Prion diseases are fatal neurodegenerative disorders characterized by long incubation periods. To investigate whether concurrent diseases can modify the clinical outcome of prion-affected subjects, we tested the effect of viral infection on the binding and internalization of PrP(Sc), essential steps of prion propagation. To this effect, we added scrapie brain homogenate or purified PrP(Sc) to fibroblasts previously infected with minute virus of mice (MVM), a mouse parvovirus. We show here that the rate of incorporation of PrP(Sc) into MVM-infected cells was significantly higher than that observed for naïve cells. Immunostaining of cells and immunoblotting of subcellular fractions using antibodies recognizing PrP and LysoTracker, a lysosomal marker, revealed that in both control and MVM-infected cells the incorporated PrP(Sc) was associated mostly with lysosomes. Interestingly, flotation gradient analysis revealed that the majority of the PrP(Sc) internalized into MVM-infected cells shifted toward raft-containing low-density fractions. Concomitantly, the MVM-infected cells demonstrated increased levels of the glycosphingolipid GM1 (an essential raft lipid component) throughout the gradient and a shift in caveolin 1 (a raft protein marker) toward lighter membrane fractions compared with noninfected cells. Our results suggest that the effect of viral infection on membrane lipid composition may promote the incorporation of exogenous PrP(Sc) into rafts. Importantly, membrane rafts are believed to be the conversion site of PrP(C) to PrP(Sc); therefore, the association of exogenous PrP(Sc) with such membrane microdomains may facilitate prion infection.
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.
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.
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