Peter Tattersall

The autonomously replicating parvoviruses of vertebrates.

Publisher Summary Various aspects of the natural history of autonomous parvoviruses are beginning to be understood in some detail, mostly through the analysis of tissue culture analogs of the pathogenic processes observed in the whole animal. This chapter reviews the current state of knowledge of autonomous parvovirus structure and replication. The chapter explores the strategies employed by these viruses—at the molecular and cellular levels—to parasitize their various hosts. Members of the autonomous parvovirus group are capable of productive replication without the aid of a helper virus in the majority of host cells. Cell cycling—although necessary—is not sufficient for the lytic, productive replication of individual parvovirus strains. The differentiated state of the host cell is of paramount importance. It has also been reported that the surface structure of the viral particle—as monitored by the expression or absence of certain antigenic configurations—may have a dramatic influence on the ability of the virus to replicate in a particular host cell type, and that this capsid-mediated specificity may well involve intracellular interactions with host cell factors, as well as, or rather than, differences in binding to a specific cell surface receptor.

The family Parvoviridae

A set of proposals to rationalize and extend the taxonomy of the family Parvoviridae is currently under review by the International Committee on Taxonomy of Viruses (ICTV). Viruses in this family infect a wide range of hosts, as reflected by the longstanding division into two subfamilies: the Parvovirinae, which contains viruses that infect vertebrate hosts, and the Densovirinae, encompassing viruses that infect arthropod hosts. Using a modified definition for classification into the family that no longer demands isolation as long as the biological context is strong, but does require a near-complete DNA sequence, 134 new viruses and virus variants were identified. The proposals introduce new species and genera into both subfamilies, resolve one misclassified species, and improve taxonomic clarity by employing a series of systematic changes. These include identifying a precise level of sequence similarity required for viruses to belong to the same genus and decreasing the level of sequence similarity required for viruses to belong to the same species. These steps will facilitate recognition of the major phylogenetic branches within genera and eliminate the confusion caused by the near-identity of species and viruses. Changes to taxon nomenclature will establish numbered, non-Latinized binomial names for species, indicating genus affiliation and host range rather than recapitulating virus names. Also, affixes will be included in the names of genera to clarify subfamily affiliation and reduce the ambiguity that results from the vernacular use of “parvovirus” and “densovirus” to denote multiple taxon levels.

Functional implications of the structure of the murine parvovirus, minute virus of mice

BACKGROUND Minute virus of mice (MVM) is a single-stranded (ss) DNA-containing, murine parvovirus with a capsid built up of 60 icosahedrally related polypeptide chains, each of which consists of the C-terminal region common to two structural proteins, VP1 and VP2. In infectious virions, most VP2 molecules are cleaved to VP3 by the removal of about 20 amino acids from the N terminus. Of the 587 amino acids in VP2, approximately half are identical to those in the analogous capsid protein of the antigenically distinct canine parvovirus (CPV), the crystal structure of which has previously been determined. The three-dimensional structure determination of MVMi (the immunosuppressive strain of MVM) was previously reported to 3.5 A resolution. RESULTS We report here an analysis of the MVMi virus structure and provide insights into tissue tropism, antigenicity and DNA packaging. Amino acids determining MVM tissue tropism were found to cluster on, or near, the viral surface. A conserved, glycine-rich, N-terminal peptide was seen to run through a cylindrical channel along each fivefold axis and may have implications for antigenicity. Density within the virion was interpreted as 29 ssDNA nucleotides per icosahedral asymmetric unit, and accounts for over one-third of the viral genome. CONCLUSIONS The presence of the glycine-rich sequence in the fivefold channels of MVMi provides a possible mechanism to explain how the unique N-terminal region of VP1 becomes externalized in infectious parvovirions. Residues that determine tropism may form an attachment recognition site for a secondary host-cell factor that modulates tissue specificity. The ordering of nucleotides in a similar region of the interior surface in the CPV and MVMi capsids suggests the existence of a genomic DNA-recognition site within the parvoviral capsid.

Parvoviral host range and cell entry mechanisms.

Parvoviruses elaborate rugged nonenveloped icosahedral capsids of approximately 260 A in diameter that comprise just 60 copies of a common core structural polypeptide. While serving as exceptionally durable shells, capable of protecting the single-stranded DNA genome from environmental extremes, the capsid also undergoes sequential conformational changes that allow it to translocate the genome from its initial host cell nucleus all the way into the nucleus of its subsequent host. Lacking a duplex transcription template, the virus must then wait for its host to enter S-phase before it can initiate transcription and usurp the cells synthetic pathways. Here we review cell entry mechanisms used by parvoviruses. We explore two apparently distinct modes of host cell specificity, first that used by Minute virus of mice, where subtle glycan-specific interactions between host receptors and residues surrounding twofold symmetry axes on the virion surface mediate differentiated cell type target specificity, while the second involves novel protein interactions with the canine transferrin receptor that allow a mutant of the feline leukopenia serotype, Canine parvovirus, to bind to and infect dog cells. We then discuss conformational shifts in the virion that accompany cell entry, causing exposure of a capsid-tethered phospholipase A2 enzymatic core that acts as an endosomolytic agent to mediate virion translocation across the lipid bilayer into the cell cytoplasm. Finally, we discuss virion delivery into the nucleus, and consider the nature of transcriptionally silent DNA species that, escaping detection by the cell, might allow unhampered progress into S-phase and hence unleash the parvoviral Trojan horse.

Targeting Human Glioblastoma Cells: Comparison of Nine Viruses with Oncolytic Potential

ABSTRACT Brain tumors classified as glioblastomas have proven refractory to treatment and generally result in death within a year of diagnosis. We used seven in vitro tests and one in vivo trial to compare the efficacy of nine different viruses for targeting human glioblastoma. Green fluorescent protein (GFP)-expressing vesicular stomatitis (VSV), Sindbis virus, pseudorabies virus (PRV), adeno-associated virus (AAV), and minute virus of mice i-strain (MVMi) and MVMp all infected glioblastoma cells. Mouse and human cytomegalovirus, and simian virus 40 showed only low levels of infection or GFP expression. VSV and Sindbis virus showed strong cytolytic actions and high rates of replication and spread, leading to an elimination of glioblastoma. PRV and both MVM strains generated more modest lytic effects and replication capacity. VSV showed a similar oncolytic profile on U-87 MG and M059J glioblastoma. In contrast, Sindbis virus showed strong preference for U-87 MG, whereas MVMi and MVMp preferred M059J. Sindbis virus and both MVM strains showed highly tumor-selective actions in glioblastoma plus fibroblast coculture. VSV and Sindbis virus were serially passaged on glioblastoma cells; we isolated a variant, VSV-rp30, that had increased selectivity and lytic capacity in glioblastoma cells. VSV and Sindbis virus were very effective at replicating, spreading within, and selectively killing human glioblastoma in an in vivo mouse model, whereas PRV and AAV remained at the injection site with minimal spread. Together, these data suggest that four (VSV, Sindbis virus, MVMi, and MVMp) of the nine viruses studied merit further analysis for potential therapeutic actions on glioblastoma.

Seroepidemiology of Human Bocavirus Defined Using Recombinant Virus-Like Particles

BACKGROUND Human bocavirus (HBoV) is a newly identified human parvovirus for which seroepidemiology and antigenic properties remain undefined. METHODS The HBoV VP2 gene, expressed from a baculovirus vector, produced virus-like particles (VLPs), which were used to raise rabbit anti-HBoV antisera and to develop an enzyme-linked immunosorbent assay (ELISA). The VLP-based ELISA was used to screen for HBoV-specific immunoglobulin G antibodies in a convenience sample of 270 serum specimens, mostly from children, obtained at Yale-New Haven Hospital; 208 specimens were also screened for erythrovirus B19-specific antibodies by a B19 VLP-based ELISA. RESULTS Immunofluorescence and ELISA showed that human parvoviruses HBoV and B19 are antigenically distinct. By the HBoV VLP-based ELISA, 91.8% and 63.6% of serum specimens from infants in the first and second months of life, respectively, were found to be seropositive, as were 45.4% from 3-month-old infants and 25.0% from 4-month-old infants. The percentages of HBoV-seropositive children increased to 40.7%-60.0% for children 5-47 months of age and to >85% for individuals >or=48 months old. However, the overall percentage of B19-seropositive individuals was <40.5% for all age groups screened. CONCLUSIONS HBoV infection is common during childhood, but a minority of children and young adults screened have evidence of B19 infection.

cis Requirements for the Efficient Production of Recombinant DNA Vectors Based on Autonomous Parvoviruses

The replication of viral genomes and the production of recombinant viral vectors from infectious molecular clones of parvoviruses MVMp and H1 were greatly improved by the introduction of a consensus NS-1 nick site at the junction between the left-hand viral terminus and the plasmid DNA. Progressive deletions of up to 1600 bp in the region encoding the structural genes as well as insertions of foreign DNA in replacement of those sequences did not appreciably affect the replication ability of the recombinant H1 virus genomes. In contrast, the incorporation of these genomes into recombinant particles appeared to depend on in cis-provided structural gene sequences. Indeed, the production of H1 viral vectors by cotransfection of recombinant clones and helper plasmids providing the structural proteins (VPs) in trans, drastically decreased when more than 800 bp was removed from the VP transcription unit. Furthermore, titers of viral vectors, in which most of the VP-coding region was replaced by an equivalent-length sequence consisting of reporter cDNA and stuffer DNA, were reduced more than 50 times in comparison with recombinant vectors in which stuffer DNA was not substituted for the residual VP sequence. In addition, viral vector production was restricted by the overall size of the genome, with a mere 6% increase in DNA length leading to an approximately 10 times lower encapsidation yield. Under conditions fulfilling the above-mentioned requirements for efficient packaging, titers of virus vectors from improved recombinant molecular DNA clones amounted to 5 x 10(7) infectious units per milliliter of crude extract. These titers should allow the assessment of the therapeutic effect of recombinant parvoviruses expressing small transgenes in laboratory animals.

Sequence homology between the structural polypeptides of minute virus of mice.

The DNA-containing (full) particles of minute virus of mice contain three polypeptide species, designated A (Mr=83,000), B (Mr=64,000) and C (Mr=61,000). These three proteins are compared here by tryptic and chymotryptic fingerprinting after radio-iodination of their tyrosyl residues in vitro. Polypeptide B and C digests are almost identical, thus confirming the precursor-product relationship between them suggested by previous kinetic studies. Both types of fingerprint show one peptide which occurs in the C polypeptide but not in B, indicating that the cleavage in vivo may not occur at either a tryptic or chymotryptic site. In addition to the relationship between B and C, all of the sequence of B is present in the largest polypeptide A, which constitutes≈16% of the total virion protein. The A polypeptide contains additional tyrosyl peptides, comprising about 20% of the total, which do not occur in either B or C. Proteolytic digestion of intact full particles in vitro shows that the cleavage of B in vivo can be closely mimicked by trypsin and to a lesser extent by chymotrypsin. However, the B polypeptide in the empty virion is resistant to cleavage by either enzyme in vitro, indicating that it adopts a different conformation in each particle type. This correlates well with the in vivo observation that empty particles contain polypeptide B, in addition to A, and do not contain any polypeptide C. The A polypeptide is completely resistant to cleavage by either enzyme in either particle, suggesting that the conformation of the common sequence in polypeptide A may be similar to that adopted by polypeptide B in empty virions. A scheme for the maturation of infectious parvovirus virions is described.

Parvovirus initiator protein NS1 and RPA coordinate replication fork progression in a reconstituted DNA replication system.

ABSTRACT We show here that the DNA helicase activity of the parvoviral initiator protein NS1 is highly directional, binding to the single strand at a recessed 5′ end and displacing the other strand while progressing in a 3′-to-5′ direction on the bound strand. NS1 and a cellular site-specific DNA binding factor, PIF, also known as glucocorticoid modulating element binding protein, bind to the left-end minimal replication origin of minute virus of mice, forming a ternary complex. In this complex, NS1 is activated to nick one DNA strand, becoming covalently attached to the 5′ end of the nick in the process and providing a 3′ OH for priming DNA synthesis. In this situation, the helicase activity of NS1 did not displace the nicked strand, but the origin duplex was distorted by the NS1-PIF complex, as assayed by its sensitivity to KMnO4 oxidation, and a stretch of about 14 nucleotides on both strands of the nicked origin underwent limited unwinding. Addition of Escherichia coli single-stranded DNA binding protein (SSB) did not lead to further unwinding. However, addition of recombinant human single-stranded DNA binding protein (RPA) to the initiation reaction catalyzed extensive unwinding of the nicked origin, suggesting that RPA may be required to form a functional replication fork. Accordingly, the unwinding mediated by NS1 and RPA promoted processive leading-strand synthesis catalyzed by recombinant human DNA polymerase δ, PCNA, and RFC, using the minimal left-end origin cloned in a plasmid as a template. The requirement for RPA, rather than SSB, in the unwinding reaction indicated that specific NS1-RPA protein interactions were formed. NS1 was tested by enzyme-linked immunosorbent assay for binding to two- or three-subunit RPA complexes expressed from recombinant baculoviruses. NS1 efficiently bound each of the baculovirus-expressed complexes, indicating that the small subunit of RPA is not involved in specific NS1 binding. No NS1 interactions were observed with E. coli SSB or other proteins included as controls.

Alternate splicing in a parvoviral nonstructural gene links a common amino-terminal sequence to downstream domains which confer radically different localization and turnover characteristics

Minute virus of mice (MVM) encodes two groups of nonstructural proteins, the 83-kDa NS-1 polypeptides encoded from a contiguous sequence in the left half of the genome and the 25-kDa NS-2 polypeptides, which share a common amino-terminal domain with NS-1 but are multiply spliced. Peptide-specific antibodies were used to demonstrate that three alternatively spliced forms of NS-2 are synthesized when synchronized A9 cells are infected with the prototype strains of MVM, MVM(p), and that each of these species migrates as two bands on sodium dodecyl sulfate-gel electrophoresis, due to the presence of both phosphorylated and unphosphorylated forms. While most NS-1 molecules are located in the nucleus, all three species of NS-2 are predominantly cytoplasmic, and their phosphorylated forms are exclusively cytoplasmic. Although both NS-1 and NS-2 molecules are synthesized early in infection, all forms of NS-2 are synthesized and accumulate three to four times as NS-1 molecules, making them the predominant virally coded proteins in the cell at this time. Despite their common amino-terminal domain, NS-2 molecules turn over rapidly while NS-1 polypeptides persist for many hours. Apart from the fact that the three NS-2 gene products are synthesized in different molar amounts, we were unable to detect any differences in the expression, stability, distribution, or phosphorylation of the various molecular forms, suggesting that these latter characteristics are mediated by their common internal exon.