Parvoviruses infect and kill tumor cells in vivo and in vitro more efficiently than normal cells. Infection of transformed cells by the parvovirus minute virus of mice (MVM) results in high expression of the major non-structural cytolytic viral protein NS1, which induces a cell death modulated by cellular factors. In this work, we show that MVMp infection and/or NS1 protein expression in permissive transformed rat fibroblast cells leads to apoptosis in wild type and p53(-/-) cells. Apoptotic cell morphology correlates with mitochondrial membrane permeabilization and activation of caspases 9 and 3 but not caspase 8. Thus, further characterization of the antitumor activity of MVMp and its NS1 protein may contribute to the eradication of tumors, including those lacking p53.
Fischer rat fibroblasts, naturally resistant to killing by the fibrotropic strain of minute virus of mice [(parvovirus MVM(p)], became sensitive to MVM when transformed by polyomavirus. This sensitization did not involve an increase in the percentage of cells which synthesized viral capsid antigens or in the percentage of cells which produced infectious virus. The addition of anti-MVM antiserum to the growth medium of MVM-infected cells had only a small effect on their survival rates, indicating that the majority of the killing effect of MVM occurs in a single cycle of infection. The data indicate that cell killing by MVM is independent of infectious virus production and thus support the notion that the preferential cytolytic effect is affected by viral cytotoxic gene products which accumulate to intolerable levels in transformed cells but not in normal ones. Finally, using cells transformed with polyomavirus and genomic and subgenomic clones of polyomavirus, we showed that the extent of sensitization to killing by MVM depended on the transforming agent used.
PBCV-1 belongs to a family of large viruses that replicate in the exsymbiont green algae Chlorella strain NC64A. The viral, 330-kb DNA genome encodes a relatively large number of functionally active proteins including restriction and modification enzymes, DNA polymerase, glycosylation, and cell wall degrading enzymes. Sequencing of the viral DNA, now in progress, revealed many major open reading frames (ORF), which resemble known genes in sequence data bases and which have not previously been found in viral genomes. Here we report on the identification and characterization of one such gene, aspartate transcarbamylase (ATCase), an enzyme that catalyzes the committing step in the de novo biosynthetic pathway of pyrimidines. The cloned gene is highly homologous to a variety of plant ATCases and includes the typical ATCase catalytic motif. When cloned into the pGEX-2T expression vector, a fusion protein with ATCase activity could be demonstrated and distinguished from the host ATCase activity. The viral enzyme is expressed early and transiently in the infection. To our knowledge, this is the first virus known to encode and express its own de novo nucleotide precursors' synthetic enzymes.
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.
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