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.

A novel rodent Chapparvovirus in feces of wild rats.

Chapparvovirus, a recently determined new genus in the family Parvoviridae, can infect many species of animals including bats, chickens, and pigs. Here, using viral metagenomics method, we identified a novel Chapparvovirus from feces of wild rats and designated it as rat parvovirus 2 (RPV2). The nearly complete genome of RPV2 is 4222-nt long and includes two ORFs encoding a 654-aa nonstructural protein 1 (NS1) and a 472-aa capsid protein (VP), respectively. Phylogenetic analysis over the amino acid sequence of the NS1 showed that RPV2 clustered with Eidolon helvum parvovirus 2 (EHPV2), porcine parvovirus 7 (PPV7), and turkey parvovirus 1 (TP1), forming a separate clade. Sequence analysis indicated that the NS1 protein of RPV2 shared the highest amino acid sequence identity (51 %) with that of EHPV2. According to the genetic distance-based criteria, RPV2 identified here belongs to a novel species of Chapparvovirus.

Role of capsid proteins in parvoviruses infection.

The parvoviruses are widely spread in many species and are among the smallest DNA animal viruses. The parvovirus is composed of a single strand molecule of DNA wrapped into an icosahedral capsid. In a viral infection, the massy capsid participates in the entire viral infection process, which is summarized in this review. The capsid protein VP1 is primarily responsible for the infectivity of the virus, and the nuclear localization signal (NLS) of the VP1 serves as a guide to assist the viral genome in locating the nucleus. The dominant protein VP2 provides an "anti-receptor", which interacts with the cellular receptor and leads to the further internalization of virus, and, the N-terminal of VP2 also cooperates with the VP1 to prompt the process of nucleus translocation. Additionally, a cleavage protein VP3 is a part of the capsid, which exists only in several members of the parvovirus family; however, the function of this cleavage protein remains to be fully determined. Parvoviruses can suffer from the extreme environmental conditions such as low pH, or even escape from the recognition of pattern recognition receptors (PRRs), due to the protection of the stable capsid, which is thought to be an immune escape mechanism. The applications of the capsid proteins to the screening and the treatment of diseases are also discussed. The processes of viral infection should be noted, because understanding the virus-host interactions will contribute to the development of therapeutic vaccines.

Complementation for an essential ancillary non-structural protein function across parvovirus genera.

Parvoviruses encode a small number of ancillary proteins that differ substantially between genera. Within the genus Protoparvovirus, minute virus of mice (MVM) encodes three isoforms of its ancillary protein NS2, while human bocavirus 1 (HBoV1), in the genus Bocaparvovirus, encodes an NP1 protein that is unrelated in primary sequence to MVM NS2. To search for functional overlap between NS2 and NP1, we generated murine A9 cell populations that inducibly express HBoV1 NP1. These were used to test whether NP1 expression could complement specific defects resulting from depletion of MVM NS2 isoforms. NP1 induction had little impact on cell viability or cell cycle progression in uninfected cells, and was unable to complement late defects in MVM virion production associated with low NS2 levels. However, NP1 did relocate to MVM replication centers, and supports both the normal expansion of these foci and overcomes the early paralysis of DNA replication in NS2-null infections.

Expression, purification, characterization and subcellular localization of the goose parvovirus rep1 protein.

The goose parvovirus (GPV) Rep1 protein is both essential for viral replication and a potential target for GPV diagnosis, but its protein characterization and intracellular localization is not clear. We constructed a recombinant plasmid, pET28a/GPV-Rep1, and expressed the Rep1 gene in BL21 (DE3) Escherichia coli. A protein approximately 75 kDa in size was obtained from lysates of E. coli cells expressing the recombinant plasmid. SDS-PAGE analysis showed that after induction with 0.6 mM isopropyl ß-D-thiogalactosidase (IPTG) at 30°C for 5 h, the Rep1 protein was highly overexpressed. Two methods used to purify proteins, a salinity-gradient elution and Ni-NTA affinity chromatography, were performed. The amount of Rep1 protein obtained by Ni-NTA affinity chromatography was 41.23 mg, while 119.9 mg of Rep1 protein was obtained by a salinity-gradient elution from a 1 L E. coli BL21 (DE3) culture. An immunogenicity analysis showed that the protein could significantly elicit a specific antibody response in immunized goslings compared to control groups. Antibody titers peaked to 1:5120 (optical density (OD) 450 = 3.9) on day 28 after immunization but had mean titers of 1:10,240 (OD450 = 4.2) in gosling groups immunized with a commercially available GPV-attenuated vaccine strain. Experiments examining subcellular localization showed that the Rep1 protein appeared to associate predominantly with the nuclear membrane, especially during later times of infection. This work provides a basis for biochemical and structural studies on the GPV Rep1 protein.

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.

Trans-activation of the long terminal repeat of human immunodeficiency virus type 1 by the parvovirus B19 NS1 gene product.

Persistent parvovirus B19 infections in human immunodeficiency virus type 1 (HIV-1)-infected patients have been reported. The two viruses could share common target cells. The NS1 protein of B19 regulates B19 expression and we have investigated its possible effect on the long terminal repeat (LTR) of HIV-1. In transient transfection experiments, NS1 trans-activated the expression of reporter genes under the control of the HIV-1 LTR. The effect of NS1 was apparent only in the presence of the HIV-1 Tat protein, and required intact TAR and TATA box sequences.

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.

Assembly of empty capsids by using baculovirus recombinants expressing human parvovirus B19 structural proteins.

Empty parvovirus B19 capsids were isolated from insect cells infected with a recombinant baculovirus expressing parvovirus B19 VP2 alone and also with a double-recombinant baculovirus expressing both VP1 and VP2. That VP2 alone can assemble to form capsids is a phenomenon not previously observed in parvoviruses. The stoichiometry of the capsids containing both VP1 and VP2 was similar to that previously observed in parvovirus B19-infected cells. The capsids were similar to native capsids in size and appearance, and their antigenicity was demonstrated by immunoprecipitation and enzyme-linked immunosorbent assay with B19-specific antibodies.

Interaction of virally coded protein and a cell cycle-regulated cellular protein with the bovine parvovirus left terminus ori.

Replication of parvoviruses requires cis signals located in terminal palindromes that function as origins of replication in conjunction with trans-acting viral and cellular proteins. A gel retardation assay was used to identify proteins in crude nuclear extracts of bovine parvovirus (BPV)-infected bovine fetal lung cells that interact with the hairpinned left end (3' OH terminus of the viral minus strand in the flop conformation) of BPV. Three specific DNA-protein complexes formed. One complex was shown to involve a BPV structural protein(s) by inhibiting its formation when antiserum specific for these BPV proteins was used. By specific competition with serum containing antibodies against the BPV nonstructural proteins, a second complex was shown to involve a BPV nonstructural protein. A third complex contained protein of cellular origin and was also formed with extracts of uninfected bovine fetal lung cells. DNA competition assays suggest that the viral proteins do not bind to the right hairpin, which differs in sequence and secondary structure from the left terminus, or to a BPV terminus that lacks the first 52 nucleotides, preventing formation of the stem of the hairpin. The cellular protein is regulated in a cell cycle-dependent fashion, with its binding activity increased in uninfected, actively dividing cells compared with contact-inhibited cells. Since autonomous parvovirus replication requires an S-phase factor for progeny formation, the terminal binding protein demonstrated here is a candidate for this factor.

The production of human parvovirus capsid proteins in Escherichia coli and their potential as diagnostic antigens.

We have expressed a number of polypeptides derived from the capsid proteins of the human parvovirus B19 in Escherichia coli. These include native VP1 (84K) and VP2 (58K) proteins and also fusions to beta-galactosidase containing differing amounts of the amino terminus of the VP1/2 polypeptide. Although each of these was expressed at high levels and the majority were produced as full-length proteins, only one was soluble. This soluble polypeptide, p132, is a beta-galactosidase fusion protein that includes 145 amino acids from B19 which are entirely derived from the region unique to VP1. Despite containing such a small portion of VP1, which itself constitutes only 4% of total capsid protein, p132 reacted with all our known anti-B19 IgM-positive human serum samples. We conclude that this region contains epitopes which must be prominently exposed on the intact virus. We have demonstrated the use of this recombinant antigen in a simple diagnostic assay for B19-specific antibodies which can be used for initial screening of human serum samples. In a survey of 103 serum specimens, our ELISA positively identified all samples (19/19) which were positive by IgM antibody capture radioimmunoassay. The recombinant p132 antigen is efficiently produced and readily purified from E. coli, and its use as a diagnostic antigen should increase the availability of routine clinical testing for human parvovirus infection.

Antigenic parvovirus B19 coat proteins VP1 and VP2 produced in large quantities in a baculovirus expression system.

Two baculovirus expression vectors derived from Autographica californica nuclear polyhedrosis virus (AcNPV) were prepared containing the complete 2.5 kb coding region for parvovirus B19 coat protein VP1 (AcB19VP1L) and the 1.8 kb coding region for VP2 (AcB19VP2L), placed under the control of the polyhedrin promoter. The recombinant viruses were used to infect Spodoptera frugiperda cells and the proteins expressed were analysed using appropriate antibodies. AcB19VP1L-infected cells produced B19 VP1 as shown by its reaction with 13 human sera containing B19-specific antibodies in Western blot analysis and indirect immunofluorescence. The signal seen with VP1 in immunofluorescence makes it suitable for the development of a diagnostic assay based on this technique. VP1 also reacted with two monoclonal antibodies (mAbs) specific for the B19 protein part of a 196 kDa beta-galactosidase B19 fusion protein expressed in E. coli. Cells infected with AcB19VP2L produced B19 VP2 which reacted with the same human sera in indirect immunofluorescence and with five of the 13 sera in Western blots. VP2 did not react with the fusion protein-specific mAbs. The large amounts of viral antigen produced in this system means the development of widely available diagnostic tests for B19 infection and the further characterization of the B19 structural proteins are within reach.

The terminal protein of minute virus of mice is an 83 kilodalton polypeptide linked to specific forms of double-stranded and single-stranded viral DNA.

A new assay (label transfer from DNA to protein) enabled the identification of a terminal protein (TP) in nucleoprotein complexes extracted from cells infected with the parvovirus, minute virus of mice, MVM. In SDS-PAGE, TP migrates as a major band at 83 kDa, with a minor 65 kDa component, each of which exactly co-migrates with the cellular forms of the virally coded polypeptide NS-1. In parallel, the analysis of nucleoproteins by SDS-agarose gel electrophoresis allowed us to observe that the major species of viral DNA molecules (mRF, dRF and ssDNA) are all present in the form of DNA-protein complexes. Three forms of mRF DNA were identified, two of which are protein-associated and one which appears to be protein-free.

Bovine parvovirus DNA-binding proteins: identification by a combined DNA hybridization and immunodetection assay.

We have investigated the interaction between bovine parvovirus (BPV) capsid and non-capsid proteins and restriction fragments of the BPV genome by a combined DNA hybridization and immunodetection assay. 32P-labelled DNA was bound to nitrocellulose membranes bearing lysates of mock-infected and virus-infected cells whose proteins had been separated by SDS-polyacrylamide gel electrophoresis. The position of bound DNA was determined by autoradiography. The proteins on the membrane were still accessible to specific antibodies, allowing confirmation of the DNA-binding species by an immunodetection reaction. In 0.2 M-NaCl, BPV capsid proteins VP2 (72,000 daltons) and VP3 (62,000 daltons) bound the 0 to 16 map unit EcoRI fragment of BPV DNA which contained label in either the minus or plus strand. At higher salt concentration (0.5 M), only VP2 still bound DNA. Within this fragment, the capsid protein binding was restricted to those nucleotides between map units 0 and 4. No binding to capsid proteins was seen with the fragment spanning the middle of the genome and minor binding to VP3 was seen with the 5' end. Binding to the BPV non-capsid protein NP-1 was observed with the 0 to 16 map unit fragment when label was in the virion strand and to other possibly BPV-coded proteins when label was in the plus strand. The NP-1 binding was localized to map units 4 to 16. We did not detect binding to the BPV homologue(s) of the autonomous parvovirus non-capsid protein NS1, due in part to its low concentration in the cell lysates used. Points of the parvovirus replication cycle at which DNA-binding proteins may serve controlling functions are discussed.

Parvovirus H-1 expression: mapping of the abundant cytoplasmic transcripts and identification of promoter sites and overlapping transcription units.

The 5.2-kilobase (kb) genome of the autonomous parvovirus H-1 was transcribed in the rightward direction, yielding steady-state polyadenylated transcripts of 4.8, 3.2, and 2.9 kb. Detailed mapping of these transcripts demonstrated that the H-1 genome contained two overlapping transcription units: the larger unit extended from 4 map units (5' end) to 96 map units (3' end), and the smaller unit extended from 40 map units (5' end) to 96 map units (3' end). The 4.8- and 3.2-kb transcripts were derived from the larger transcription unit and differed by a 1,500-nucleotide segment (10 to 40 map units) which was present in the 4.8-kb transcript but was spliced from the 3.2-kb transcript. The 2.9-kb transcript, the most abundant of the three known H-1 transcripts, was derived from the smaller transcription unit. The sequence at each initiation site was consistent with the presence of a class II (RNA polymerase II) promoter, and cell-free transcription of parvovirus H-1 restriction fragments containing either promoter resulted in transcription of the correct DNA strand and produced 5' ends identical to those seen in vivo. All three transcripts contained a small but heterogeneous splice at 45 to 47 map units. Minor differences in splicing at this site may result in the synthesis of different viral proteins.

The NS-1 polypeptide of the autonomous parvovirus MVM is a nuclear phosphoprotein.

Cells infected with the autonomous parvovirus MVM synthesize a major virally coded non-structural protein which accumulates in the nucleus and is phosphorylated to a significant extent. Peptide map analysis shows that this in vivo product has the same primary sequence as the NS-1 protein previously identified in the in vitro translation products of MVM encoded mRNA, and as such is the product of the largest messenger RNA species, a spliced 4.8 kb (R1) transcript. In vivo NS-1 exists in two predominant forms, a phosphorylated 84-85 kDa species and an unphosphorylated or poorly phosphorylated 83 kDa form. Both forms are preferentially extracted from the nucleus using an extraction procedure which enriches for replication complexes.

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.

Identification and characterization of a porcine parvovirus nonstructural polypeptide.

Sera from porcine parvovirus (PPV)-infected swine fetuses immunoprecipitated and 84- to 86-kilodalton polypeptide in addition to the A and B virion structural proteins. This polypeptide, designated NS-1, was present in PPV-infected cell lysates but not in purified virions. Partial proteolysis mapping revealed that NS-1 was not related to the A and B viral structural proteins. All three proteins in infected cells were phosphorylated at serine residues, and NS-1 also contained phosphothreonine. From pulse-labeling experiments with either 32Pi or [35S]methionine, NS-1 was found to first appear 5 to 7 h postinfection, whereas the viral structural polypeptides were first synthesized 9 to 11 h postinfection. Pulse-chase experiments revealed that NS-1 initially appeared as an 84-kilodalton protein and was subsequently structurally modified to forms of slower electrophoretic mobilities. The time of appearance of NS-1 after virus infection coincided with the initiation of viral DNA synthesis, suggesting that this polypeptide (and the modified forms thereof) may be involved in PPV replication.

Inhibition and recovery of the replication of depurinated parvovirus DNA in mouse fibroblasts.

Apurinic sites were introduced in the single-stranded DNA of parvovirus minute-virus-of-mice (MVM) and their effect on viral DNA synthesis was measured in mouse fibroblasts. Approximately one apurinic site per viral genome, is sufficient to block its replication in untreated cells. The exposure of host cells to a sublethal dose of UV-light 15 hours prior to virus infection, enhances their ability to support the replication of depurinated MVM. Cell preirradiation induces the apparent overcome of 10-15% of viral DNA replication blocks. These results indicate that apurinic sites prevent mammalian cells from replicating single-stranded DNA unless a recovery process is activated by cell UV-irradiation.

Biochemical characterization of a human parvovirus.

The buoyant density, nucleic acid, and proteins of the human serum parvovirus-like agent were investigated. Evidence is presented which suggests that the virus has genomic single-stranded DNA, and that complementary strands may be encapsidated in separate virions. Three proteins of 48 000, 68 000 and 80 000 mol. wt. were found to co-purify with viral antigen at a density of 1.43 g/ml on CsCl gradients. On the basis of these properties it is suggested that this virus is a parvovirus.