Pathogenicity of fibroblast- and lymphocyte-specific variants of minute virus of mice.

We tested two strains of the minute virus of mice (MVM) for pathogenic effects and patterns of infection in laboratory mice. The two strains differ in their ability to infect differentiated cultured cells: the prototype virus, MVMp, infects only fibroblasts, while its variant, MVMi, is restricted to lymphocytes. We find that neither strain has any demonstrable effects on the T-cell function of mice infected as adults. In contrast, MVMi, but not MVMp, is able to induce a runting syndrome accompanied by mild immune deficiencies upon the infection of newborn mice. After neonatal infection, MVMi spreads to many organs, and the presence of viral replicative form DNA is evident in nucleic acid hybridization experiments. In contrast, replication of MVMp can be detected only by the seroconversion of infected animals. Newborn mice that grow abnormally as a result of MVMi infection also have low circulating antibody titers to the virus. This phenomenon may be a consequence of the lymphotropism of MVMi.     

Evolution to pathogenicity of the parvovirus minute virus of mice in immunodeficient mice involves genetic heterogeneity at the capsid domain that determines tropism

Very little is known about the role that evolutionary dynamics plays in diseases caused by mammalian DNA viruses. To address this issue in a natural host model, we compared the pathogenesis and genetics of the attenuated fibrotropic and the virulent lymphohematotropic strains of the parvovirus minute virus of mice (MVM), and of two invasive fibrotropic MVM (MVMp) variants carrying the I362S or K368R change in the VP2 major capsid protein, in the infection of severe combined immunodeficient (SCID) mice. By 14 to 18 weeks after oronasal inoculation, the I362S and K368R viruses caused lethal leukopenia characterized by tissue damage and inclusion bodies in hemopoietic organs, a pattern of disease found by 7 weeks postinfection with the lymphohematotropic MVM (MVMi) strain. The MVMp populations emerging in leukopenic mice showed consensus sequence changes in the MVMi genotype at residues G321E and A551V of VP2 in the I362S virus infections or A551V and V575A changes in the K368R virus infections, as well as a high level of genetic heterogeneity within a capsid domain at the twofold depression where these residues lay. Amino acids forming this capsid domain are important MVM tropism determinants, as exemplified by the switch in MVMi host range toward mouse fibroblasts conferred by coordinated changes of some of these residues and by the essential character of glutamate at residue 321 for maintaining MVMi tropism toward primary hemopoietic precursors. The few viruses within the spectrum of mutants from mice that maintained the respective parental 321G and 575V residues were infectious in a plaque assay, whereas the viruses with the main consensus sequences exhibited low levels of fitness in culture. Consistent with this finding, a recombinant MVMp virus carrying the consensus sequence mutations arising in the K368R virus background in mice failed to initiate infection in cell lines of different tissue origins, even though it caused rapid-course lethal leukopenia in SCID mice. The parental consensus genotype prevailed during leukopenia development, but plaque-forming viruses with the reversion of the 575A residue to valine emerged in affected organs. The disease caused by the DNA virus in mice, therefore, involves the generation of heterogeneous viral populations that may cooperatively interact for the hemopoietic syndrome. The evolutionary changes delineate a sector of the surface of the capsid that determines tropism and that surrounds the sialic acid receptor binding domain.     

Replication of minute virus of mice DNA is critically dependent on accumulated levels of NS2

Following transfection of murine fibroblasts, the lymphotropic strain of minute virus of mice (MVMi) does not efficiently produce progeny single-strand DNA (ssDNA). However, changing a single nucleotide in the MVMi 3' splice site to that found in the fibrotropic strain MVMp enabled full DNA replication and production of ssDNA. This change enhanced excision of the large intron and the production of NS2, likely by improving interaction, in fibroblasts with the branch point-binding U2 snRNA. One function of NS2 involves interaction with the nuclear export protein Crm1. The defect in production of MVMi ssDNA in fibroblasts can also be overcome by introducing a mutation in MVMi NS2 that enhances its interaction with Crm1. Although MVMi contains a 3' splice site that performs poorly in fibroblasts, MVMi generated at least as much R2 and NS2 in murine lymphocytes as did MVMp in fibroblasts. Therefore, it appears that MVMp has acquired a mutation that improves the excision of the large intron, as it adapted to fibroblasts to accommodate the need for NS2 for replication in these cells, and that the ratio of NS1 to NS2 may play a larger role in the host range of MVM than previously appreciated.     

Identification of the sialic acid structures recognized by minute virus of mice and the role of binding affinity in virulence adaptation

Sialic acid binding is required for infectious cell surface receptor recognition by parvovirus minute virus of mice (MVM). We have utilized a glycan array consisting of approximately 180 different carbohydrate structures to identify the specific sialosides recognized by the prototype (MVMp) and immunosuppressive (MVMi) strains of MVM plus three virulent mutants of MVMp, MVMp-I362S, MVMp-K368R, and MVMp-I362S/K368R. All of the MVM capsids specifically bound to three structures with a terminal sialic acid-linked alpha2-3 to a common Galbeta1-4GlcNAc motif: Neu5Acalpha2-3Galbeta1-4GlcNAcbeta1-4Galbeta1-4GlcNAc (3'SiaLN-LN), Neu5Acalpha2-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc (3'SiaLN-LN-LN), and Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)-GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAc (sLe(x)-Le(x)-Le(x)). In addition, MVMi also recognized four multisialylated glycans with terminal alpha2-8 linkages: Neu5Acalpha2-8Neu5Acalpha2-8Neu5Acalpha ((Sia)(3)), Neu5Acalpha2-8Neu5Acalpha2-3Galbeta1-4Glc (GD3), Neu5Acalpha2-8Neu5Acalpha2-8Neu5Acalpha2-3Galbeta1-4Glc (GT3), and Neu5Acalpha2-8Neu5Acalpha2-3(GalNAcbeta1-4)Galbeta1-4Glc (GD2). Interestingly, the virulent MVMp-K368R mutant also recognized GT3. Analysis of the relative binding affinities using a surface plasmon resonance biospecific interaction (BIAcore) assay showed the wild-type MVMp and MVMi capsids binding with higher affinity to selected glycans compared with the virulent MVMp mutants. The reduced affinity of the virulent MVMp mutants are consistent with previous in vitro cell binding assays that had shown weaker binding to permissive cells compared with wild-type MVMp. This study identifies the sialic acid structures recognized by MVM. It also provides rationale for the tropism of MVM for malignant transformed cells that contain sLe(x) motifs and the neurotropism of MVMi, which is likely mediated via interactions with multisialylated glycans known to be tumor cell markers. Finally, the observations further implicate a decreased binding affinity for sialic acid in the in vivo adaptation of MVMp to a virulent phenotype.     

Host cell specificity of minute virus of mice in the developing mouse embryo

Productive infection by the murine autonomous parvovirus minute virus of mice (MVM) depends on a dividing cell population and its differentiation state. We have extended the in vivo analysis of the MVM host cell type range into the developing embryo by in utero inoculation followed by further gestation. The fibrotropic p strain (MVMp) and the lymphotropic i strain (MVMi) did not productively infect the early mouse embryo but were able to infect overlapping sets of cell types in the mid- or late-gestation embryo. Both MVMp and MVMi infected developing bone primordia, notochord, central nervous system, and dorsal root ganglia. MVMp exhibited extensive infection in fibroblasts, in the epithelia of lung and developing nose, and, to a lesser extent, in the gut. MVMi also infected endothelium. The data indicated that the host ranges of the two MVM strains consist of overlapping sets of cell types that are broader than previously known from neonate and in vitro infection experiments. The correlation between MVM host cell types and the cell types that activate the transgenic P4 promoter is consistent with the hypothesis that activation of the incoming viral P4 promoter by the host cell is one of the host range determinants of MVM.     

Targeting human glioblastoma cells: comparison of nine viruses with oncolytic potential

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.     

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.     

Virulent variants emerging in mice infected with the apathogenic prototype strain of the parvovirus minute virus of mice exhibit a capsid with low avidity for a primary receptor

The mechanisms involved in the emergence of virulent mammalian viruses were investigated in the adult immunodeficient SCID mouse infected by the attenuated prototype strain of the parvovirus Minute Virus of Mice (MVMp). Cloned MVMp intravenously inoculated in mice consistently evolved during weeks of subclinical infection to variants showing altered plaque phenotypes. All the isolated large-plaque variants spread systemically from the oronasal cavity and replicated in major organs (brain, kidney, liver), in sharp contrast to the absolute inability of the MVMp and small-plaque variants to productively invade SCID organs by this natural route of infection. The virulent variants retained the MVMp capacity to infect mouse fibroblasts, consistent with the lack of genetic changes across the 220-to-335 amino acid sequence of VP2, a capsid domain containing main determinants of MVM tropism. However, the capsid of the virulent variants shared a lower affinity than the wild type for a primary receptor used in the cytotoxic infection. The capsid gene of a virulent variant engineered in the MVMp background endowed the recombinant virus with a large-plaque phenotype, lower affinity for the receptor, and productive invasiveness by the oronasal route in SCID mice, eventually leading to 100% mortality. In the analysis of virulence in mice, both MVMp and the recombinant virus similarly gained the bloodstream 1 to 2 days postoronasal inoculation and remained infectious when adsorbed to blood cells in vitro. However, the wild-type MVMp was cleared from circulation a few days afterwards, in contrast to the viremia of the recombinant virus, which was sustained for life. Significantly, attachment to an abundant receptor of primary mouse kidney epithelial cells by both viruses could be quantitatively competed by wild-type MVMp capsids, indicating that virulence is not due to an extended receptor usage in target tissues. We conclude that the selection of capsid-receptor interactions of low affinity, which favors systemic infection, is a major evolutionary process in the adaptation of parvoviruses to new hosts and in the cause of disease.     

The capsid determinant of fibrotropism for the MVMp strain of minute virus of mice functions via VP2 and not VP1.

The minute virus of mice, prototype strain MVMp, productively infects cultured murine fibroblasts but not T cells. The immunosuppressive strain, MVMi, shows the converse tropism. These reciprocal tropisms are mediated by the viral capsids, in which their determinants have been mapped to a few specific amino acids in the primary sequence shared by VP1 and VP2. Which of these proteins is relevant in presenting these determinants during infection is not known. We have approached this question using a recombinant parvovirus system in which a LuIII-derived transducing genome, containing the luciferase reporter in place of viral coding sequences, can be packaged by capsid proteins from separate helper sources. We generated transducing virions by using helper constructs expressing either VP1 or VP2, containing the MVMp or MVMi tropic determinant region, in various combinations. The virions were used to infect human NB324K cells and murine A9 fibroblasts. Transduction of the human cells (permissive for both MVMp and MVMi) required both VP1 and VP2 and was successful with all combinations of these proteins. In contrast, significant transducing activity for A9 cells was detected only with recombinant virions containing VP2 of MVMp, while the use of either source of VP1 had little effect. We conclude that VP2 from MVMp is necessary to enable infection of murine A9 fibroblasts.     

Structural determinants of tissue tropism and in vivo pathogenicity for the parvovirus minute virus of mice

Two strains of the parvovirus minute virus of mice (MVM), the immunosuppressive (MVMi) and the prototype (MVMp) strains, display disparate in vitro tropism and in vivo pathogenicity. We report the crystal structures of MVMp virus-like particles (MVMp(b)) and native wild-type (wt) empty capsids (MVMp(e)), determined and refined to 3.25 and 3.75 A resolution, respectively, and their comparison to the structure of MVMi, also refined to 3.5 A resolution in this study. A comparison of the MVMp(b) and MVMp(e) capsids showed their structures to be the same, providing structural verification that some heterologously expressed parvovirus capsids are indistinguishable from wt capsids produced in host cells. The structures of MVMi and MVMp capsids were almost identical, but local surface conformational differences clustered from symmetry-related capsid proteins at three specific domains: (i) the icosahedral fivefold axis, (ii) the "shoulder" of the protrusion at the icosahedral threefold axis, and (iii) the area surrounding the depression at the icosahedral twofold axis. The latter two domains contain important determinants of MVM in vitro tropism (residues 317 and 321) and forward mutation residues (residues 399, 460, 553, and 558) conferring fibrotropism on MVMi. Furthermore, these structural differences between the MVM strains colocalize with tropism and pathogenicity determinants mapped for other autonomous parvovirus capsids, highlighting the importance of common parvovirus capsid regions in the control of virus-host interactions.     

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.     

Growth of the parvovirus minute virus of mice MVMp3 in EL4 lymphocytes is restricted after cell entry and before viral DNA amplification: cell-specific differences in virus uncoating in vitro.

Two murine parvoviruses with genomic sequences differing only in 33 nucleotides (8 amino acids) in the region coding for the capsid proteins show different host cell specificities: MVMi grows in EL4 T lymphocytes and MVMp3 grows in A9 fibroblasts. In this study we compared the courses of infections with these two viruses in EL4 cells in order to investigate at which step(s) the infection process of MVMp3 is interrupted. The two viruses bound equally well to EL4 cells, and similar amounts of MVMi and MVMp3 input virion DNA appeared in the nuclear fractions of EL4 cells 1 h after infection. However, double-stranded replicative-form (RF) DNA of the two viruses appeared at different times, at 10 h postinfection with MVMi and at 24 h postinfection with MVMp3. The amount of MVMp3 RF DNA detected at 24 h was very small because it was produced only in a tiny subset of the population of EL4 cells that proved to be permissive for MVMp3. Replication of double-stranded viral DNA in EL4 cells was measured after transfection of purified RF DNA, cloned viral DNA, and cloned viral DNA with a mutation preventing synthesis of the capsid proteins. In each of these cases, DNA replication was comparable for MVMi and MVMp3. Production of virus particles also appeared to be similar after transfection of the two types of RF DNA into EL4 cells. Conversion of incoming 32P-labeled single-stranded MVM DNA to 32P-labeled double-stranded RF DNA was detected only after RF DNA amplification, indicating that few molecules serve as templates for viral DNA amplification. We showed that extracts of EL4 cells contain a factor which can destabilize MVMi virions but not MVMp3 by testing the sensitivity of viral DNA to DNase and by CsCl gradient analyses of viral particles. We therefore conclude that the MVMp3 life cycle is arrested after the transport of virions to the nucleus and prior to the replication of RF DNA, most likely at the stage of viral decapsidation.     

DNA sequence comparison between two tissue-specific variants of the autonomous parvovirus, minute virus of mice.

We have determined the complete nucleotide sequence of the DNA of the immunosuppressive variant of the parvovirus minute virus of mice (MVMi) and compared it to the published sequence (12) of the fibroblast-specific strain (MVMp). We have found 175 differences between the two viruses, most of which affect single nucleotides. Despite these differences, the genomic organization of MVMp and MVMi is identical. There are 29 amino-acid changes between the putative viral gene products of MVMi and MVMp, 16 of which are conservative. We discuss the possibility that the differential tissue-specificity of the two variants is linked to differences within the non-transcribed region near the 5' end of the viral genomes.     

Profiling of Glycan Receptors for Minute Virus of Mice in Permissive Cell Lines Towards Understanding the Mechanism of Cell Recognition

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3′SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3′SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3′SIA-Le^X identified in a previous glycan microarray screen.     

NS2 is required for efficient translation of viral mRNA in minute virus of mice-infected murine cells.

Detailed analysis of five NS2 mutants of the autonomous parvovirus minute virus of mice (MVMp) has revealed the following. At low multiplicities of infection, NS2 mutants killed NB324K cells as well as wild-type (wt) MVM did and grew to high titers, while in contrast they grew poorly and did not readily kill murine A9 cells. Following CaPO4 transfection of murine fibroblasts, NS2 mutant infectious clones generated approximately 10-fold less monomer replicative-form DNA than wt and no detectable progeny single-stranded DNA. On nonmurine semipermissive NB324K cells, however, these mutant plasmid clones generated near wt levels of all replicative DNA forms. After infection of highly synchronized murine fibroblasts by NS2 mutant virus at inputs equivalent to those of the wt, mutant monomer replicative-form DNA was decreased 5- to 10-fold compared with that of the wt, and progeny single-stranded DNA accumulation was decreased to an even greater extent. Both total and cytoplasmic NS2 mutant RNA was decreased, but the amount of total viral mRNA generated, relative to accumulated viral DNA in the same experiments, was similar to that seen in wt infection. The accumulation of virus-generated proteins was also decreased in NS2 mutant infection; however, the magnitude of this decrease, compared with that of wt infections, was significantly greater than the concomitant decrease in mutant-generated levels of accumulated cytoplasmic RNA, and this effect was most dramatic for VP2. There was no such disparity between the relative accumulation of mutant-generated RNA and protein in cells permissive for the growth of these mutants. These results suggest that translation of MVM viral RNA is specifically reduced in NS2 mutant infection of restrictive cells. Because the affected viral proteins are required for the efficient production of viral replicative DNA forms, these results reveal a fundamental, although perhaps not the only, role for NS2 in parvovirus infection.     

Myeloid depression follows infection of susceptible newborn mice with the parvovirus minute virus of mice (strain i).

The in vivo myelosuppressive capacity of strain i of the parovirus minute virus of mice (MVMi) was investigated in newborn BALB/c mice inoculated with a lethal intranasal dose. MVMi infection reached maximum levels of DNA synthesis and infectious titers in lymphohemopoietic organs at 4 to 6 days postinoculation and was restricted by an early neutralizing humoral immune response. After viral control (by 10 days postinoculation), a significant decrease in femoral and splenic cellularity, as well as in granulocyte-macrophage colony-forming unit and erythroid burst-forming unit hemopoietic progenitors, was observed in most inoculated animals. This delayed myeloid depression, although it may be not a major cause of the lethality of the infection, implies indirect pathogenic mechanisms induced by MVMi infection in a susceptible host.     

The NS2 proteins of parvovirus minute virus of mice are required for efficient nuclear egress of progeny virions in mouse cells

The small nonstructural NS2 proteins of parvovirus minute virus of mice (MVMp) were previously shown to interact with the nuclear export receptor Crm1. We report here the analysis of two MVM mutant genomic clones generating NS2 proteins that are unable to interact with Crm1 as a result of amino acid substitutions within their nuclear export signal (NES) sequences. Upon transfection of human and mouse cells, the MVM-NES21 and MVM-NES22 mutant genomic clones were proficient in synthesis of the four virus-encoded proteins. While the MVM-NES22 clone was further able to produce infectious mutant virions, no virus could be recovered from cells transfected with the MVM-NES21 clone. Whereas the defect of MVM-NES21 appeared to be complex, the phenotype of MVM-NES22 could be traced back to a novel distinct NS2 function. Infection of mouse cells with the MVM-NES22 mutant led to stronger nuclear retention not only of the NS2 proteins but also of infectious progeny MVM particles. This nuclear sequestration correlated with a severe delay in the release of mutant virions in the medium and with prolonged survival of the infected cell populations compared with wild-type virus-treated cultures. This defect could explain, at least in part, the small size of the plaques generated by the MVM-NES22 mutant when assayed on mouse indicator cells. Altogether, our data indicate that the interaction of MVMp NS2 proteins with the nuclear export receptor Crm1 plays a critical role at a late stage of the parvovirus life cycle involved in release of progeny viruses.     

Minute virus of mice small nonstructural protein NS2 interacts and colocalizes with the Smn protein

The small nonstructural protein NS2 of the minute virus of mice (MVM) is required for efficient viral replication, although its mode of action is unclear. Here we demonstrate that NS2 and survival motor neuron protein (Smn) interact in vitro and in vivo. NS2 and Smn also colocalize in infected nuclei at late times following MVM infection.     

Experimentally induced infection with autonomous parvoviruses, minute virus of mice and H-1, in the African multimammate mouse (Mastomys coucha)

To determine whether the multimammate mouse (Mastomys coucha) could be used to evaluate rodent parvovirus-based vectors, neonates were subcutaneously inoculated with minute virus of mice (prototype strain, MVMp) or rat parvovirus H-1. The course of infection with both viruses was similar. Seroconversion occurred within two weeks after virus inoculation, as detected by use of hemagglutination-inhibition assays, and antibody titers remained high for the entire observation period of 12 months. Viral DNA and infective virions were detected in several organs of inoculated animals prior to seroconversion, as measured by use of Southern blotting and plaque assays, respectively. Infective particles subsequently became undetectable, whereas viral DNA imprints persisted in distinct organs for at least nine months. Clinical signs of parvovirus infection appeared around six weeks after virus inoculation, and consisted of hemorrhages, stunted growth, and transient hair color changes. Sudden death occurred in a significant fraction of animals infected with MVMp, but not H-1 virus, at the time of weaning. Altogether, MVMp, which is innocuous to its natural host, the mouse, and H-1 virus, which is poorly pathogenic to the rat, appear to be pathogenic for Mastomys coucha.     

Minute virus of mice: antibody response, viral shedding, and persistence of viral DNA in multiple strains of mice

Minute virus of mice (MVM) is a major concern for laboratory animal facilities because it remains with considerably high prevalence despite strict barrier systems. The aim of this study was to elucidate potential risks associated with MVM infection by investigating the role of the genetic background on antibody production and persistence as well as viral shedding. Mice of various strains and stocks were inoculated oronasally with the immunosuppressive strain MVMi; in addition, natural infection was modeled through contact exposure. As determined by serology, seroconversion and serum levels of IgG differed considerably among strains and stocks, especially in the contact-exposed group. For example, C57BL/6J mice responded well to exposure in contrast to FVB/N, NMRI, ICR, and C3H/HeN mice. Titration studies indicated that the viral dose necessary to induce seroconversion was strain-dependent. Experiments to dissect the role of the major histocompatibility complex haplotype in the response to MVMi gave inconclusive results. To detect viral persistence, spleens and feces were analyzed by PCR at 16 wk after exposure, and the infectivity of PCR-positive spleens was investigated by IP and oronasal inoculation of naive mice. Although DNA was detected in the spleens of some mice, feces remained negative, and naive mice were not infected by inoculation. In addition, viral shedding declined rapidly after day 20 postinoculation. In summary, the data show that seroconversion and antibody response to MVMi infection depend on the genetic background of mice, with the infective dose being a critical factor. The role of viral DNA in chronically infected mice will require further elucidation.