José M. Almendral

Restriction site map of African swine fever virus DNA

Treatment of African swine fever virus DNA (about 170 kbp) with the restriction endonucleases SalI, EcoRI, KpnI, PvuI, and SmaI yielded 14, 31, 17, 13, and 11 fragments, respectively. The order of the restriction fragments produced by each nuclease was established by identifying the crosslinked EcoRI and SalI terminal fragments and then finding overlapping fragments. The five restriction fragment maps were integrated into a single map by locating SalI, KpnI, PvuI, and SmaI sites in cloned EcoRI fragments, and orienting each fragment in the overall map.

Transcription and translation maps of African swine fever virus

A transcription map of African swine fever (ASF) virus DNA was obtained by hybridization of 32P-labeled early and late RNAs synthesized in Vero cells infected with ASF virus to dot-blots containing cloned restriction fragments spanning the viral genome. Early RNAs synthesized in infected cells in the presence of protein or DNA synthesis inhibitors hybridized preferentially to four regions in the genome, with coordinates E1 (0-51.9 kbp), E3 (63.7-75.2 kbp), E5 (100.1-111.6 kbp), and E7 (150-170 kbp). Late RNA present in infected cells after DNA replication hybridized with essentially all the genome. The RNA synthesized in vitro by the RNA polymerase associated with ASF virions hybridized to the same DNA regions than early RNA. After hybridization selection with DNA restriction fragments and translation in reticulocyte lysates the RNA synthesized in vitro produced the same proteins as early RNA. These results suggest that early RNA is synthesized in the infected cells by the virion-associated RNA polymerase. Maps of early and late proteins of ASF virus were constructed by cell-free translation of early or late RNAs selected by hybridization to cloned restriction fragments of virus DNA. About 100 early and 100 late polypeptide bands were mapped on the ASF virus genome.

Ex vivo expansion and selection of retrovirally transduced bone marrow: an efficient methodology for gene-transfer to murine lympho-haemopoietic stem cells

An efficient procedure for the insertion of genetic markers into a large proportion of the mouse haemopoietic system was developed, based on the in vitro, expansion of retrovirally infected bone marrow and selection of the transduced cells. Bone marrow cells harvested 4 d after 5‐FU treatment were incubated under IL‐3/SCF stimulation and their growth dynamic, susceptibility to retroviral infection and reconstitution capacity evaluated throughout the incubation period. On the third day of culture a maximum expansion in the CFU‐GM and CFU‐S12 progenitor pools was observed (130‐ and 15‐fold, respectively), with no apparent impairment in long‐term repopulating precursors. This expansion was, however, accompanied by a net decrease in the CFU‐GM susceptibility to the infection by supernatants containing a Moloney‐derived ecotropic retroviral vector carrying the neo’gene. The designed protocol thus involved the infection of freshly harvested 5‐FU‐treated bone marrow, followed by expansion under IL‐3/SCF stimulation and selection for resistance to G418. This procedure allowed us to harvest up to 780 CFU‐GM and 50 CFU‐S12 per 105 bone marrow cells, free from non‐genetically marked progenitors. Most of the animals reconstituted with the transduced marrow bore, for at least 5 months, a very high proportion of bone marrow, spleen and thymus cells tagged with the reporter gene. These results, together with the high percentage of haemopoietic precursors bearing the neo’gene and expressing resistance to G418 5 months after the transplantation indicates that long‐term lympho‐haemopoietic repopulating cells were efficiently transduced and selected in vitro under conditions that preserve their self‐renewal and differentiation properties. This gene‐transfer methodology may improve the development of gene therapy protocols where the purging of non‐transduced precursors would guarantee a lasting and uniform expression of exogenous genes.

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. 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.

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. 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.

Evolution to Pathogenicity of the Parvovirus Minute Virus of Mice in Immunodeficient Mice Involves Genetic Heterogeneity at the Capsid Domain That Determines Tropism

ABSTRACT 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.

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. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life cycles. This junction may determine the characteristic parvovirus tropism for proliferative and cancer cells, and its disturbance could critically contribute to persistence in host tissues.

Cytotoxic infection of hematopoietic stem and committed progenitor cells by the parvovirus minute virus of mice. Propagation of an acute myelosuppression in culture.

We have investigated the ability of two strains of the parvovirus minute virus of mice to impair mouse hematopoiesis in vitro. We found that the BFU-E and CFU-GM committed progenitors, CFU-Mix pluripotent progenitor, as well as the CFU-S12d, one of the most primitive hematopoietic precursors of the stem cell compartment detectable by colony technique, were similarly inhibited in their proliferative capacity by the immunosuppressive strain MVMi, but not by the prototype virus MVMp. The inhibitory effect correlated with the input of purified MVMi and was reversed by neutralizing MVM antiserum, showing that cytotoxic mechanisms underlying infectious MVMi replication and not operating in MVMp-infected cells were responsible for the reproductive death of hematopoietic precursors. In agreement with this, myeloid nonadherent cells of long-term bone marrow cultures were selectively permissive for MVMi but not for MVMp replication, as judged by viral DNA synthesis, the expression of the nonstructural cytotoxic NS-1 protein, and virus propagation in these cells. Altogether, the suppressive effects mediated by the MVMi cytotoxic infection define a wide lympho-myelotropism not previously reported for this virus. The MVM-mouse model highlights the role that unsuspected virus-hematopoietic compartment interactions may play in bone marrow failures of immunocompromised animal or human hosts.

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. 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.