The small nonstructural protein (NS2) of the parvovirus minute virus of mice is required for efficient DNA replication and infectious virus production in a cell-type-specific manner.

Seven mutations which affect only the small nonstructural protein NS2 were introduced into the infectious clone of the autonomous parvovirus, minute virus of mice (MVM). The majority of these mutants were severely defective for replication following transfection of normal host murine A9 fibroblasts; however, all were found to replicate more efficiently and produce infectious virus in certain other cell types, including human NB324K. The isolation of viral stocks from NB324K cells permitted a more detailed analysis of the mutant defect on A9 cells. NS2 mutant NS2-2018 was shown to be approximately 10-fold deficient for viral monomer replicative-form DNA production within a single-burst cycle in infected A9 cells and produced a reduced amount of progeny single strand. Mutant NS2-2018 generated wild-type levels of monomer replicative-form DNA on NB324K cells but made reduced levels of progeny single strand and small plaques on these cells. The accumulation of NS1 is reduced late in NS2-2018 infection of A9 cells, but NS1 accumulates to wild-type levels late in NB324K cell infections. NS1 nuclear localization is not dependent on NS2 in A9 or NB324K cells. These results indicate that NS2 participates in MVM DNA replication and is required for efficient viral growth. The requirement for NS2 during MVM replication is also host cell specific. This requirement is significantly more pronounced in the normal host murine A9 cells than in certain other cell types, including NB324K.     

Interaction between parvovirus NS2 protein and nuclear export factor Crm1 is important for viral egress from the nucleus of murine cells

A mutation that disrupts the interaction between the NS2 protein of minute virus of mice and the nuclear export factor Crm1 results in a block to egress of mutant-generated full virions from the nucleus of infected murine cells. These mutants produce wild-type levels of monomer and dimer replicative DNA forms but are impaired in their ability to generate progeny single-stranded DNA in restrictive murine cells in the first round of infection. The NS2-Crm1 interaction mutant can be distinguished phenotypically from an NS2-null mutant and reveals a role for the Crm1-mediated export pathway at a late step in viral infection.     

Directed integration of minute virus of mice DNA into episomes.

Recent studies with adeno-associated virus (AAV) have shown that site-specific integration is directed by DNA sequence motifs that are present in both the viral replication origin and the chromosomal preintegration DNA and that specify binding and nicking sites for the viral regulatory Rep protein. This finding raised the question as to whether other parvovirus regulatory proteins might direct site-specific recombination with DNA targets that contain origin sequences functionally equivalent to those described for AAV. To investigate this question, active and inactive forms of the minute virus of mice (MVM) 3' replication origin, derived from a replicative-form dimer-bridge intermediate, were propagated in an Epstein-Barr virus-based shuttle vector which replicates as an episome in a cell-cycle-dependent manner in mammalian cells. Upon MVM infection of these cells, the infecting genome integrated into episomes containing the active-origin sequence reported to be efficiently nicked by the MVM regulatory protein NS1. In contrast, MVM did not integrate into episomes containing either the inactive form of the origin sequence reported to be inefficiently nicked by NS1 or the active form from which the NS1 consensus nick site had been deleted. The structure of the cloned MVM episomal recombinants displayed several features previously described for AAV episomal and chromosomal recombinants. The findings indicate that the rules which govern AAV site-specific recombination also apply to MVM and suggest that site-specific chromosomal insertions may be achievable with different autonomous parvovirus replicator proteins which recognize binding and nicking sites on the target DNA.     

Genome Replication and Postencapsidation Functions Mapping to the Nonstructural Gene Restrict the Host Range of a Murine Parvovirus in Human Cells

The infection outcome of the Parvoviridae largely relies on poorly characterized intracellular factors modulated by proliferation, differentiation, and transformation of host cells. We have studied the interactions displayed by the highly homologous p and i strains of the murine parvovirus minute virus of mice (MVM), with a series of transformed cells of rat (C6) and human (U373, U87, SW1088, SK-N-SH) nervous system origin, seeking for molecular mechanisms governing parvovirus host range. The MVMp infection of C6 and U373 cells was cytotoxic and productive, whereas the other nervous cells behaved essentially as resistant to this virus. In contrast, MVMi did not complete its life cycle in any of the human nervous cells, though it efficiently killed the astrocytic tumor cells by two types of nonproductive infections: (i) normal synthesis of all viral macromolecules with a late defect in infectious virion maturation and release to the medium in U373; and (ii) high levels of accumulation of the full set of viral messenger RNAs and of both nonstructural (NS-1) and structural (VP-1 and VP-2) proteins, under a very low viral DNA amplification, in U87 and SW1088 cells. Further analyses showed that U87 was permissive for nuclear transport of MVMi proteins, leading to efficient assembly of empty viral capsids with a normal phosphorylation and VP1-to-VP2 ratio. The DNA amplification blockade in U87 occurred after conversion of the incoming MVMi genome to the monomeric replicative form, and it operated independently of the delivery pathway used by the viral particle, since it could not be overcome by transfection with cloned infectious viral DNA. Significantly, a chimeric MVMi virus harboring the coding region of the nonstructural (NS) gene replaced with that of MVMp showed a similar pattern of restriction in U87 cells as the parental MVMi virus, and it attained in U373 cultures an infectious titer above 100-fold higher under equal levels of DNA amplification and genome encapsidation. The results suggest that the activity of complexes formed by the NS polypeptides and recruited cellular factors restrict parvovirus DNA amplification in a cell type-dependent manner and that NS functions may in addition determine MVM host range acting at postencapsidation steps of viral maturation. These data are relevant for understanding the increased multiplication of autonomous parvovirus in some transformed cells and the transduction efficacy of nonreplicative parvoviral vectors, as well as a general remark on the mechanisms by which NS genes may regulate viral tropism and pathogenesis.     

Parvovirus minute virus of mice induces a DNA damage response that facilitates viral replication

Infection by DNA viruses can elicit DNA damage responses (DDRs) in host cells. In some cases the DDR presents a block to viral replication that must be overcome, and in other cases the infecting agent exploits the DDR to facilitate replication. We find that low multiplicity infection with the autonomous parvovirus minute virus of mice (MVM) results in the activation of a DDR, characterized by the phosphorylation of H2AX, Nbs1, RPA32, Chk2 and p53. These proteins are recruited to MVM replication centers, where they co-localize with the main viral replication protein, NS1. The response is seen in both human and murine cell lines following infection with either the MVMp or MVMi strains. Replication of the virus is required for DNA damage signaling. Damage response proteins, including the ATM kinase, accumulate in viral-induced replication centers. Using mutant cell lines and specific kinase inhibitors, we show that ATM is the main transducer of the signaling events in the normal murine host. ATM inhibitors restrict MVM replication and ameliorate virus-induced cell cycle arrest, suggesting that DNA damage signaling facilitates virus replication, perhaps in part by promoting cell cycle arrest. Thus it appears that MVM exploits the cellular DNA damage response machinery early in infection to enhance its replication in host cells.     

Parvovirus replication in normal and transformed human cells correlates with the nuclear translocation of the early protein NS1.

The parvovirus H-1 infection of the normal human diploid fibroblast strain MRC-5 produces a cytopathic effect, but no increase in infectious virus has been observed. Previously, we reported that large amounts of empty capsids are assembled in the nucleus of H-1 infected MRC-5 cells (S. Singer and S. Rhode, in D. Ward and P. Tattersall, ed., Replication of Mammalian Parvoviruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1978). The level of viral replicative-form DNA synthesis as shown by metabolic labeling is markedly reduced in these cells. Synthesis of the early protein NS1 is normal or slightly decreased, and the usual amount of the 92,000-molecular-weight (92K) posttranslationally modified NS1 was seen. The second deficient parameter that we have observed in the abortive infection is the nuclear translocation of NS1. In contrast, the simian virus 40-transformed MRC-5 cell line MRC-5 V1 and the simian virus 40-transformed human kidney cell line NB undergo a productive infection by H-1 accompanied by more efficient translocation of NS1 to the nucleus. The results indicate that there is an association between defective translocation of the NS1 rep protein to the nucleus and defective amplification of parvovirus replicative-form DNA. The nuclear translocation of specific proteins seems to be a function that is altered by development or neoplastic transformation.     

Process of infection with bacteriophage phi chi 174. XL. Viral DNA replication of phi chi 174 mutants blocked in progeny single-stranded DNA synthesis.

Mutation in several different cistrons of bacteriophage phi chi 174 blocks net progeny single-stranded DNA synthesis at the late period of infection (15). For the study of the functions of these cistrons in single-stranded DNA synthesis, asymmetric replication of replicative form DNA was examined at the late period of infection with amber mutants of these cistrons. While the normal, rapid process of asymmetric single-stranded viral DNA synthesis is blocked at the late period of these mutant infections, an asymmetric synthesis of the viral strand of replicative-form DNA is observed in this period, though at a reduced level, together with degradation of prelabeled viral strand. Some intermediate replicative-form molecules were also detected. Asymmetric synthesis of the viral strand of replicative-form DNA at the late period of phi chi infection is completely inhibited in the presence of a low concentration (35mug/ml) of chloramphenicol (which also blocks net single-stranded viral DNA synthesis). These results are discussed in terms of the possible role of the specific viral proteins for normal single-stranded DNA synthesis.     

Characterization of a metastable, partially replicated dimeric intermediate of minute virus of mice

Intracellular, replicative-form DNA of minute virus of mice was characterized by agarose gel electrophoresis, velocity sedimentation, electron microscopy, restriction endonuclease digestion, and sensitivity to the single-stranded nuclease S1. This analysis demonstrated the presence in murine cells infected with minute virus of mice of a 10.0-kilobase pair dimer replicative form, a 5-kilobase pair monomer replicative form, as well as a 5-kilobase viral single-stranded DNA species. Two additional viral DNA species that migrated in 0.5% agarose gels with apparent sizes of 8.0 and 5.5 kilobase pairs were also observed. Further investigation indicated that the 8.0-kilobase pair DNA represents a novel class of metastable, partially replicated, dimeric intermediates. This finding has important implications for the mechanism of parvovirus DNA replication.     

Mutation of lysine 405 to serine in the parvovirus H-1 NS1 abolishes its functions for viral DNA replication, late promoter trans activation, and cytotoxicity.

A consensus sequence in parvovirus nonstructural protein NS1 has been predicted to be an ATP-binding domain associated with an ATPase and a DNA helicase activity. To investigate the function of NS1 in viral gene expression, a site-directed mutagenesis converting NS1 lysine 405 to serine in parvovirus H-1 was carried out by the polymerase chain reaction. As shown previously, a parvovirus genome containing a deleted NS1 gene was excised from a bacterial plasmid and replicated when a wild-type NS1 gene was provided in trans but failed to be excised and replicate when the mutant NS1 gene was supplied. Interestingly, the serine 405 mutation totally lost the activity of trans activation on the virus late promoter (P38) in a chloramphenicol acetyltransferase (CAT) assay and it lost evidence for cytotoxicity in two tumor cell lines (HeLa Gey and NB324K). The serine 405 NS1 protein was translocated normally to the nucleus. These results suggest that the NS1 lysine 405 of H-1 in its putative purine nucleotide-binding site is essential for viral DNA replication and that this domain may be involved in the regulation of the P38 promoter by an unknown mechanism. The loss of NS1 cytotoxicity on tumor cells suggests that NS1 expression is the major cause of cell killing by parvoviruses, which may facilitate further study of the mechanism of oncosuppression by parvoviruses.     

Two amino acid substitutions within the capsid are coordinately required for acquisition of fibrotropism by the lymphotropic strain of minute virus of mice.

Nucleotide changes at both codons 317 and 321 in the VP2 capsid gene of the immunosuppressive strain of the murine parvovirus minute virus of mice, MVM(i), are required to create a virus capable of growing in A9 fibroblasts. This double mutant virus, ILB1, has growth characteristics very similar to those of the prototype fibrotropic strain MVM(p) in both single- and multiple-round infections of fibroblasts and is about 100-fold better at infecting fibroblasts than MVM(i). When only one nucleotide position is changed, either in codon 317 (as in ILB2) or in codon 321 (as in ILB3), the resulting viruses are less than twice as efficient as their parent MVM(i) at infecting fibroblasts. In the restrictive infection of A9 cells by the single mutants and MVM(i), gene expression and DNA replication were markedly reduced compared with ILB1 infection of the same cells or compared with infections of permissive hybrid cells by each of the viruses. This suggests that restriction acts predominantly at an early step in the infection. Since the phenotypes of ILB2 and ILB3 are essentially indistinguishable in restrictive infections, it is most likely that the individual loci affect the same step in the viral life cycle. The dramatic increase in fibroblast infectivity shown by ILB1 indicates a synergistic interaction between these two amino acid residues in the same rate-limiting process in fibroblast infection.     

Nuclear export of the nonenveloped parvovirus virion is directed by an unordered protein signal exposed on the capsid surface

It is uncertain whether nonenveloped karyophilic virus particles may actively traffic from the nucleus outward. The unordered amino-terminal domain of the VP2 major structural protein (2Nt) of the icosahedral parvovirus minute virus of mice (MVM) is internal in empty capsids, but it is exposed outside of the shell through the fivefold axis of symmetry in virions with an encapsidated single-stranded DNA genome, as well as in empty capsids subjected to a heat-induced structural transition. In productive infections of transformed and normal fibroblasts, mature MVM virions were found to efficiently exit from the nucleus prior to cell lysis, in contrast to the extended nuclear accumulation of empty capsids. Newly formed mutant viruses lacking the three phosphorylated serine residues of 2Nt were hampered in their exit from the human transformed NB324K nucleus, in correspondence with the capacity of 2Nt to drive microinjected phosphorylated heated capsids out of the nucleus. However, in normal mouse A9 fibroblasts, in which the MVM capsid was phosphorylated at similar sites but with a much lower rate, the nuclear exit of virions and microinjected capsids harboring exposed 2Nt required the infection process and was highly sensitive to inhibition of the exportin CRM1 in the absence of a demonstrable interaction. Thus, the MVM virion exits the nucleus by accessing nonconventional export pathways relying on cell physiology that can be intensified by infection but in which the exposure of 2Nt remains essential for transport. The flexible 2Nt nuclear transport signal may illustrate a common structural solution used by nonenveloped spherical viruses to propagate in undamaged host tissues.     

The putative metal coordination motif in the endonuclease domain of human Parvovirus B19 NS1 is critical for NS1 induced S phase arrest and DNA damage

The non-structural proteins (NS) of the parvovirus family are highly conserved multi-functional molecules that have been extensively characterized and shown to be integral to viral replication. Along with NTP-dependent helicase activity, these proteins carry within their sequences domains that allow them to bind DNA and act as nucleases in order to resolve the concatameric intermediates developed during viral replication. The parvovirus B19 NS1 protein contains sequence domains highly similar to those previously implicated in the above-described functions of NS proteins from adeno-associated virus (AAV), minute virus of mice (MVM) and other non-human parvoviruses. Previous studies have shown that transient transfection of B19 NS1 into human liver carcinoma (HepG2) cells initiates the intrinsic apoptotic cascade, ultimately resulting in cell death. In an effort to elucidate the mechanism of mammalian cell demise in the presence of B19 NS1, we undertook a mutagenesis analysis of the protein's endonuclease domain. Our studies have shown that, unlike wild-type NS1, which induces an accumulation of DNA damage, S phase arrest and apoptosis in HepG2 cells, disruptions in the metal coordination motif of the B19 NS1 protein reduce its ability to induce DNA damage and to trigger S phase arrest and subsequent apoptosis. These studies support our hypothesis that, in the absence of replicating B19 genomes, NS1-induced host cell DNA damage is responsible for apoptotic cell death observed in parvoviral infection of non-permissive mammalian cells.     

Reverse genetic system for the analysis of parvovirus telomeres reveals interactions between transcription factor binding sites in the hairpin stem

The left-hand or 3'-terminal hairpin of minute virus of mice (MVM) contains sequence elements essential for both viral DNA replication at the left-hand origin (oriL) and for the modulation of the P4 promoter, from which the viral nonstructural gene cassette is transcribed. This hairpin sequence has proven difficult to manipulate in the context of the viral genome. Here we describe a system for generating mutant viruses using synthetic hairpin oligonucleotides and a truncated form of the infectious clone. This allows manipulation of the sequence of the left-hand hairpin and examination of the effects in the context of the viral life cycle. We have confirmed the requirement for a functional parvovirus initiation factor (PIF) binding site and determined that an optimized PIF binding site, with 6 bases between the half-sites, was actually detrimental to viral growth. The distal PIF half-site overlaps a cyclic AMP-responsive element (CRE), which was shown to play an important role in initiating infection, particularly in 324K simian virus 40-transformed human fibroblasts. Interestingly, reducing the spacing of the PIF half-sites, and thus the affinity of the binding site for PIF, increased viral fitness relative to wild type in 324K cells, but not in murine A9 cells. These results indicate that the relative importance of factor binding to the CRE and PIF sites during the establishment of an infection differs markedly between these two host cells and suggest that the suboptimal spacing of PIF half-sites found in wild-type virus represents a necessary reduction in the affinity of the PIF interaction in favor of CRE function.     

Asymmetric resolution of a parvovirus palindrome in vitro.

Cell extracts from murine A9 or human HeLa cells containing wild-type copies the NS1 polypeptide of minute virus of mice (MVM), produced from a recombinant vaccinia virus, can support the resolution of viral 3' termini from palindromic junction fragments of dimeric, replicative-form MVM DNA. Resolution resulted in the generation of two new viral termini, one associated with each arm of the junction palindrome. Telomeres were created in two configurations, "extended" forms, which were covalently associated with NS1 molecules, and smaller "turn-around" forms in which a single arm of the palindrome terminated at the axis of dyad symmetry in a covalent bond which cross-linked the two strands. The in vitro resolution reaction was asymmetric, generating predominantly extended-form termini from one arm of the palindrome but predominantly turn-around forms from the other. This asymmetry was independent of the type of cell used to prepare the in vitro extract or the orientation of the palindrome in the plasmid and was obtained for all cloned junction sequences of 156 bp or more. Two modified forms of the duplex junction fragment, which appeared to be intermediates in the resolution process since they were nicked, covalently linked to NS1, and associated with newly synthesized DNA, were identified. The structures of these intermediates suggest that resolution is initiated by preferential nicking at one of the two candidate resolution sites. The asymmetric nature of this resolution reaction is discussed in terms of current models of MVM DNA replication.     

Segregation of a single outboard left-end origin is essential for the viability of parvovirus minute virus of mice

During DNA replication, the hairpin telomeres of Minute Virus of Mice (MVM) are extended and copied to create imperfectly palindromic duplex junction sequences that bridge adjacent genomes in concatameric replicative-form DNA. These are resolved by the viral initiator protein, NS1, but mechanisms employed at the two telomeres differ. Left-end:left-end junctions are resolved asymmetrically at a single site, OriLTC, by NS1 acting in concert with a host factor, parvovirus initiation factor (PIF). Replication segregates doublet and triplet sequences, initially present as unpaired nucleotides in the bubble region of the left-end hairpin stem, to either side of the junction. These act as spacers between the NS1 and PIF binding sites, and their asymmetric distribution sets up active (OriLTC) and inactive (OriLGAA) forms of OriL. We used a reverse genetic approach to disrupt this asymmetry and found that neither opposing doublets nor triplets in the hairpin bubble were tolerated. Viable mutants were isolated at low frequency and found to contain second-site mutations that either restored the asymmetry or crippled one PIF binding site. These mutations either inactivated the inboard or activated the outboard form of OriL, a polarity that strongly suggests that, in the genus Parvovirus, an active inboard OriL is lethal.     

Molecular characterization of a newly recognized mouse parvovirus.

Mouse parvovirus (MPV), formerly known as orphan parvovirus, is a newly recognized rodent parvovirus distinct from both serotypes of minute virus of mice (MVM). Restriction analysis of the MPV genome indicated that many restriction sites in the capsid region were different from those of MVM, but most sites in the nonstructural (NS) region of the genome were conserved. MPV resembled MVM in genome size, replication intermediates, and NS proteins. Replication intermediates in infected cells were the same for MPV and MVM, including packaging of the 5-kb minus (V) strand. Furthermore, the MPV NS proteins were the same size as and present at the same ratio as the MVM(i) proteins in infected cells. Cloning and sequencing of the MPV genome revealed a genome organization closely resembling that of MVM, with conservation of open reading frames, promoter sequences, and splice sites. The left terminal hairpin was identical to that of MVM(i), but the right terminus was not conserved. Also, the MPV genome was unique in that it contained 1.8 copies of the terminal repeat sequence rather than the 1 or 2 copies found in other parvoviruses. The predicted amino acid sequence of the NS proteins of MPV and MVM(i) were nearly identical. In contrast, the predicted amino acid sequence of the capsid proteins of MPV was different from sequences of other parvoviruses. These results confirm that MPV is a distinct murine parvovirus and account for the antigenic differences between MPV and MVM.