Assembly of Structurally Simple Icosahedral Viruses.

Icosahedral viruses exhibit elegant pathways of capsid assembly and maturation regulated by symmetry principles. Assembly is a dynamic process driven by consecutive and genetically programmed morphogenetic interactions between protein subunits. The non-symmetric capsid subunits are gathered by non-covalent contacts and interactions in assembly intermediates, which serve as blocks to build a symmetric capsid.

Assessment of molecular modulation by multifrequency electromagnetic pulses to preferably eradicate tumorigenic cells.

Physics methods of cancer therapy are extensively used in clinical practice, but they are invasive and often confront undesired side effects. A fully new equipment that allows sustained emission of intense and time-controlled non-ionizing multifrequency electromagnetic pulse (MEMP), has been applied to eukaryotic cells in culture. The equipment discriminates the overall electronegative charge of the cell cultures, and its subsequent proportional emission may thereby become higher and lethal to cancer cells of generally high metabolic activity.

VEGF-Virus Interactions: Pathogenic Mechanisms and Therapeutic Applications.

Many types of viruses directly or indirectly target the vascular endothelial growth factor (VEGF) system, which is a central regulator of vasculogenesis and angiogenesis in physiological homeostasis, causing diverse pathologies. Other viruses have been developed into effective therapeutic tools for VEGF modulation in conditions such as cancer and eye diseases. Some viruses may alter the levels of VEGF in the pathogenesis of respiratory syndromes, or they may encode VEGF-like factors, promoting vascular disruption and angiogenesis to enable viruses' systemic spread.

Intracellular virion traffic to the endosome driven by cell type specific sialic acid receptors determines parvovirus tropism.

Parvoviruses are promising anticancer and gene therapy agents, but a deep knowledge of the entry process is crucial to exploit their therapeutic potential. We addressed this issue while attempting to retarget the oncolytic parvovirus minute virus of mice (MVMp) to the tumor vasculature. Residues at three functional domains of the icosahedral capsid were substituted by rational design with peptides competing with the vascular endothelial growth factor.

Viral targeting of glioblastoma stem cells with patient-specific genetic and post-translational p53 deregulations.

Cancer therapy urges targeting of malignant subsets within self-renewing heterogeneous stem cell populations. We dissect the genetic and functional heterogeneity of human glioblastoma stem cells (GSCs) within patients by their innate responses to non-pathogenic mouse parvoviruses that are tightly restrained by cellular physiology. GSC neurospheres accumulate assembled capsids but restrict viral NS1 cytotoxic protein expression by an innate PKR/eIF2α-P response counteractable by electric pulses.

Antiangiogenic Vascular Endothelial Growth Factor-Blocking Peptides Displayed on the Capsid of an Infectious Oncolytic Parvovirus: Assembly and Immune Interactions.

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.

Differential phosphorylation and n-terminal configuration of capsid subunits in parvovirus assembly and viral trafficking.

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.

Protoparvovirus Cell Entry.

The (PtPV) genus of the family of viruses includes important animal pathogens and reference molecular models for the entire family. Some virus members of the PtPV genus have arisen as promising tools to treat tumoral processes, as they exhibit marked oncotropism and oncolytic activities while being nonpathogenic for humans. The PtPVs invade and replicate within the nucleus making extensive use of the transport, transcription and replication machineries of the host cells.

The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly.

It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition.

A slender tract of glycine residues is required for translocation of the VP2 protein N-terminal domain through the parvovirus MVM capsid channel to initiate infection.

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.

Assembly of simple icosahedral viruses.

Icosahedral viruses exhibit elegant pathways of capsid assembly and maturation regulated by symmetry principles. Assembly is a dynamic process driven by consecutive and genetically programmed morphogenetic interactions between protein subunits. The non-symmetric capsid subunits are gathered by hydrophobic contacts and non-covalent interactions in assembly intermediates, which serve as blocks to build a symmetric capsid.

Essential role of the unordered VP2 n-terminal domain of the parvovirus MVM capsid in nuclear assembly and endosomal enlargement of the virion fivefold channel for cell entry.

The unordered N-termini of parvovirus capsid proteins (Nt) are translocated through a channel at the icosahedral five-fold axis to serve for virus traffick. Heterologous peptides were genetically inserted at the Nt of MVM to study their functional tolerance to manipulations. Insertion of a 5T4-single-chain antibody at VP2-Nt (2Nt) yielded chimeric capsid subunits failing to enter the nucleus.

Translation control by protein kinase R restricts minute virus of mice infection: role in parvovirus oncolysis.

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.

Viral oncolysis that targets Raf-1 signaling control of nuclear transport.

The central role of Raf protein kinase isoforms in human cancer demands specific anti-Raf therapeutic inhibitors. Parvoviruses are currently used in experimental cancer therapy due to their natural oncotropism and lytic life cycle. In searching for mechanisms underlying parvovirus oncolysis, we found that trimers of the major structural protein (VP) of the parvovirus minute virus of mice (MVM), which have to be imported into the nucleus for capsid assembly, undergo phosphorylation by the Raf-1 kinase.

A supraphysiological nuclear export signal is required for parvovirus nuclear export.

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.

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.

Minute virus of mice, a parvovirus, in complex with the Fab fragment of a neutralizing monoclonal antibody.

The structure of virus-like particles of the lymphotropic, immunosuppressive strain of minute virus of mice (MVMi) in complex with the neutralizing Fab fragment of the mouse monoclonal antibody (MAb) B7 was determined by cryo-electron microscopy to 7-A resolution. The Fab molecule recognizes a conformational epitope at the vertex of a three-fold protrusion on the viral surface, thereby simultaneously engaging three symmetry-related viral proteins in binding. The location of the epitope close to the three-fold axis is consistent with the previous analysis of MVMi mutants able to escape from the B7 antibody.

Nuclear transport of trimeric assembly intermediates exerts a morphogenetic control on the icosahedral parvovirus capsid.

The connection between nuclear transport and morphogenesis of a large macromolecular entity has been investigated using the karyophylic capsid of the parvovirus minute virus of mice (MVM) as a model. The VP1 (82 kDa) and VP2 (63 kDa) proteins forming the T = 1 icosahedral MVM capsid at the respective 1:5 molar ratio of synthesis, could be covalently cross-linked with dimethyl suberimidate into two types of oligomeric assemblies, which were present at stoichiometric amounts in infected cell extracts and purified viral particles. The larger species contained VP1 and corresponded in size (200 kDa) to a heterotrimer of one VP1 and two VP2 subunits.

Host-selected amino acid changes at the sialic acid binding pocket of the parvovirus capsid modulate cell binding affinity and determine virulence.

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.

Low pH-dependent endosomal processing of the incoming parvovirus minute virus of mice virion leads to externalization of the VP1 N-terminal sequence (N-VP1), N-VP2 cleavage, and uncoating of the full-length genome.

Minute virus of mice (MVM) enters the host cell via receptor-mediated endocytosis. Although endosomal processing is required, its role remains uncertain. In particular, the effect of low endosomal pH on capsid configuration and nuclear delivery of the viral genome is unclear.

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.

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.

Functional relevance of amino acid residues involved in interactions with ordered nucleic acid in a spherical virus.

In the spherical virion of the parvovirus minute virus of mice, several amino acid side chains of the capsid were previously found to be involved in interactions with the viral single-stranded DNA molecule. We have individually truncated by mutation to alanine many (ten) of these side chains and analyzed the effects on capsid assembly, stability and conformation, viral DNA encapsidation, and virion infectivity. Mutation of residues Tyr-270, Asp-273, or Asp-474 led to a drastic reduction in infectivity.