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.

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.

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.

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.

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.

The three-dimensional structure of canine parvovirus and its functional implications.

The three-dimensional atomic structure of a single-stranded DNA virus has been determined. Infectious virions of canine parvovirus contain 60 protein subunits that are predominantly VP-2. The central structural motif of VP-2 has the same topology (an eight-stranded antiparallel beta barrel) as has been found in many other icosahedral viruses but represents only about one-third of the capsid protein. There is a 22 angstrom (A) long protrusion on the threefold axes, a 15 A deep canyon circulating about each of the five cylindrical structures at the fivefold axes, and a 15 A deep depression at the twofold axes. By analogy with rhinoviruses, the canyon may be the site of receptor attachment. Residues related to the antigenic properties of the virus are found on the threefold protrusions. Some of the amino termini of VP-2 run to the exterior in full but not empty virions, which is consistent with the observation that some VP-2 polypeptides in full particles can be cleaved by trypsin. Eleven nucleotides are seen in each of 60 symmetry-related pockets on the interior surface of the capsid and together account for 13 percent of the genome.

Purification and characterization of the infectious hypodermal and haematopoietic necrosis virus of penaeid shrimps.

Infectious hypodermal and haematopoietic necrosis (IHHN) is one of the most important viral diseases of cultured penaeid shrimps and is potentially a limiting factor in the development of farming projects for some species of these shrimps. Although the IHHN agent was recognized early as being viral in origin, attempts to characterize it were inconclusive because of difficulties in obtaining sufficient amounts of purified virions to permit its characterization. Recent improvements of purification procedures have allowed the physicochemical characterization of this virus. Purified IHHNV is a non-enveloped icosahedral particle averaging 22 nm in diameter, exhibiting a mean buoyant density of 1.40 g/ml in CsCl. The genome is a single molecule of ssDNA with an estimated size of 4.1 kb by molecule length measurement in transmission electron microscopy. As determined by SDS-PAGE, the particle contains four polypeptides with Mrs of 74K, 47K, 39K and 37.5K, respectively. From its characteristics, this virus could be a member of the Parvoviridae family.

Characterization of newly isolated rat viruses from asymptomatic laboratory rats.

We surveyed the extent of rat virus (RV) infections in Japan and isolated new RV strains. The new strains were similar to the prototype RV strain in stability, morphology in electron microscopy and structural polypeptides. There were slight differences, however, in hemagglutination activity and the antigenicity.

Immune response to B19 parvovirus and an antibody defect in persistent viral infection.

B19 parvovirus has been shown to persist in some immunocompromised patients, and treatment with specific antibodies can lead to decreased quantities of circulating virus and hematologic improvement. A defective immune response to B19 parvovirus in these patients was shown by comparison of results using a capture RIA and immunoblotting. In normal individuals, examination of paired sera showed that the dominant humoral immune response during early convalescence was to the virus major capsid protein (58 kD) and during late convalescence to the minor capsid species (83 kD). In patients with persistent parvovirus infection, variable titers against intact particles were detected by RIA, but the sera from these patients had minimal or no IgG to capsid proteins determined by Western analysis. Competition experiments suggested that this discrepancy was not explicable on the basis of immune complex formation alone and that these patients may have a qualitative abnormality in antibody binding to virus. In neutralization experiments, in which erythroid colony formation in vitro was used as an assay of parvovirus activity, sera from patients with poor reactivity on immunoblotting were also inadequate in inhibiting viral infectivity. A cellular response to purified B19 parvovirus could not be demonstrated using proliferation assays and PBMC from individuals with serologic evidence of exposure to virus. These results suggest that production of neutralizing antibody to capsid protein plays a major role in limiting parvovirus infection in man.

Infectious anemia caused by a parvovirus-like virus in Georgia broilers.

Pale chicks with necrotic dermatitis, small bursas of Fabricius (BFs), small thymuses, pale bone marrow, and watery blood were suspected of having parvovirus-like virus- (PVLV) associated disease. Histologic lesions included atrophy or hypoplasia of thymuses and BFs, and septic necrotizing clostridial dermatitis and hepatitis. Clostridium perfringens was cultured from skin and liver. A PVLV was isolated in a Marek's disease tumor cell line (MDCC-MSB1) culture and was identified by physicochemical, immunofluorescent, and morphologic features. This isolate was named GA-1 PVLV. Specific-antibody-negative chicks and embryos infected with heat- or chloroform-treated GA-1 PVLV developed anemia at the same rate. Control chicks never were anemic. This is the first isolation of PVLV from clinically ill chickens in the United States and the first report of PVLV-induced anemia in chickens in the Western Hemisphere.

Human parvovirus (HPV/B19) infection with purpura.

A 33-year-old man complained of purpura (petechial hemorrhage) in chelidons, poples, axillae, and bilateral chest in addition to other symptoms such as lumbago, arthralgia, muscular pain, and fever. On the next day of the onset, human parvovirus (HPV/B19) antigen and HPV/B19 DNA were detected in his serum, and twelve days later IgM antibody to HPV/B19 became detectable. This case supports the relationship between purpura and HPV/B19 infection.

Size and antigenic comparisons among the structural proteins of selected autonomous parvoviruses.

The size and antigenic relationships among structural proteins (VPs) of canine parvovirus (CPV), feline parvovirus (FPV), porcine parvovirus (PPV), minute virus of mice (MVM) and bovine parvovirus (BPV) were determined by SDS-PAGE of radiolabelled, purified virus and immunoprecipitated viral proteins. Mature virions of CPV, FPV, PPV and MVM were composed of three VPs designated VP1, VP2 and VP3. The corresponding proteins of each virus were similar in molecular weight [79,000 to 82,500 (VP1), 65,000 to 66,000 (VP2), 62,000 to 63,500 (VP3)]. Additional similarities among VPs were indicated by antigenic relationships which included precipitation of VPs of CPV, FPV and PPV by both homologous antisera and antisera raised to each of the other two viruses and by precipitation of VPs of MVM by cat anti-FPV sera. A non-structural protein identified in lysates of cells infected with FPV and CPV was precipitated by cat anti-FPV and dog anti-CPV sera only. Mature virions of BPV were composed of four VPs [74,500 (VP1), 67,000 (VP2), 60,000 (VP3), 57,500 (VP4)] which were antigenically unrelated to those of the other parvoviruses tested. However, the possibility that swine are sometimes infected with a virus which is antigenically related to BPV was suggested by the finding that sera from conventionally raised swine, irrespective of their serological status for PPV, precipitated VPs of BPV, whereas neither pre-exposure sera nor anti-PPV sera from gnotobiotic pigs did so.

An electron microscopical investigation of faecal small round viruses.

A retrospective study of small round featureless viruses (SRVs) initially identified by negative-staining electron microscopy of stool samples was performed. A variety of technique, including immunoelectron microscopy and caesium chloride gradient centrifugation, was applied in an attempt to classify further these viruses. Over a four-year period, 64 SRV-positive samples were reported (1.8% of the stool samples sent for electron microscopy and 6.2% of the total number of positive samples), of which 53 were available for further study. A significant degree of misclassification was found. Viruses previously identified as SRVs were shown to be astrovirus (n = 14), calicivirus (n = 2), and "Norwalk-like" virus (n = 1). The majority of the 36 remaining samples were identified as parvovirus-like (n = 27) (75%), 14 of which were associated with the presence of adenovirus particles. Enteroviruses (n = 3) and hepatitis A virus (n = 1) were infrequently detected. The remaining viruses (n = 5) could not be adequately classified. Parvovirus may be the predominant SRV associated with acute diarrhoeal disease in childhood.

An eight-year study of the viral agents of acute gastroenteritis in humans: ultrastructural observations and seasonal distribution with a major emphasis on coronavirus-like particles.

During an 8-yr period, 862 stool specimens from patients with gastroenteritis were examined by electron microscopy after negative staining with 2% phosphotungstic acid (pH 6.5). Forty-one percent of the specimens submitted over an 8-yr period were determined to be positive for virus or viruslike particles belonging to one or more of seven morphologically distinct viral groups. Coronavirus-like particles (CVLPs) were present in 69.8% of the positive stool specimens. Membranous profiles containing "complement-type" holes (10 nm in diameter) were identified in some preparations containing CVLPs. The second most prevalent viral agent found in stool specimens was the rotavirus (17% of all positive stools). The incidence of other viruses identified in the survey were as follows: adenovirus 4.5%, picorna/parvovirus agents 2.9%, Norwalk-like agent 2.9%, astrovirus 1.9%, and calicivirus 0.5%. Unclassified small round viruses (approximately 25-30 nm in diameter) represented 0.5%. It was also determined that there was a seasonal distribution in excretion of all viruses except for CVLPs. A greater number of viruses were identified in the cooler, drier months of the year.

Proteins tightly associated with the termini of replicative form DNA of Kilham rat virus, an autonomous parvovirus.

Revie et al. [Revie, D., Tseng, B. Y., Grafstrom, R. H. & Goulian, M. (1979) Proc. Natl. Acad. Sci. USA 76, 5539-5543] have proposed that the double-stranded replicative form (RF) DNA of the autonomous rodent parvovirus H-1 has protein of 60 kDa covalently bound at its 5' termini. We present evidence that the RF DNA of a similar rodent parvovirus, Kilham rat virus (KRV), also has covalently bound protein. NaDodSO4/polyacrylamide gel electrophoresis of purified, 125I-labeled RF DNA shows that proteins of 68-72, 66, 64, and 55 kDa copurify with the DNA during velocity and equilibrium sedimentation in the presence of detergents and 4 M guanidine HCl. Phenol extraction in the presence of 2-mercaptoethanol removes the 68- to 72-kDa proteins, but the 66-, 64-, and 55-kDa proteins remain tightly, but noncovalently, bound. The latter polypeptides also appear to associate with protease-treated RF DNA when mixed with uninfected cell extract. Following removal of these proteins by electrophoresis in NaDodSO4/agarose gels, two proteins (called RF TP-90 and RF TP-40), of about 90 and 40 kDa, become evident. These remain bound to the DNA and are released only after nuclease digestion of the DNA. These two proteins, apparently not of viral origin, are associated with terminal restriction fragments of the RF DNA and appear to be covalently bound to the 5' termini of both strands.

Viruses and virus-like particles in the faeces of dogs with and without diarrhoea.

Negative staining electron microscopy was used to identify viruses in 157 normal and 29 diarrhoeal faecal samples collected from 156 dogs admitted to an animal shelter during an 8 month period (March to October) in 1982. Seven distinct viral types were detected: 21-26 nm parvovirus-like particles, 28-31 nm astrovirus-like particles, a previously undescribed 34-35 nm "round" virus particle, coronavirus, coronavirus-like particles ( CVLP ), rotavirus and papova-like virus. Parvovirus-like particles alone were detected in 14 diarrhoeal and 50 normal faeces, astrovirus-like particles in 3 normal faeces, "round" viruses in 4 normal faeces, coronavirus in 2 diarrhoeal and 5 normal faeces, CVLP in one diarrhoeal and one normal faeces, rotavirus in 2 normal faeces, papova-like virus in one normal faeces, both parvovirus-like particles and coronavirus in 2 diarrhoeal and 2 normal faeces, parvovirus-like particles and rotavirus in one normal faeces and parvovirus-like and papova-like virus in one normal faeces. The significance of these findings in canine and human disease is discussed.

Characterization of a virus that causes transient aplastic crisis.

Transient aplastic crisis in children with congenital hemolytic anemias has been linked epidemiologically to infection with a serum parvovirus-like virus (SPLV). The virus is found in the blood in the early stages of the crisis, and serum containing SPLV inhibits erythroid colony formation in vitro. After sedimentation of virus-containing sera through a sucrose density gradient, colony inhibitory activity is present in the particulate fraction and separate from serum immunoglobulins. No inhibitory activity can be recovered from convalescent-phase sera after similar fractionation procedures. Inhibition of erythroid colony formation in vitro is not a feature of sera from other viral infections. The pattern of resistance of SPLV activity to chemicals and enzymes is compatible with it being a parvovirus. By using replating techniques, a target of SPLV has been identified as a late erythroid progenitor cell. Neither SPLV antigen nor anti-SPLV IgM was present in the sera of patients with other forms of bone marrow failure.

Aplastic crisis due to parvovirus infection in pyruvate kinase deficiency.

A thirteen-year-old boy with congenital haemolytic anaemia due to pyruvate kinase (PK) deficiency had an aplastic crisis. A serum parvovirus-like virus (SPLV) was demonstrated in the blood by electron microscopy and, subsequently, IgM and IgG antibodies to the prototype SPLV B19 were detected. In an attempt to define the level of erythropoiesis that is involved in parvovirus-induced bone marrow suppression, the levels of circulating early erythroid progenitors (burst forming units erythroid, BFU-E) were monitored during the crisis and recovery period. The virus-containing plasma inhibited the formation of BFU-Es from non-immune subjects and this effect was neutralised by convalescent serum. Colony forming units granulocyte-macrophage (CFU-GM) were also inhibited but this was probably non-specific since neutralisation did not reverse the effect. These experiments, together with the clinical data, suggest a selective effect of SPLV at the stage of erythroid progenitors.