RESUMO
Single-molecule experimental techniques and theoretical approaches reveal that important aspects of virus biology can be understood in biomechanical terms at the nanoscale. A detailed knowledge of the relationship in virus capsids between small structural changes caused by single-point mutations and changes in mechanical properties may provide further physics-based insights into virus function; it may also facilitate the engineering of viral nanoparticles with improved mechanical behavior. Here, we used the minute virus of mice to undertake a systematic experimental study on the contribution to capsid stiffness of amino acid side chains at interprotein interfaces and the specific noncovalent interactions they establish. Selected side chains were individually truncated by introducing point mutations to alanine, and the effects on local and global capsid stiffness were determined using atomic force microscopy. The results revealed that, in the natural virus capsid, multiple, mostly hydrophobic, side chains buried along the interfaces between subunits preserve a comparatively low stiffness of most (S2 and S3) regions. Virtually no point mutation tested substantially reduced stiffness, whereas most mutations increased stiffness of the S2/S3 regions. This stiffening was invariably associated with reduced virus yields during cell infection. The experimental evidence suggests that a comparatively low stiffness at S3/S2 capsid regions may have been biologically selected because it facilitates capsid assembly, increasing infectious virus yields. This study demonstrated also that knowledge of individual amino acid side chains and biological pressures that determine the physical behavior of a protein nanoparticle may be used for engineering its mechanical properties.
Assuntos
Aminoácidos/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Vírus Miúdo do Camundongo/química , Vírus Miúdo do Camundongo/patogenicidade , Infecções por Parvoviridae/virologia , Aminoácidos/química , Vírus Miúdo do Camundongo/isolamento & purificação , Vírus Miúdo do Camundongo/fisiologiaRESUMO
Engagement of innate viral sensors elicits a robust antiviral program via the induction of type I interferons (IFNs). Innate defense mechanisms against ssDNA viruses are not well defined. Here, we examine type I IFN induction and effectiveness in controlling a ssDNA virus. Using mouse embryonic fibroblasts (MEFs), we found that a murine parvovirus, minute virus of mice (MVMp), induced a delayed but significant IFN response. MEFs deficient in mitochondrial antiviral signaling protein (MAVS) mounted a wild-type IFN response to MVMp infection, indicating that RIG-I-dependent RNA intermediate recognition is not required for innate sensing of this virus. However, MVMp-induced IFNs, as well recombinant type I IFNs, were unable to inhibit viral replication. Finally, MVMp infected cells became unresponsive to Poly (I:C) stimulation. Together, these data suggest that the MVMp efficiently evades antiviral immune mechanisms imposed by type I IFNs, which may in part explain their efficient transmission between mice.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antivirais/imunologia , Fibroblastos/virologia , Interferon Tipo I/imunologia , Vírus Miúdo do Camundongo/imunologia , Vírus Miúdo do Camundongo/patogenicidade , Infecções por Parvoviridae/imunologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antivirais/metabolismo , Feminino , Fibroblastos/imunologia , Imunidade Inata , Interferon Tipo I/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Vírus Miúdo do Camundongo/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Infecções por Parvoviridae/virologia , RNA Polimerase III , Receptores de Superfície Celular , Replicação Viral/imunologiaRESUMO
Cellular factors associated with the parvovirus minute virus of mice (MVM) during infection are thought to play important roles in the MVM life cycle but only a few of these have been identified. Here we used a proteomic-based approach in order to identify host-binding partners of MVM. Using purified MVM as bait for immunoprecipitation assays, a total of 150 proteins were identified in MVM immunoprecipitates by quantitative liquid chromatography-tandem mass spectrometry. Galectin-3 was one of six proteins showing a statistically significant enrichment across replicates. Small interfering RNA depletion studies revealed an important role for galectin-3 in MVM endocytosis and infectivity in LA9 mouse fibroblast cells. Galectin-3-depleted cells were less susceptible to MVM infection than control cells and showed a significant reduction of MVM cellular uptake, but not of MVM binding to the cell surface. Our results indicate an important role for galectin-3 in the cellular uptake of MVM. We propose that galectin-3 facilitates the access of MVM to its receptor(s) at the plasma membrane and in this way promotes MVM endocytosis.
Assuntos
Galectina 3/fisiologia , Vírus Miúdo do Camundongo/fisiologia , Receptores Virais/fisiologia , Internalização do Vírus , Animais , Endocitose/fisiologia , Camundongos , Vírus Miúdo do Camundongo/genética , Vírus Miúdo do Camundongo/patogenicidade , Proteômica , Replicação Viral/genéticaRESUMO
Parvoviruses are small, nonenveloped, single-stranded DNA viruses which replicate in the nucleus of the host cell. We have previously found that early during infection the parvovirus minute virus of mice (MVM) causes small, transient disruptions of the nuclear envelope (NE). We have now investigated the mechanism used by MVM to disrupt the NE. Here we show that the viral phospholipase A2, the only known enzymatic domain on the parvovirus capsid, is not involved in causing NE disruption. Instead, the virus utilizes host cell caspases, which are proteases involved in causing NE breakdown during apoptosis, to facilitate these nuclear membrane disruptions. Studies with pharmacological inhibitors indicate that caspase-3 in particular is involved. A caspase-3 inhibitor prevents nuclear lamin cleavage and NE disruption in MVM-infected mouse fibroblast cells and reduces nuclear entry of MVM capsids and viral gene expression. Caspase-3 is, however, not activated above basal levels in MVM-infected cells, and other aspects of apoptosis are not triggered during early MVM infection. Instead, basally active caspase-3 is relocalized to the nuclei of infected cells. We propose that NE disruption involving caspases plays a role in (i) parvovirus entry into the nucleus and (ii) alteration of the compartmentalization of host proteins in a way that is favorable for the virus.
Assuntos
Caspase 3/metabolismo , Vírus Miúdo do Camundongo/patogenicidade , Membrana Nuclear/patologia , Animais , Linhagem Celular , Fibroblastos/virologia , Humanos , Camundongos , Fosfolipases A2/metabolismo , Proteínas Virais/metabolismoRESUMO
Parvoviruses infect and kill tumor cells in vivo and in vitro more efficiently than normal cells. Infection of transformed cells by the parvovirus minute virus of mice (MVM) results in high expression of the major non-structural cytolytic viral protein NS1, which induces a cell death modulated by cellular factors. In this work, we show that MVMp infection and/or NS1 protein expression in permissive transformed rat fibroblast cells leads to apoptosis in wild type and p53(-/-) cells. Apoptotic cell morphology correlates with mitochondrial membrane permeabilization and activation of caspases 9 and 3 but not caspase 8. Thus, further characterization of the antitumor activity of MVMp and its NS1 protein may contribute to the eradication of tumors, including those lacking p53.
Assuntos
Apoptose , Fibroblastos/fisiologia , Fibroblastos/virologia , Vírus Miúdo do Camundongo/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Caspase 3/metabolismo , Caspase 9/metabolismo , Células Cultivadas , Camundongos , Camundongos Knockout , Ratos , Proteína Supressora de Tumor p53/deficiênciaRESUMO
Infections with the autonomous parvovirus Minute virus of mice (MVM) are generally characterized as acute and self-limiting. However, MVM remains with considerably high prevalence rates in laboratory mouse colonies impeding rodent based research. The objective of this study was to assess whether the immunosuppressive variant of MVM (MVMi) establishes a persistent infection in immunocompetent adult mice. Therefore, we approached the question whether replicating and/or infectious virus is present in mice after the decline of viral shedding and whether immunosuppression might modify the infection. Dissection or induction of immunosuppression of individually housed mice was performed at 8 weeks post inoculation after fecal samples tested negative for viral DNA for at least 2 subsequent weeks as determined by weekly PCR analyses. MVMi mRNA was detected by both, RT-PCR and in situ RT-PCR in spleens at 8 weeks post inoculation with positive cells resembling lymphocytes and macrophages. These findings and the use of explant cultures strongly indicated the presence of replicating virus in spleens at 8 weeks post inoculation. Following immunosuppression (by irradiation), an induction of viral shedding was observed. Additionally, an increase in the amount of viral DNA was detected by real-time qPCR in mesenteric lymph nodes after irradiation. In summary, our data support the notion that MVMi persists in lymphoid tissue of immunocompetent adult mice despite the onset of host immunity.
Assuntos
Vírus Miúdo do Camundongo/imunologia , Infecções por Parvoviridae/veterinária , Doenças dos Roedores/virologia , Imunidade Adaptativa/imunologia , Animais , DNA Viral/análise , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Masculino , Camundongos , Vírus Miúdo do Camundongo/patogenicidade , Infecções por Parvoviridae/imunologia , Infecções por Parvoviridae/virologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Doenças dos Roedores/imunologia , Baço/virologia , Eliminação de Partículas Virais/imunologiaRESUMO
Hamster parvovirus (HaPV) was isolated 2 decades ago from hamsters with clinical signs similar to those induced in hamsters experimentally infected with other rodent parvoviruses. Genetically, HaPV is most closely related to mouse parvovirus (MPV), which induces subclinical infection in mice. A novel MPV strain, MPV3, was detected recently in naturally infected mice, and genomic sequence analysis indicates that MPV3 is almost identical to HaPV. The goal of the present studies was to examine the infectivity of HaPV in mice. Neonatal and weanling mice of several mouse strains were inoculated with HaPV. Tissues, excretions, and sera were harvested at 1, 2, 4, and 8 wk after inoculation and evaluated by quantitative PCR and serologic assays specific for HaPV. Quantitative PCR detected viral DNA quantities that greatly exceeded the quantity of virus in inocula in multiple tissues of infected mice. Seroconversion to both nonstructural and structural viral proteins was detected in most immunocompetent mice 2 or more weeks after inoculation with HaPV. In neonatal SCID mice, viral transcripts were detected in lymphoid tissues by RT-PCR and viral DNA was detected in feces by quantitative PCR at 8 wk after inoculation. No clinical signs, gross, or histologic lesions were observed. These findings are similar to those observed in mice infected with MPV. These data support the hypothesis that HaPV and MPV3 are likely variants of the same viral species, for which the mouse is the natural rodent host with rare interspecies transmission to the hamster.
Assuntos
Infecções por Parvoviridae , Parvovirus/patogenicidade , Doenças dos Roedores , Animais , Cricetinae/virologia , Cricetulus/virologia , DNA Viral/análise , DNA Viral/isolamento & purificação , Feminino , Masculino , Camundongos , Camundongos Endogâmicos/virologia , Camundongos SCID/virologia , Vírus Miúdo do Camundongo/genética , Vírus Miúdo do Camundongo/patogenicidade , Infecções por Parvoviridae/transmissão , Infecções por Parvoviridae/veterinária , Parvovirus/genética , Doenças dos Roedores/transmissão , Doenças dos Roedores/virologiaRESUMO
The relevance of translational control in the gene expression and oncotropism of the autonomous parvoviruses was investigated with MVMp, the prototype strain of minute virus of mice (MVM), infecting normal and transformed rodent and human cells of different tissue origins. Mouse embryo fibroblasts (MEFs) and NIH 3T3 fibroblasts were resistant to MVMp infection, but 3T3 fibroblasts derived from double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) knockout mice (PKR(o/o)) behaved in a manner that was highly permissive to productive MVMp replication. NIH 3T3 resistance correlated with significant phosphorylation of eukaryotic translation initiation factor 2 (eIF2) occurring at early time points after infection. Permissive PKR(o/o) cells were converted to MVMp-restrictive cells after reintroduction of the PKR gene by transfection. Conversely, regulated expression of the vaccinia virus E3 protein, a PKR inhibitor, in MEFs prevented eIF2alpha phosphorylation and increased MVMp protein synthesis. In vitro-synthesized genome-length R1 mRNA of MVMp was a potent activator of PKR. Virus-resistant primary MEFs and NIH 3T3 cells responded to MVMp infection with significant increases in eIF2alpha phosphorylation. In contrast, virus-permissive mouse (PKR(o/o), BHK21, and A9) and human transformed (NB324K fibroblast, U373 glioma, and HepG2 hepatoma) cells consistently showed no significant increase in the level of eIF2alpha phosphorylation following MVMp infection. The synthesis of the viral NS1 protein was inversely correlated with the steady-state PKR levels. Our results show that the PKR-mediated antiviral response is an important mechanism for control of productive MVMp infection, and its impairment in human transformed cells allowed efficient MVMp gene expression. PKR translational control may therefore contribute to the oncolysis of MVMp and other autonomous parvoviruses.
Assuntos
Vírus Miúdo do Camundongo/imunologia , Vírus Miúdo do Camundongo/patogenicidade , Biossíntese de Proteínas , Proteínas Virais/biossíntese , Replicação Viral , eIF-2 Quinase/imunologia , eIF-2 Quinase/metabolismo , Animais , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Fibroblastos/virologia , Teste de Complementação Genética , Hepatócitos/virologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroglia/virologia , Tropismo Viral , eIF-2 Quinase/deficiênciaRESUMO
The aim of this study was to determine the susceptibility of murine embryonic stem (mESCs) to mouse hepatitis virus (MHV-A59) and mouse minute virus (MMVp) and the effect of these viruses on germline transmission (GLT) and the serological status of recipients and pups. When recipients received 10 blastocysts, each injected with 10(0) TCID(50) MHV-A59, three out of five recipients and four out of 14 pups from three litters became seropositive. When blastocysts were injected with 10(-5) TCID(50) MMVp, all four recipients and 14 pups from four litters remained seronegative. The mESCs replicated MHV-A59 but not MMVp, MHV-A59 being cytolytic for mESCs. Exposure of mESCs to the viruses over four to five passages but not for 6 h affected GLT. Recipients were seropositive for MHV-A59 but not for MMVp when mESCs were cultured with the virus over four or five passages. The data show that GLT is affected by virus-contaminated mESCs.
Assuntos
Blastocisto/virologia , Infecções por Coronavirus/virologia , Células-Tronco Embrionárias/virologia , Células Germinativas/fisiologia , Hepatite Viral Animal/transmissão , Vírus Miúdo do Camundongo/patogenicidade , Vírus da Hepatite Murina/patogenicidade , Infecções por Parvoviridae/virologia , Animais , Transferência Embrionária , Feminino , Fertilização in vitro , Citometria de Fluxo , Hepatite Viral Animal/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Vírus Miúdo do Camundongo/isolamento & purificação , Vírus Miúdo do Camundongo/metabolismo , Vírus da Hepatite Murina/isolamento & purificação , Vírus da Hepatite Murina/metabolismo , Receptores Virais/metabolismo , Estudos Soroepidemiológicos , Replicação ViralRESUMO
Well-defined tissue tropism makes Autonomous Parvoviruses a valuable model for studies of virus-cell interactions and gene therapy research. We developed a new Minute Virus of Mice variant, different from the known prototype (MVMp) and immunosuppressive (MVMi) strains. The new virus variant, designated F1, was isolated from the culture of semi-permissive Fisher Rat Fibroblasts, F111, infected with MVMp. The F1 genome carried point mutations in regions known to determine the mutually restricted host ranges of MVMp and MVMi. In F111 cells, F1 cytotoxicity, gene expression and multiplication were significantly higher compared to MVMp. Conversely the wild-type virus propagated in MVMp-permissive cells more efficiently than the F1. Reversion of the F1-specific mutations to wild-type MVMp sequence, following reverse-passaging of the mutant virus in MVMp-permissive cells, confirmed a specific adaptation of the F1 virus to F111 cells. Considerable divergence in tissue specificities between the wild-type and mutant viruses was demonstrated in vivo.
Assuntos
Vírus Miúdo do Camundongo/patogenicidade , Adaptação Fisiológica , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Linhagem Celular , Primers do DNA/genética , DNA Viral/genética , Genes Virais , Variação Genética , Interações Hospedeiro-Patógeno/genética , Humanos , Camundongos , Vírus Miúdo do Camundongo/genética , Vírus Miúdo do Camundongo/fisiologia , Modelos Moleculares , Especificidade de Órgãos , Mutação Puntual , Regiões Promotoras Genéticas , Ratos , Virulência/genéticaRESUMO
Intermittent serodetection of mouse parvovirus (MPV) infections in animal facilities occurs frequently when soiled bedding sentinel mouse monitoring systems are used. We evaluated induction of seroconversion in naïve single-caged weanling ICR mice (n = 10 per group) maintained on 5-fold serially diluted contaminated bedding obtained from SCID mice persistently shedding MPV1e. Soiled bedding from the infected SCID mice was collected, diluted, and redistributed weekly to cages housing ICR mice to represent chronic exposure to MPV at varying prevalence in a research colony. Sera was collected every other week for 12 wk and evaluated for reactivity to MPV nonstructural and capsid antigens by multiplex fluorescent immunoassay. Mice were euthanized after seroconversion, and DNA extracted from lymph node and spleen was evaluated by quantitative PCR. Cumulative incidence of MPV infection for each of the 7 soiled bedding dilution groups (range, 1:5 to 1:78125 [v/v]) was 100%, 100%, 90%, 20%, 70%, 60%, and 20%, respectively. Most seropositive mice (78%) converted within the first 2 to 3 wk of soiled bedding exposure, correlating to viral exposure when mice were 4 to 7 wk of age. Viral DNA was detected in lymphoid tissues collected from all mice that were seropositive to VP2 capsid antigen, whereas viral DNA was not detected in lymphoid tissue of seronegative mice. These data indicate seroconversion occurs consistently in young mice exposed to high doses of virus equivalent to fecal MPV loads observed in acutely infected mice, whereas seroconversion is inconsistent in mice chronically exposed to lower doses of virus.
Assuntos
Transmissão de Doença Infecciosa/veterinária , Abrigo para Animais , Vírus Miúdo do Camundongo/patogenicidade , Infecções por Parvoviridae/veterinária , Doenças dos Roedores/transmissão , Animais , DNA Viral/análise , Fezes/virologia , Feminino , Linfonodos/química , Linfonodos/virologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Infecções por Parvoviridae/sangue , Infecções por Parvoviridae/transmissão , Gravidez , Doenças dos Roedores/virologia , Testes Sorológicos/veterinária , Organismos Livres de Patógenos Específicos , Baço/química , Baço/virologia , Eliminação de Partículas ViraisRESUMO
CRM1 exports proteins that carry a short leucine-rich peptide signal, the nuclear export signal (NES), from the nucleus. Regular NESs must have low affinity for CRM1 to function optimally. We previously generated artificial NESs with higher affinities for CRM1, termed supraphysiological NESs. Here we identify a supraphysiological NES in an endogenous protein, the NS2 protein of parvovirus Minute Virus of Mice (MVM). NS2 interacts with CRM1 without the requirement of RanGTP, whereas addition of RanGTP renders the complex highly stable. Mutation of a single hydrophobic residue that inactivates regular NESs lowers the affinity of the NS2 NES for CRM1 from supraphysiological to regular. Mutant MVM harboring this regular NES is compromised in viral nuclear export and productivity. In virus-infected mouse fibroblasts we observe colocalization of NS2, CRM1 and mature virions, which is dependent on the supraphysiological NS2 NES. We conclude that supraphysiological NESs exist in nature and that the supraphysiological NS2 NES has a critical role in active nuclear export of mature MVM particles before cell lysis.
Assuntos
Núcleo Celular/metabolismo , Núcleo Celular/virologia , Vírus Miúdo do Camundongo/metabolismo , Sinais de Exportação Nuclear , Proteínas não Estruturais Virais/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Carioferinas/metabolismo , Camundongos , Vírus Miúdo do Camundongo/patogenicidade , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ligação Proteica , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas não Estruturais Virais/química , Vírion/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Proteína Exportina 1RESUMO
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.
Assuntos
Proteínas do Capsídeo/fisiologia , Variação Genética , Vírus Miúdo do Camundongo/patogenicidade , Infecções por Parvoviridae/virologia , Substituição de Aminoácidos , Animais , Proteínas do Capsídeo/genética , Células Cultivadas , Análise Mutacional de DNA , Feminino , Camundongos , Camundongos SCID , Vírus Miúdo do Camundongo/genética , Proteínas Mutantes/genética , Infecções por Parvoviridae/patologia , Ensaio de Placa Viral , VirulênciaRESUMO
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 cell's 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.
Assuntos
Infecções por Parvoviridae/fisiopatologia , Infecções por Parvoviridae/virologia , Parvovirus , Sequência de Aminoácidos , Animais , Gatos , Linhagem Celular , Cães , Humanos , Camundongos , Vírus Miúdo do Camundongo/química , Vírus Miúdo do Camundongo/patogenicidade , Vírus Miúdo do Camundongo/ultraestrutura , Modelos Moleculares , Dados de Sequência Molecular , Parvovirus/química , Parvovirus/patogenicidade , Parvovirus/ultraestrutura , Parvovirus Canino/química , Parvovirus Canino/patogenicidade , Parvovirus Canino/ultraestrutura , Ratos , Especificidade da Espécie , Vírion/química , Vírion/ultraestruturaRESUMO
The structural and functional relevance of amino acid residues surrounding cavities within the hydrophobic core of the protein subunits that form the capsid of parvoviruses has been investigated. Several of the evolutionarily conserved, hydrophobic residues that delimit these cavities in the capsid of the minute virus of mice were replaced by other hydrophobic residues that would affect the size and/or shape of the cavity. When four or more methylene-sized groups were introduced, or six or more groups removed, capsid assembly was drastically impaired. In contrast, the introduction or removal of up to three groups had no significant effect on capsid assembly or thermostability. However, many of these mutations affected a capsid conformational transition needed for viral infectivity. Replacement of some polar residues around the largest cavity showed that capsid assembly requires a carboxylate buried within this cavity, but both aspartate and glutamate are structurally accepted. Again, only the aspartate allowed the production of infectious viruses, because of a specific role in encapsidation of the viral genome. These observations provide evidence of a remarkable structural tolerance to mutation of the hydrophobic core of the protein subunits in a viral capsid, and of an involvement of core residues and internal cavities in capsid functions needed for infectivity.
Assuntos
Proteínas do Capsídeo/química , Vírus Miúdo do Camundongo/metabolismo , Substituição de Aminoácidos , Animais , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Células Cultivadas , DNA Viral/química , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Microscopia Eletrônica de Transmissão , Vírus Miúdo do Camundongo/genética , Vírus Miúdo do Camundongo/patogenicidade , Modelos Moleculares , Mutagênese Sítio-Dirigida , MutaçãoRESUMO
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.
Assuntos
Vírus Miúdo do Camundongo/metabolismo , Vírus Miúdo do Camundongo/patogenicidade , Ácido N-Acetilneuramínico/química , Sítios de Ligação , Capsídeo/química , Modelos Moleculares , Mutação , Fenótipo , Polissacarídeos/química , Ligação Proteica , VirulênciaRESUMO
Random-source DNA samples obtained from naturally infected laboratory mice (n=381) were evaluated by PCR and RFLP analysis to determine the prevalence of murine parvovirus strains circulating in contemporary laboratory mouse colonies. Mouse parvovirus (MPV) was detected in 77% of samples, Minute virus of mice (MVM) was detected in 16% of samples and both MVM and MPV were detected in 7% of samples. MVMm, a strain recently isolated from clinically ill NOD-mu chain knockout mice, was detected in 91% of MVM-positive samples, with the Cutter strain of MVM (MVMc) detected in the remaining samples. The prototypic and immunosuppressive strains of MVM were not detected in any of the samples. MPV-1 was detected in 78% of the MPV-positive samples and two newly identified murine parvoviruses, tentatively named MPV-2 and MPV-3, were detected in 21 and 1% of the samples, respectively. The DNA sequence encompassing coding regions of the viral genome and the predicted protein sequences for MVMm, MPV-2 and MPV-3 were determined and compared with those of other rodent parvovirus strains and LuIII parvovirus. The genomic organization for the newly identified viral strains was similar to that of other rodent parvoviruses, and nucleotide sequence identities indicated that MVMm was most similar to MVMc (96.1%), MPV-3 was most similar to hamster parvovirus (HaPV) (98.1%) and MPV-2 was most similar to MPV-1 (95.3%). The genetic similarity of MPV-3 and HaPV suggests that HaPV epizootics in hamsters may result from cross-species transmission, with mice as the natural rodent host for this virus.
Assuntos
Animais de Laboratório , Vírus Miúdo do Camundongo/classificação , Epidemiologia Molecular , Infecções por Parvoviridae/veterinária , Parvovirus/classificação , Doenças dos Roedores/epidemiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Viral/análise , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Vírus Miúdo do Camundongo/genética , Vírus Miúdo do Camundongo/isolamento & purificação , Vírus Miúdo do Camundongo/patogenicidade , Dados de Sequência Molecular , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/virologia , Parvovirus/genética , Parvovirus/isolamento & purificação , Parvovirus/patogenicidade , Prevalência , Doenças dos Roedores/virologia , Análise de Sequência de DNA , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Many animal viruses produce cytopathic effects in their host cells during a productive infection. While some virus infections can be assayed by the production of plaques, many viruses, while producing cytotoxicity, do not easily form plaques, or do not form plaques at all. Additionally, viruses within families such as the parvoviruses may have different preferred forms of titration making comparative virology difficult even among related groups. Porcine parvovirus (PPV), canine parvovirus (CPV), and minute virus of mice (MVM) are usually titrated using different infectivity assays. A direct comparison of infectious virus titer between these parvoviruses was sought, and a tetrazolium salt assay, MTT has been applied to measure cytopathic effect produced by viral infection for different members of the parvovirus family. Infectious PPV measured using the MTT and the TCID50 assays exhibited excellent correlation and titers for CPV and MVM were consistently duplicated using the MTT assay. The MTT assay was also applied to an unrelated virus, Sindbis, which is routinely titrated by plaque assay. MTT titration of Sindbis virus mutants was found to be valuable for preliminary screening. This assay can be adapted, by correlation to an accepted titration method, to any viral system which produces measurable cytopathic effect.
Assuntos
Colorimetria/métodos , Efeito Citopatogênico Viral , Vírus/patogenicidade , Animais , Linhagem Celular , Cricetinae , Cães , Formazans/metabolismo , Humanos , Vírus Miúdo do Camundongo/patogenicidade , Parvovirus Canino/patogenicidade , Parvovirus Suíno/patogenicidade , Reprodutibilidade dos Testes , Sindbis virus/patogenicidade , Suínos , Sais de Tetrazólio/metabolismo , Ensaio de Placa ViralRESUMO
The role of receptor recognition in the emergence of virulent viruses was investigated in the infection of severe combined immunodeficient (SCID) mice by the apathogenic prototype strain of the parvovirus minute virus of mice (MVMp). Genetic analysis of isolated MVMp viral clones (n = 48) emerging in mice, including lethal variants, showed only one of three single changes (V325M, I362S, or K368R) in the common sequence of the two capsid proteins. As was found for the parental isolates, the constructed recombinant viruses harboring the I362S or the K368R single substitutions in the capsid sequence, or mutations at both sites, showed a large-plaque phenotype and lower avidity than the wild type for cells in the cytotoxic interaction with two permissive fibroblast cell lines in vitro and caused a lethal disease in SCID mice when inoculated by the natural oronasal route. Significantly, the productive adsorption of MVMp variants carrying any of the three mutations selected through parallel evolution in mice showed higher sensitivity to the treatment of cells by neuraminidase than that of the wild type, indicating a lower affinity of the viral particle for the sialic acid component of the receptor. Consistent with this, the X-ray crystal structure of the MVMp capsids soaked with sialic acid (N-acetyl neuraminic acid) showed the sugar allocated in the depression at the twofold axis of symmetry (termed the dimple), immediately adjacent to residues I362 and K368, which are located on the wall of the dimple, and approximately 22 A away from V325 in a threefold-related monomer. This is the first reported crystal structure identifying an infectious receptor attachment site on a parvovirus capsid. We conclude that the affinity of the interactions of sialic-acid-containing receptors with residues at or surrounding the dimple can evolutionarily regulate parvovirus pathogenicity and adaptation to new hosts.
Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Vírus Miúdo do Camundongo/genética , Vírus Miúdo do Camundongo/fisiologia , Ácido N-Acetilneuramínico/metabolismo , Substituição de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteínas do Capsídeo/fisiologia , Linhagem Celular , Cristalografia por Raios X , DNA Viral/genética , Feminino , Genes Virais , Humanos , Camundongos , Camundongos SCID , Vírus Miúdo do Camundongo/patogenicidade , Modelos Moleculares , Mutagênese Sítio-Dirigida , Parvovirus , Fenótipo , Estrutura Terciária de Proteína , Receptores Virais/fisiologia , Virulência/genética , Virulência/fisiologiaRESUMO
Cylindrical projections surrounding the fivefold-symmetry axes in minute virus of mice (MVM) harbor central pores that penetrate through the virion shell. In newly released DNA-containing particles, these pores contain residues 28 to 38 belonging to a single copy of VP2, disposed so that its extreme N-terminal domain projects outside the particle. Virions are metastable, initially sequestering internally the N termini of all copies of the minor capsid protein, VP1, that is essential for entry. This VP1 domain can be externalized in vitro in response to limited heating, and we show here that the efficiency of this transition is greatly enhanced by proteolysis of VP2 N termini to yield VP3. This step also renders the VP1 rearrangement pH dependent, indicating that VP2 cleavage is a maturation step required to prime subsequent emergence of the VP1 "entry" domain. The tightest constriction within the cylinder is created by VP2 leucine 172, the five symmetry-related copies of which form a portal that resembles an iris diaphragm across the base of the pore. In MVMp, threonine substitution at this position, L172T, yields infectious particles following transfection at 37 degrees C, but these can initiate infection only at 32 degrees C, and this process can be blocked by exposing virions to a cellular factor(s) at 37 degrees C during the first 8 h after entry. At 32 degrees C, the mutant particle is highly infectious, and it remains stable prior to VP2 cleavage or following cleavage at pH 5.5 or below. However, upon exposure to neutral pH following VP2 cleavage, its VP1-specific sequences and genome are extruded even at room temperature, underscoring the significance of the VP2 cleavage step for MVM particle dynamics.