TLR-9 contributes to the antiviral innate immune sensing of rodent parvoviruses MVMp and H-1PV by normal human immune cells.

The oncotropism of Minute Virus of Mice (MVMp) is partially related to the stimulation of an antiviral response mediated by type-I interferons (IFNs) in normal but not in transformed mouse cells. The present work was undertaken to assess whether the oncotropism displayed against human cells by MVMp and its rat homolog H-1PV also depends on antiviral mechanisms and to identify the pattern recognition receptor (PRR) involved. Despite their low proliferation rate which represents a drawback for parvovirus multiplication, we used human peripheral blood mononuclear cells (hPBMCs) as normal model specifically because all known PRRs are functional in this mixed cell population and moreover because some of its subsets are among the main IFN producers upon infections in mammals. Human transformed models consisted in lines and tumor cells more or less permissive to both parvoviruses. Our results show that irrespective of their permissiveness, transformed cells do not produce IFNs nor develop an antiviral response upon parvovirus infection. However, MVMp- or H-1PV-infected hPBMCs trigger such defense mechanisms despite an absence of parvovirus replication and protein expression, pointing to the viral genome as the activating element. Substantial reduction of an inhibitory oligodeoxynucleotide (iODN) of the latter IFN production identified TLR-9 as a potential PRR for parvoviruses in hPBMCs. However, neither the iODN treatment nor an antibody-induced neutralization of the IFN-triggered effects restored parvovirus multiplication in these cells as expected by their weak proliferation in culture. Finally, given that a TLR-9 activation could also not be observed in parvovirus-infected human lines reported to be endowed with a functional TLR-9 pathway (Namalwa, Raji, and HEK293-TLR9(+/+)), our data suggest that transformed human cells do not sense MVMp or H-1PV either because of an absence of PRR expression or an intrinsic, or virus-driven defect in the endosomal sensing of the parvovirus genomes by TLR-9.

Activation of an antiviral response in normal but not transformed mouse cells: a new determinant of minute virus of mice oncotropism.

Parvovirus minute virus of mice (MVMp) is endowed with oncotropic properties so far ascribed only to the dependency of the virus life cycle on cellular factors expressed during S phase and/or modulated by malignant transformation. For other viruses oncotropism relies on their inability to circumvent type I interferon (IFN)-induced innate antiviral mechanisms, the first line of defense triggered by normal cells against viral infections. These agents propagate, therefore, preferentially in transformed/tumor cells, which often lack functional antiviral mechanisms. The present study aimed at investigating whether antiviral processes also contribute to MVMp oncotropism. Our results demonstrate that in contrast to MVMp-permissive transformed mouse A9 fibroblasts, freshly isolated normal counterparts (mouse embryonic fibroblasts [MEFs]) mount, through production and release of type I IFNs upon their infection, an antiviral response against MVMp lytic multiplication. Pretreatment of MEFs with a type I IFN-beta-neutralizing antibody, prior to MVMp infection, inhibits the virus-triggered antiviral response and improves the fulfillment of the MVMp life cycle. Our results also show that part of the A9 permissiveness to MVMp relies on the inability to produce type I IFNs upon parvovirus infection, a feature related either to an A9 intrinsic deficiency of this process or to an MVMp-triggered inhibitory mechanism, since stimulation of these cells by exogenous IFN-beta strongly inhibits the parvovirus life cycle. Taken together, our results demonstrate for the first time that parvovirus infection triggers an innate antiviral response in normal cells and suggest that the MVMp oncotropism depends at least in part on the failure of infected transformed cells to mount such a response.

Modulation of minute virus of mice cytotoxic activities through site-directed mutagenesis within the NS coding region.

Late in infection, parvovirus minute virus of mice (MVMp) induces the lysis of mouse A9 fibroblasts. This effect depends on the large nonstructural phosphoprotein NS1, which plays in addition a major role in viral DNA replication and progeny particle production. Since the NS1 C-terminal region is subjected to late phosphorylation events and protein kinase C (PKC) family members regulate NS1 replicative activities, the present study was conducted to determine the impact of PKCs on NS1 cytotoxic functions. To this end, we performed site-directed mutagenesis, substituting alanine residues for two consensus PKC-phosphorylation sites located within the NS1 C-terminal region, T585 and S588. Although these substitutions had no detectable effect on virus multiplication in a single-round infection, the NS1-585A mutant virus was significantly less toxic to A9 cells than wild-type MVMp, whereas the NS1-588A mutant virus was endowed with a higher killing potential. These alterations correlated with specific changes in the late phosphorylation pattern of the mutant NS1 proteins compared to the wild-type polypeptide. Since the mutations introduced in this region of the viral genome also made changes in the minor nonstructural protein NS2, a contribution of this polypeptide to the above-mentioned phenotypes of mutant viruses cannot be excluded at present. However, the involvement of NS1 in these phenotypes was directly supported by the respective reduced and enhanced capacity of NS1-585A and NS1-588A recombinant proteins for inducing morphological alterations and cell detachment in transfected A9 cultures. Altogether, these data suggest that late-occurring phosphorylation of NS1 specifically regulates the cytotoxic functions of the viral product and that residues T585 and S588 contribute to this control in an antagonistic way.

Specific interaction of the nonstructural protein NS1 of minute virus of mice (MVM) with [ACCA](2) motifs in the centre of the right-end MVM DNA palindrome induces hairpin-primed viral DNA replication.

The linear single-stranded DNA genome of minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate DNA. Amplification of viral RF DNA requires the structural transition of the right-end palindrome from a linear duplex into a double-hairpin structure, which serves for the repriming of unidirectional DNA synthesis. This conformational transition was found previously to be induced by the MVM nonstructural protein NS1. Elimination of the cognate NS1-binding sites, [ACCA](2), from the central region of the right-end palindrome next to the axis of symmetry was shown to markedly reduce the efficiency of hairpin-primed DNA replication, as measured in a reconstituted in vitro replication system. Thus, [ACCA](2) sequence motifs are essential as NS1-binding elements in the context of the structural transition of the right-end MVM palindrome.