extensive when NH4Cl was present from 30 min preinfection, whereas no significant inhibition was observed when the cells were treated after 2 hr postinfection. Thus, the results indicate that CPV requires exposure to low pH in an endosomal compartment to initiate a productive infection.

Fatal disease in nursing puppies associated with minute virus of canines.

Thirteen cases of a previously undescribed parvoviral infection affecting puppies ranging in age from 5 to 21 days is described. The cases were originally thought to represent an unusual pathologic manifestation of canine parvovirus-2 (CPV-2) infection. However, failure to confirm CPV-2 infection in any of the cases suggested a different parvovirus was involved. Minute virus of canines (MVC) was subsequently isolated from a case by using the Walter Reed Canine Cell Line, the only cell line which will support the growth of MVC. The pathologic and virologic findings for these 13 cases are described in this report.

Preliminary X-ray crystallographic investigation of human parvovirus B19.

Crystals that diffract X rays to at least 8 A resolution have been grown from human B19 parvovirus empty capsids. These particles consist of VP-2 derived from a baculovirus expression system. This is possibly the first time that a self-assembled empty viral capsid, grown in other than normal host cells, has been crystallized. Partial X-ray diffraction data have been collected using synchrotron radiation. The space group is P2(1)3 with a = 362 A. The particle position in the crystal cell is given, at least roughly, from packing considerations.

Nonstructural protein NS2 of parvovirus H-1 is required for efficient viral protein synthesis and virus production in rat cells in vivo and in vitro.

We generated a mutation in the gene for the nonstructural protein NS2 of parvovirus H-1 in which the highly conserved dinucleotide AG at the 3' splice acceptor site of NS2 intron 1 was mutated to CG. The mutation does not change the amino acid sequence for NS1. The splice acceptor (SA) mutant gene was introduced into the H-1 virus (H-1SA) and an infectious clone of LuIII (pLuH1SA). The R2 transcripts encoding NS2 were absent by both Northern blot and primer extension analysis in the LuH1SA or H-1SA virus-infected cells and the NS2 protein was undetectable in the infected cell lysate by immunoprecipitation. These NS2 null mutant viruses were capable of lytic growth in cell lines that were derived from human, hamster, and dog, but they produced lower virus titers than wild-type H-1. The H-1SA virus nonproductively infected Rat2 rat fibroblasts and transformed Rat2 cell lines. Analysis of synchronized infections of rat fibroblasts demonstrated that H-1SA viral duplex replicative form DNA replication was reduced and that single-stranded progeny DNA was deficient compared to wild-type H-1. In addition, H-1SA viral protein synthesis was about 10% of wild-type virus and virions were not detectable in rat fibroblasts. However, H-1SA mRNAs R1 and R3 accumulated to wild-type levels. NS2 was also required for productive infection in newborn rats but not in newborn hamsters. These results indicate that NS2 plays an important role in the regulation of viral protein synthesis in rat cells in vivo and in vitro.

Mapping specific functions in the capsid structure of canine parvovirus and feline panleukopenia virus using infectious plasmid clones.

DNA sequences between 0 and 98.8 genome map units (m.u.) from canine parvovirus (CPV) and feline panleukopenia virus (FPV) were cloned into plasmid vectors to form infectious molecular clones. Those plasmids were transfected into permissive cells and viruses recovered were shown to contain intact genomes, having regenerated the complete viral 5' ends up to 100 m.u. The viruses derived from the plasmids were compared to the original viruses, and shown to be indistinguishable in antigenic type, hemagglutination (HA) type and host range. The plasmid origin of the viruses was shown by preparing recombinant clones between CPV and FPV, and demonstrating the recombinant nature of the resulting viruses by restriction mapping and by sequencing viral DNA across the recombination sites. The sequences of our wild-type isolates CPV-d and FPV-b were completed, revealing 50 nucleotide sequence differences, of which 16 determined coding changes--5 in NS-1,2 in NS-2, and 9 in VP-2 protein. The sequences of the 5' ends (95.3-100 m.u.) of both viruses were also determined. Analysis of recombinant viruses mapped both CPV- and FPV-specific antigenic epitopes, the pH dependence of HA, and sequences affecting canine host range of the viruses within the VP-1 and VP-2 structural protein genes. Most of the specific changes were shown to be either on, or within one amino acid of, the surface of the virus capsid, indicating that the exposed surface of the parvovirus capsid plays an important role in determining a number of virus functions. The specific epitopes were affected by differences in a raised area on the capsid ("threefold spike"), while the pH dependence of HA difference was adjacent to a depression in the surface of the capsid at the twofold axis of symmetry.

Adaptation of the viral haemorrhagic disease virus of rabbits to the DJRK cell strain.

Liver emulsion of rabbits which had died of viral haemorrhagic disease (VHD) was inoculated onto DJRK cell culture. After two passages, specific cytopathic effect was observed. Immunofluorescence was found in the nucleus at the early stage of infection and later also in the cytoplasm. The virus propagated in cell culture at the fifth, tenth and sixteenth passages was found to cause typical VHD. Electron microscopy showed that there were numerous virions in the infected cells. The cultured virus, inactivated with formaldehyde, could elicit haemagglutination inhibition antibodies in the inoculated rabbits and protect them against the challenge of virulent VHD virus.

In vitro propagation of parvovirus B19 in primary foetal liver culture.

The culture of parvovirus B19 in foetal liver tissue has been described recently. We have established the technique in our laboratory and studied parameters affecting the yield of B19 virus. Replication of the virus was detected by radioimmunoassay for B19 antigen and dot blot hybridization assay of B19 DNA, and the virus was localized by immunofluorescence and thin section electron microscopy. B19 DNA and antigen production became detectable at day 2 and reached a maximum at day 5. Virus particles were seen mainly in cell nuclei, but some cytoplasmic membranes were lined with virus particles. The amount of virus produced depended on the age of the foetus and the cell culture and the concentration of erythropoietin and interleukin 3 in the culture medium.