RESUMEN
The oncolytic autonomous parvovirus Minute Virus of Mice (MVM) establishes infection in the nuclear environment by usurping host DNA damage signaling proteins in the vicinity of cellular DNA break sites. MVM replication induces a global cellular DNA Damage Response (DDR) that is dependent on signaling by the ATM kinase and inactivates the cellular ATR-kinase pathway. However, the mechanism of how MVM generates cellular DNA breaks remains unknown. Using single molecule DNA Fiber Analysis, we have discovered that MVM infection leads to a shortening of host replication forks as infection progresses, as well as induction of replication stress prior to the initiation of virus replication. Ectopically expressed viral non-structural proteins NS1 and NS2 are sufficient to cause host-cell replication stress, as is the presence of UV-inactivated non-replicative MVM genomes. The host single-stranded DNA binding protein Replication Protein A (RPA) associates with the UV-inactivated MVM genomes, suggesting MVM genomes might serve as a sink for cellular stores of RPA. Overexpressing RPA in host cells prior to UV-MVM infection rescues DNA fiber lengths and increases MVM replication, confirming that MVM genomes deplete RPA stores to cause replication stress. Together, these results indicate that parvovirus genomes induce replication stress through RPA exhaustion, rendering the host genome vulnerable to additional DNA breaks.
Asunto(s)
Virus Diminuto del Ratón , Infecciones por Parvoviridae , Parvovirus , Animales , Ratones , Virus Diminuto del Ratón/genética , Proteína de Replicación A/genética , Parvovirus/genética , Replicación Viral/genética , Infecciones por Parvoviridae/genética , Replicación del ADN/genéticaRESUMEN
Parvoviruses are single-stranded DNA viruses that utilize host proteins to vigorously replicate in the nuclei of host cells, leading to cell cycle arrest. The autonomous parvovirus, minute virus of mice (MVM), forms viral replication centers in the nucleus which are adjacent to cellular DNA damage response (DDR) sites, many of which are fragile genomic regions prone to undergoing DDR during the S phase. Since the cellular DDR machinery has evolved to transcriptionally suppress the host epigenome to maintain genomic fidelity, the successful expression and replication of MVM genomes at these cellular sites suggest that MVM interacts with DDR machinery distinctly. Here, we show that efficient replication of MVM requires binding of the host DNA repair protein MRE11 in a manner that is independent of the MRE11-RAD50-NBS1 (MRN) complex. MRE11 binds to the replicating MVM genome at the P4 promoter, remaining distinct from RAD50 and NBS1, which associate with cellular DNA break sites to generate DDR signals in the host genome. Ectopic expression of wild-type MRE11 in CRISPR knockout cells rescues virus replication, revealing a dependence on MRE11 for efficient MVM replication. Our findings suggest a new model utilized by autonomous parvoviruses to usurp local DDR proteins that are crucial for viral pathogenesis and distinct from those of dependoparvoviruses, like adeno-associated virus (AAV), which require a coinfected helper virus to inactivate the local host DDR. IMPORTANCE The cellular DNA damage response (DDR) machinery protects the host genome from the deleterious consequences of DNA breaks and recognizes invading viral pathogens. DNA viruses that replicate in the nucleus have evolved distinct strategies to evade or usurp these DDR proteins. We have discovered that the autonomous parvovirus, MVM, which is used to target cancer cells as an oncolytic agent, depends on the initial DDR sensor protein MRE11 to express and replicate efficiently in host cells. Our studies reveal that the host DDR interacts with replicating MVM molecules in ways that are distinct from viral genomes being recognized as simple broken DNA molecules. These findings suggest that autonomous parvoviruses have evolved distinct mechanisms to usurp DDR proteins, which can be used to design potent DDR-dependent oncolytic agents.
Asunto(s)
Proteína Homóloga de MRE11 , Virus Diminuto del Ratón , Infecciones por Parvoviridae , Animales , Ratones , Proteínas de Ciclo Celular/metabolismo , Receptores con Dominio Discoidina/genética , Receptores con Dominio Discoidina/metabolismo , Daño del ADN , Replicación del ADN , Virus Diminuto del Ratón/genética , Infecciones por Parvoviridae/genética , Replicación Viral/fisiología , Proteína Homóloga de MRE11/metabolismoRESUMEN
The biological function of macromolecular complexes depends not only on large-scale transitions between conformations, but also on small-scale conformational fluctuations at equilibrium. Information on the equilibrium dynamics of biomolecular complexes could, in principle, be obtained from local resolution (LR) data in cryo-electron microscopy (cryo-EM) maps. However, this possibility had not been validated by comparing, for a same biomolecular complex, LR data with quantitative information on equilibrium dynamics obtained by an established solution technique. In this study we determined the cryo-EM structure of the minute virus of mice (MVM) capsid as a model biomolecular complex. The LR values obtained correlated with crystallographic B factors and with hydrogen/deuterium exchange (HDX) rates obtained by mass spectrometry (HDX-MS), a gold standard for determining equilibrium dynamics in solution. This result validated a LR-based cryo-EM approach to investigate, with high spatial resolution, the equilibrium dynamics of biomolecular complexes. As an application of this approach, we determined the cryo-EM structure of two mutant MVM capsids and compared their equilibrium dynamics with that of the wild-type MVM capsid. The results supported a previously suggested linkage between mechanical stiffening and impaired equilibrium dynamics of a virus particle. Cryo-EM is emerging as a powerful approach for simultaneously acquiring information on the atomic structure and local equilibrium dynamics of biomolecular complexes.
Asunto(s)
Aminoácidos , Cápside , Microscopía por Crioelectrón , Sustancias Macromoleculares , Aminoácidos/química , Cápside/química , Microscopía por Crioelectrón/métodos , Conformación Proteica , Sustancias Macromoleculares/química , Virus Diminuto del Ratón/química , Virus Diminuto del Ratón/genéticaRESUMEN
Icosahedral viruses are under a micrometer in diameter, their infectious genome encapsulated by a shell assembled by a multiscale process, starting from an integer multiple of 60 viral capsid or coat protein (VP) monomers. We predict and validate inter-atomic hotspot interactions between VP monomers that are important for the assembly of 3 types of icosahedral viral capsids: Adeno Associated Virus serotype 2 (AAV2) and Minute Virus of Mice (MVM), both T = 1 single stranded DNA viruses, and Bromo Mosaic Virus (BMV), a T = 3 single stranded RNA virus. Experimental validation is by in-vitro, site-directed mutagenesis data found in literature. We combine ab-initio predictions at two scales: at the interface-scale, we predict the importance (cruciality) of an interaction for successful subassembly across each interface between symmetry-related VP monomers; and at the capsid-scale, we predict the cruciality of an interface for successful capsid assembly. At the interface-scale, we measure cruciality by changes in the capsid free-energy landscape partition function when an interaction is removed. The partition function computation uses atlases of interface subassembly landscapes, rapidly generated by a novel geometric method and curated opensource software EASAL (efficient atlasing and search of assembly landscapes). At the capsid-scale, cruciality of an interface for successful assembly of the capsid is based on combinatorial entropy. Our study goes all the way from resource-light, multiscale computational predictions of crucial hotspot inter-atomic interactions to validation using data on site-directed mutagenesis' effect on capsid assembly. By reliably and rapidly narrowing down target interactions, (no more than 1.5 hours per interface on a laptop with Intel Core i5-2500K @ 3.2 Ghz CPU and 8GB of RAM) our predictions can inform and reduce time-consuming in-vitro and in-vivo experiments, or more computationally intensive in-silico analyses.
Asunto(s)
Proteínas de la Cápside , Cápside , Ensamble de Virus/genética , Cápside/química , Cápside/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Simulación por Computador , Dependovirus/química , Dependovirus/genética , Dependovirus/metabolismo , Virus Diminuto del Ratón/química , Virus Diminuto del Ratón/genética , Virus Diminuto del Ratón/metabolismo , Mutagénesis Sitio-DirigidaRESUMEN
The autonomous parvovirus Minute Virus of Mice (MVM) localizes to cellular DNA damage sites to establish and sustain viral replication centers, which can be visualized by focal deposition of the essential MVM non-structural phosphoprotein NS1. How such foci are established remains unknown. Here, we show that NS1 localized to cellular sites of DNA damage independently of its ability to covalently bind the 5' end of the viral genome, or its consensus DNA binding sequence. Many of these sites were identical to those occupied by virus during infection. However, localization of the MVM genome to DNA damage sites occurred only when wild-type NS1, but not its DNA-binding mutant was expressed. Additionally, wild-type NS1, but not its DNA binding mutant, could localize a heterologous DNA molecule containing the NS1 binding sequence to DNA damage sites. These findings suggest that NS1 may function as a bridging molecule, helping the MVM genome localize to cellular DNA damage sites to facilitate ongoing virus replication.
Asunto(s)
Daño del ADN , Virus Diminuto del Ratón/genética , Virus Diminuto del Ratón/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Línea Celular , Replicación del ADN , ADN Viral/genética , Proteínas de Unión al ADN/genética , Femenino , Genoma Viral , Humanos , Masculino , Ratones , Infecciones por Parvoviridae/genética , Infecciones por Parvoviridae/virología , Parvovirus/genética , Replicación ViralRESUMEN
Suppression of the CpG dinucleotide is widespread in RNA viruses infecting vertebrates and plants, and in the genomes of retroviruses and small mammalian DNA viruses. The functional basis for CpG suppression in the latter was investigated through the construction of mutants of the parvovirus, minute virus of mice (MVM) with increased CpG or TpA dinucleotides in the VP gene. CpG-high mutants displayed extraordinary attenuation in A9 cells compared to wild-type MVM (>six logs), while TpA elevation showed no replication effect. Attenuation was independent of Toll-like receptor 9 and STING-mediated DNA recognition pathways and unrelated to effects on translation efficiency. While translation from codon-optimized VP RNA was enhanced in a cell-free assay, MVM containing this sequence was highly attenuated. Further mutational analysis indicated that this arose through its increased numbers of CpG dinucleotides (7â70) and separately from its increased G+C content (42.3â57.4 %), which independently attenuated replication. CpG-high viruses showed impaired NS mRNA expression by qPCR and reduced NS and particularly VP protein expression detected by immunofluorescence and replication in A549 cells, effects reversed in zinc antiviral protein (ZAP) knockout cells, even though nuclear relocalization of VP remained defective. The demonstrated functional basis for CpG suppression in MVM and potentially other small DNA viruses and the observed intolerance of CpGs in coding sequences, even after codon optimization, has implications for the use of small DNA virus vectors in gene therapy and immunization.
Asunto(s)
Fosfatos de Dinucleósidos/metabolismo , Virus Diminuto del Ratón/fisiología , Replicación Viral , Células A549 , Composición de Base , Codón , Fosfatos de Dinucleósidos/genética , Humanos , Virus Diminuto del Ratón/genética , Mutación , ARN Viral/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismoRESUMEN
As many tumor cells synthetize vascular endothelial growth factors (VEGF) that promote neo-vascularization and metastasis, frontline cancer therapies often administer anti-VEGF (α-VEGF) antibodies. To target the oncolytic parvovirus minute virus of mice (MVM) to the tumor vasculature, we studied the functional tolerance, evasion of neutralization, and induction of α-VEGF antibodies of chimeric viruses in which the footprint of a neutralizing monoclonal antibody within the 3-fold capsid spike was replaced by VEGF-blocking peptides: P6L (PQPRPL) and A7R (ATWLPPR). Both peptides allowed viral genome replication and nuclear translocation of chimeric capsid subunits. MVM-P6L efficiently propagated in culture, exposing the heterologous peptide on the capsid surface, and evaded neutralization by the anti-spike monoclonal antibody. In contrast, MVM-A7R yielded low infectious titers and was poorly recognized by an α-A7R monoclonal antibody. MVM-A7R showed a deficient assembly pattern, suggesting that A7R impaired a transitional configuration that the subunits must undergo in the 3-fold axis to close up the capsid shell. The MVM-A7R chimeric virus consistently evolved in culture into a mutant carrying the P6Q amino acid substitution within the A7R sequence, which restored normal capsid assembly and infectivity. Consistent with this finding, anti-native VEGF antibodies were induced in mice by a single injection of MVM-A7R empty capsids, but not by MVM-A7R virions. This fundamental study provides insights to endow an infectious parvovirus with immune antineovascularization and evasion capacities by replacing an antibody footprint in the capsid 3-fold axis with VEGF-blocking peptides, and it also illustrates the evolutionary capacity of single-stranded DNA (ssDNA) viruses to overcome engineered capsid structural restrictions.IMPORTANCE Targeting the VEGF signaling required for neovascularization by vaccination with chimeric capsids of oncolytic viruses may boost therapy for solid tumors. VEGF-blocking peptides (VEbp) engineered in the capsid 3-fold axis endowed the infectious parvovirus MVM with the ability to induce α-VEGF antibodies without adjuvant and to evade neutralization by MVM-specific antibodies. However, these properties may be compromised by structural restraints that the capsid imposes on the peptide configuration and by misassembly caused by the heterologous peptides. Significantly, chimeric MVM-VEbp resolved the structural restrictions by selecting mutations within the engineered peptides that restored efficient capsid assembly. These data show the promise of antineovascularization vaccines using chimeric VEbp-icosahedral capsids of oncolytic viruses but also raise safety concerns regarding the genetic stability of manipulated infectious parvoviruses in cancer and gene therapies.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/metabolismo , Virus Diminuto del Ratón/inmunología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/genética , Proteínas de la Cápside/genética , Ratones Endogámicos BALB C , Virus Diminuto del Ratón/genética , Virus Diminuto del Ratón/crecimiento & desarrollo , Virus Oncolíticos/genética , Virus Oncolíticos/crecimiento & desarrollo , Virus Oncolíticos/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Carga Viral , Ensamble de Virus , Acoplamiento Viral , Internalización del VirusRESUMEN
We used a combination of DOP-PCR with high throughput sequencing (HTS) to study infected cell cultures over time to assess the feasibility of using this technique to provide a read-out other than cytopathic effect in cell culture infectivity assays. Because DOP-PCR primers feature a short constant sequence at their 3' terminus, the procedure yields a reproducible representational library of products from a given PCR template, including viral nucleic acids. Using SV40- and MVM-infected cultures harvested at different times, we show that the number of viral matches among DOP-PCR products parallels the quantity of virus as shown by real-time PCR, and further show that HTS analysis of specific DOP-PCR products that increase in quantity over time could be used to identify the infecting virus with a sensitivity similar to that of typical cell-culture assays that rely on cytopathic effect.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Virus Diminuto del Ratón/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa/métodos , Virus 40 de los Simios/crecimiento & desarrollo , Cultivo de Virus/métodos , Animales , Células CHO , Chlorocebus aethiops , Cricetulus , Virus Diminuto del Ratón/genética , Virus 40 de los Simios/genética , Células Vero , Carga ViralRESUMEN
We have developed a generally adaptable, novel high-throughput Viral Chromosome Conformation Capture assay (V3C-seq) for use in trans that allows genome-wide identification of the direct interactions of a lytic virus genome with distinct regions of the cellular chromosome. Upon infection, we found that the parvovirus Minute Virus of Mice (MVM) genome initially associated with sites of cellular DNA damage that in mock-infected cells also exhibited DNA damage as cells progressed through S-phase. As infection proceeded, new DNA damage sites were induced, and virus subsequently also associated with these. Sites of association identified biochemically were confirmed microscopically and MVM could be targeted specifically to artificially induced sites of DNA damage. Thus, MVM established replication at cellular DNA damage sites, which provide replication and expression machinery, and as cellular DNA damage accrued, virus spread additionally to newly damaged sites to amplify infection. MVM-associated sites overlap significantly with previously identified topologically-associated domains (TADs).
Asunto(s)
Daño del ADN , Virus Diminuto del Ratón/fisiología , Animales , Reparación del ADN , Ingeniería Genética , Genoma Viral , Histonas/metabolismo , Masculino , Ratones , Virus Diminuto del Ratón/genética , Ratas , Replicación ViralRESUMEN
The influence of mouse strain, immune competence and age on the pathogenesis of a field strain of minute virus of mice (MVMm) was examined in BALB/c, C3H, C57BL/6 and SCID mice experimentally infected as neonates, weanlings and adults. Sera, bodily excretions and tissues were harvested at 7, 14, 28 and 56 days after inoculation and evaluated by serology, quantitative PCR and histopathology. Seroconversion to recombinant viral capsid protein 2 was consistently observed in all immunocompetent strains of mice, regardless of the age at which they were inoculated, while seroconversion to the viral nonstructural protein 1 was only consistently detected in neonate inoculates. Viral DNA was detected by quantitative PCR in multiple tissues of immunocompetent mice at each time point after inoculation, with the highest levels being observed in neonate inoculates at 7 days after inoculation. In contrast, viral DNA levels in tissues and bodily excretions increased consistently over time in immunodeficient SCID mice, regardless of the age at which they were inoculated, with mortality being observed in neonatal inoculates between 28 and 56 days after inoculation. Overall, productive infection was observed more frequently in immunocompetent mice inoculated as neonates as compared to those inoculated as weanlings or adults, and immunodeficient SCID mice developed persistent, progressive infection, with mortality being observed in mice inoculated as neonates. Importantly, the clinical syndrome observed in experimentally infected SCID neonatal mice recapitulates the clinical presentation reported for the naturally infected immunodeficient NOD µ-chain knockout mice from which MVMm was initially isolated.
Asunto(s)
Virus Diminuto del Ratón/fisiología , Infecciones por Parvoviridae/veterinaria , Enfermedades de los Roedores/virología , Factores de Edad , Animales , Femenino , Especificidad del Huésped , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Virus Diminuto del Ratón/clasificación , Virus Diminuto del Ratón/genética , Virus Diminuto del Ratón/aislamiento & purificación , Infecciones por Parvoviridae/inmunología , Infecciones por Parvoviridae/virología , Recombinación Genética , Enfermedades de los Roedores/inmunología , Proteínas Virales/genéticaRESUMEN
The T1 parvovirus Minute Virus of Mice (MVM) was used to study the roles that phosphorylation and N-terminal domains (Nt) configuration of capsid subunits may play in icosahedral nuclear viruses assembly. In synchronous MVM infection, capsid subunits newly assembled as two types of cytoplasmic trimeric intermediates (3VP2, and 1VP1:2VP2) harbored a VP1 phosphorylation level fivefold higher than that of VP2, and hidden Nt. Upon nuclear translocation at S phase, VP1-Nt became exposed in the heterotrimer and subsequent subviral assembly intermediates. Empty capsid subunits showed a phosphorylation level restored to VP1:VP2 stoichiometry, and the Nt concealed in their interior. However ssDNA-filled virus maturing at S/G2 lacked VP1 phosphorylation and one major VP2 phosphopeptide, and exposed VP2-Nt. Endosomal VP2-Nt cleavage resulted in VP3 subunits devoid of any phospholabel, implying that incoming viral particles specifically harbor a low phosphorylation status. Phosphorylation provides a mechanistic coupling of parvovirus nuclear assembly to the cell cycle.
Asunto(s)
Proteínas de la Cápside/metabolismo , Virus Diminuto del Ratón/fisiología , Ensamble de Virus/fisiología , Animales , Anticuerpos Antivirales , Proteínas de la Cápside/genética , Línea Celular , Regulación Viral de la Expresión Génica/fisiología , Humanos , Ratones , Virus Diminuto del Ratón/genética , Fosforilación , Dominios Proteicos , Subunidades de Proteína , ConejosRESUMEN
Murine parvovirus is one of the most prevalent infectious pathogens in mouse colonies. A specific primer pair targeting the VP2 gene of minute virus of mice (MVM) and mouse parvovirus (MPV) was utilized for high resolution melting (HRM) analysis. The resulting melting curves could distinguish these two virus strains and there was no detectable amplification of the other mouse pathogens which included rat parvovirus (KRV), ectromelia virus (ECT), mouse adenovirus (MAD), mouse cytomegalovirus (MCMV), polyoma virus (Poly), Helicobactor hepaticus (H. hepaticus) and Salmonella typhimurium (S. typhimurium). The detection limit of the standard was 10 copies/µL. This study showed that the PCR-HRM assay could be an alternative useful method with high specificity and sensitivity for differentiating murine parvovirus strains MVM and MPV.
Asunto(s)
Virus Diminuto del Ratón/aislamiento & purificación , Infecciones por Parvoviridae/diagnóstico , Parvovirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Temperatura de Transición , Animales , Proteínas de la Cápside/genética , Diagnóstico Diferencial , Ratones , Virus Diminuto del Ratón/genética , Infecciones por Parvoviridae/virología , Parvovirus/genética , Sensibilidad y EspecificidadRESUMEN
The protoparvovirus early promoters, e.g. P4 of Minute Virus of Mice (MVM), play a critical role during infection. Initial P4 activity depends on the host transcription machinery only. Since this is cell-type dependent, it is hypothesized that P4 is a host cell-type range determinant. Yet host range determinants have mapped mostly to capsid, never P4. Here we test the hypothesis using the mouse embryo as a model system. Disruption of the CRE element of P4 drastically decreased infection levels without altering range. However, when we swapped promoter elements of MVM P4 with those from equivalent regions of the closely related H1 virus, we observed elimination of infection in fibroblasts and chondrocytes and the acquisition of infection in skeletal muscle. We conclude that P4 is a host range determinant and a target for modifying the productive infection potential of the virus - an important consideration in adapting these viruses for oncotherapy.
Asunto(s)
Virus Diminuto del Ratón/fisiología , Infecciones por Parvoviridae/virología , Regiones Promotoras Genéticas , Enfermedades de los Roedores/virología , Proteínas no Estructurales Virales/genética , Animales , Regulación Viral de la Expresión Génica , Especificidad del Huésped , Ratones , Virus Diminuto del Ratón/genética , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
UNLABELLED: Although the mechanism is not well understood, growing evidence indicates that the nonenveloped parvovirus minute virus of mice (MVM) may actively egress before passive release through cell lysis. We have dissected the late maturation steps of the intranuclear progeny with the aims of confirming the existence of active prelytic egress and identifying critical capsid rearrangements required to initiate the process. By performing anion-exchange chromatography (AEX), we separated intranuclear progeny particles by their net surface charges. Apart from empty capsids (EC), two distinct populations of full capsids (FC) arose in the nuclei of infected cells. The earliest population of FC to appear was infectious but, like EC, could not be actively exported from the nucleus. Further maturation of this early population, involving the phosphorylation of surface residues, gave rise to a second, late population with nuclear export potential. While capsid surface phosphorylation was strictly associated with nuclear export capacity, mutational analysis revealed that the phosphoserine-rich N terminus of VP2 (N-VP2) was dispensable, although it contributed to passive release. The reverse situation was observed for the incoming particles, which were dephosphorylated in the endosomes. Our results confirm the existence of active prelytic egress and reveal a late phosphorylation event occurring in the nucleus as a selective factor for initiating the process. IMPORTANCE: In general, the process of egress of enveloped viruses is active and involves host cell membranes. However, the release of nonenveloped viruses seems to rely more on cell lysis. At least for some nonenveloped viruses, an active process before passive release by cell lysis has been reported, although the underlying mechanism remains poorly understood. By using the nonenveloped model parvovirus minute virus of mice, we could confirm the existence of an active process of nuclear export and further characterize the associated capsid maturation steps. Following DNA packaging in the nucleus, capsids required further modifications, involving the phosphorylation of surface residues, to acquire nuclear export potential. Inversely, those surface residues were dephosphorylated on entering capsids. These spatially controlled phosphorylation-dephosphorylation events concurred with the nuclear export-import potential required to complete the infectious cycle.
Asunto(s)
Transporte Activo de Núcleo Celular , Núcleo Celular/virología , Virus Diminuto del Ratón/fisiología , Ensamble de Virus , Liberación del Virus , Animales , Cápside , Línea Celular , Núcleo Celular/metabolismo , Fibroblastos/virología , Humanos , Ratones , Virus Diminuto del Ratón/genética , Virus Diminuto del Ratón/ultraestructura , Mutación , Infecciones por Parvoviridae/virología , Fosforilación , Virión/fisiología , Replicación ViralRESUMEN
Galectin-3 has previously been found to be required by the parvovirus minute virus of mice prototype strain (MVMp) for infection of mouse fibroblast cells. Since MVMp is an oncotropic virus, and galectin-3 is a multifunctional protein implicated in cancer metastasis, we hypothesized that galectin-3 and Mgat5, the Golgi enzyme that synthesizes high-affinity glycan ligands of galectin-3, might play a role in MVMp infection. Using siRNA-mediated knockdown of galectin-3 in mouse cells transformed with polyomavirus middle T antigen and Mgat5(-/-) mouse mammary tumor cells, we found that galectin-3 and Mgat5 are both necessary for efficient MVMp cell entry and infection, but not for cell binding. Moreover, we found that human cancer cells expressing higher levels of galectin-3 were more efficiently infected with MVMp than cell lines expressing lower galectin-3 levels. We conclude that galectin-3 and Mgat5 are involved in MVMp infection, and propose that galectin-3 is a determinant of MVMp oncotropism.
Asunto(s)
Galectina 3/metabolismo , Virus Diminuto del Ratón/fisiología , Infecciones por Parvoviridae/veterinaria , Enfermedades de los Roedores/metabolismo , Animales , Línea Celular , Galectina 3/genética , Humanos , Ratones , Virus Diminuto del Ratón/genética , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Infecciones por Parvoviridae/genética , Infecciones por Parvoviridae/metabolismo , Infecciones por Parvoviridae/virología , Enfermedades de los Roedores/genética , Enfermedades de los Roedores/virologíaRESUMEN
Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins.
Asunto(s)
Roturas del ADN de Cadena Simple , ADN Helicasas/química , ADN Helicasas/metabolismo , Virus Diminuto del Ratón/enzimología , Transactivadores/química , Transactivadores/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/química , Animales , Secuencia de Bases , ADN Helicasas/genética , Replicación del ADN , Ratones , Virus Diminuto del Ratón/química , Virus Diminuto del Ratón/genética , Modelos Moleculares , Infecciones por Parvoviridae/veterinaria , Infecciones por Parvoviridae/virología , Unión Proteica , Estructura Terciaria de Proteína , Origen de Réplica , Enfermedades de los Roedores/virología , Transactivadores/genética , Proteínas no Estructurales Virales/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The P4 promoter of the autonomous parvovirus Minute Virus of Mice (MVM) drives the production of its non-structural proteins, NS1 and NS2. The NS2 isoforms are without enzymatic activity but interact with cellular proteins. While NS2 is crucial to the viral life cycle in cultured murine cells, NS2-null mutant virus productively infects transformed host cells of other species. In the mouse, sensitivity to MVM infection is age dependent, exhibiting limited subclinical infections in adults, but sustained and potentially lethal infection in embryos. We therefore questioned whether the species-dependent requirement for NS2 function in vitro would be retained in utero. We report here that it is not. NS2-null mutant MVMp is capable of mounting a productive, albeit much reduced, infection of normal embryonic mouse cells in vivo. Based on the data, we hypothesize that NS2 may bear an as-yet undescribed immunosuppressive function.
Asunto(s)
Embrión de Mamíferos/virología , Regulación Viral de la Expresión Génica/fisiología , Virus Diminuto del Ratón/fisiología , Infecciones por Parvoviridae/virología , Proteínas no Estructurales Virales/metabolismo , Animales , Línea Celular , Femenino , Eliminación de Gen , Humanos , Ratones , Virus Diminuto del Ratón/genética , Embarazo , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
Parvoviruses halt cell cycle progression following initiation of their replication during S-phase and continue to replicate their genomes for extended periods of time in arrested cells. The parvovirus minute virus of mice (MVM) induces a DNA damage response that is required for viral replication and induction of the S/G2 cell cycle block. However, p21 and Chk1, major effectors typically associated with S-phase and G2-phase cell cycle arrest in response to diverse DNA damage stimuli, are either down-regulated, or inactivated, respectively, during MVM infection. This suggested that parvoviruses can modulate cell cycle progression by another mechanism. In this work we show that the MVM-induced, p21- and Chk1-independent, cell cycle block proceeds via a two-step process unlike that seen in response to other DNA-damaging agents or virus infections. MVM infection induced Chk2 activation early in infection which led to a transient S-phase block associated with proteasome-mediated CDC25A degradation. This step was necessary for efficient viral replication; however, Chk2 activation and CDC25A loss were not sufficient to keep infected cells in the sustained G2-arrested state which characterizes this infection. Rather, although the phosphorylation of CDK1 that normally inhibits entry into mitosis was lost, the MVM induced DDR resulted first in a targeted mis-localization and then significant depletion of cyclin B1, thus directly inhibiting cyclin B1-CDK1 complex function and preventing mitotic entry. MVM infection thus uses a novel strategy to ensure a pseudo S-phase, pre-mitotic, nuclear environment for sustained viral replication.
Asunto(s)
Ciclina B1/metabolismo , Virus Diminuto del Ratón/metabolismo , Mitosis , Infecciones por Parvoviridae/metabolismo , Animales , Proteína Quinasa CDC2 , Línea Celular , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Ciclina B1/genética , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Activación Enzimática/genética , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Humanos , Ratones , Virus Diminuto del Ratón/genética , Infecciones por Parvoviridae/genética , Infecciones por Parvoviridae/patología , Puntos de Control de la Fase S del Ciclo Celular/genética , Fosfatasas cdc25/genética , Fosfatasas cdc25/metabolismoRESUMEN
The 121-nucleotide left-end telomere of Minute Virus of Mice (MVM) can be folded into a Y-shaped hairpin with short axial ears that are highly conserved within genus Parvovirus. To explore their potential role(s) during infection, we constructed infectious plasmid clones that lacked one or other ear. Although these were nonviable when transfected into A9 cells, excision of the viral genome and DNA amplification appeared normal, and viral transcripts and proteins were expressed, but progeny virion production was minimal, supporting the idea of a potential role for the ears in genome packaging. To circumvent the absence of progeny that confounded further analysis of these mutants, plasmids were transfected into 293T cells both with and without an adenovirus helper construct, generating single bursts of progeny. These virions bound to A9 cells and were internalized but failed to initiate viral transcription, protein expression, or DNA replication. No defects in mutant virion stability or function could be detected in vitro. Significantly, mutant capsid gene expression and DNA replication could be rescued by coinfection with wild-type virions carrying a replication-competent, capsid-gene-replacement vector. To pinpoint where such complementation occurred, prior transfection of plasmids expressing only MVM nonstructural proteins was explored. NS1 alone, but not NS2, rescued transcription and protein expression from both P4 and P38 promoters, whereas NS1 molecules deleted for their C-terminal transactivation domain did not. These results suggest that the mutant virions reach the nucleus, uncoat, and are converted to duplex DNA but require an intact left-end hairpin structure to form the initiating transcription complex.
Asunto(s)
Genoma Viral , Virus Diminuto del Ratón/genética , Infecciones por Parvoviridae/virología , Parvovirus/genética , Transcripción Genética , Replicación Viral , Animales , Emparejamiento Base , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Cartilla de ADN/química , Cartilla de ADN/genética , ADN Viral/genética , Vectores Genéticos , Ratones , Infecciones por Parvoviridae/genética , Infecciones por Parvoviridae/metabolismo , Transfección , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Viruses constitute paradigms to study conformational dynamics in biomacromolecular assemblies. Infection by the parvovirus MVM (minute virus of mice) requires a conformational rearrangement that involves the intracellular externalization through capsid channels of the 2Nt (N-terminal region of VP2). We have investigated the role in this process of conserved glycine residues in an extended glycine-rich tract located immediately after 2Nt. Based on the virus structure, residues with hydrophobic side chains of increasing volume were substituted for glycine residues 31 or 33. Mutations had no effect on capsid assembly or stability, but inhibited virus infectivity. All mutations, except those to alanine residues which had minor effects, impaired 2Nt externalization in nuclear maturing virions and in purified virions, to an extent that correlated with the side chain size. Different biochemical and biophysical analyses were consistent with this result. Importantly, all of the tested glycine residue replacements impaired the capacity of the virion to initiate infection, at ratios correlating with their restrictive effects on 2Nt externalization. Thus small residues within the evolutionarily conserved glycine-rich tract facilitate 2Nt externalization through the capsid channel, as required by this virus to initiate cell entry. The results demonstrate the exquisite dependence on geometric constraints of a biologically relevant translocation event in a biomolecular complex.