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

Abstract

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. Permissive PKR(o/o) cells were converted to MVMp-restrictive cells after reintroduction of the PKR gene by transfection. Conversely, regulated expression of the vaccinia virus E3 protein, a PKR inhibitor, in MEFs prevented eIF2alpha phosphorylation and increased MVMp protein synthesis. In vitro-synthesized genome-length R1 mRNA of MVMp was a potent activator of PKR. Virus-resistant primary MEFs and NIH 3T3 cells responded to MVMp infection with significant increases in eIF2alpha phosphorylation. In contrast, virus-permissive mouse (PKR(o/o), BHK21, and A9) and human transformed (NB324K fibroblast, U373 glioma, and HepG2 hepatoma) cells consistently showed no significant increase in the level of eIF2alpha phosphorylation following MVMp infection. The synthesis of the viral NS1 protein was inversely correlated with the steady-state PKR levels. Our results show that the PKR-mediated antiviral response is an important mechanism for control of productive MVMp infection, and its impairment in human transformed cells allowed efficient MVMp gene expression. PKR translational control may therefore contribute to the oncolysis of MVMp and other autonomous parvoviruses.

FIG. 1.

Analysis of MVMp replication in NIH 3T3 and 3T3 PKR^o/o cells. (A) IF of MVMp-infected cells probed at 24 hpi with specific antibodies for detection of total virus structural proteins (α-VPs) or the protein subunits assembled in capsids (α-capsids). (B) MVMp yield in wild-type 3T3 and 3T3 PKR^o/o cells. Supernatants of infected cultures (MOI, 0.1) were sampled, and extracellular viral yield was quantified by a plaque assay. A representative result obtained at 48 hpi is shown. (C) Effect of MVMp infection (MOI, 5) on NIH 3T3 and 3T3 PKR^o/o cell growth rates. The number of viable cells measured by trypan blue exclusion at the indicated days postinfection (dpi) is shown. (D) Micrographs showing the cytopathic effect provoked by MVMp on the cultures outlined in panel C at 3 dpi.

FIG. 2.

MVMp gene expression and PKR activity in 3T3 fibroblasts. (A) Time course accumulation of MVMp proteins and eIF2α phosphorylation in wild-type 3T3 and PKR^o/o cells. Infected cells were lysed in sample buffer and analyzed by Western blotting at the indicated time points. Once the anti-phospho eIF2α blot was carried out, the membrane was stripped and reprobed with anti-total eIF2α. (B) Protein synthesis in MVMp-infected 3T3 cells. Cells were pulse-labeled with [³⁵S]Met (25 μCi/ml) for 30 min at the indicated time points (hpi), and labeled proteins were resolved by 10% SDS-PAGE and autoradiography. −, mock-infected culture. (C) IF analysis of eIF2α phosphorylation in MVMp-infected 3T3 cells. At 15 hpi, cells were fixed and simultaneously probed with anticapsid (red) and anti-phospho eIF2α (green). Arrows point to infected 3T3 cells showing some MVMp input viral particles accumulated in endosomal vesicles.

FIG. 3.

The PKR gene blocks MVMp replication in 3T3 PKR^o/o cells. (A) PKR^o/o cells were transfected with the pcPKR or pcβ-Gal plasmid. Twenty-four hours afterward, cells were infected with MVMp (MOI, 5) and processed at 24 hpi for IF with the anti-myc and anti-NS1 antibodies. Representative cell fields are shown for each experiment. Note that a fraction of the PKR protein expressed from transfected plasmid is accumulated into the nucleus as described before (73). (B) Statistical analysis of the effect of PKR expression on MVMp replication. About 300 cells from 10 randomly selected fields were scored for each sample. The percentages of transfected cells expressing NS1 (left) or expressing both myc-tagged (PKR or β-Gal) proteins and the viral NS1 protein (right) are shown. The experiment was performed twice, with similar outcomes. The percentage of untransfected cells productively infected with MVMp under these conditions, tested in parallel by NS1 expression, was about 40%.

FIG. 4.

Inhibition of PKR activity enhances MVMp protein synthesis. Tet-Off MEFs expressing the vaccinia virus E3 protein (vvE3) were induced overnight by tetracycline withdrawal (ON) and infected with MVMp at an MOI of 10. At the indicated time points (hpi), the cells were harvested and analyzed by Western blotting with the indicated antibodies. Uninduced cells (24) were maintained with 1 μg/ml tetracycline during the experiment.

FIG. 5.

In vitro activation of PKR by the MVMp R1 genomic messenger. Purified, myc-tagged PKR was assayed for its ability to phosphorylate eIF2α in the presence of increasing concentrations of in vitro-transcribed MVMp R1 mRNA (5 kb) or of dsRNA poly(I:C) of heterogeneous molecular masses (Mw) (100 to 185 kDa). Proteins were resolved by 10% SDS-PAGE and transferred to an Immobilon-P membrane. The membrane was exposed to autoradiography (upper panel) and subsequently probed with three different antisera to detect myc-tagged PKR, phosphorylated eIF2α at serine 51 (eIF2α-P), and total eIF2α (middle panels). For the lower panel, the amount of ³²P incorporated into eIF2α was quantified with a BAS-1500 phosphorimager (Fujifilm) and is presented as the fold induction value compared to the value obtained when no RNA was present in the reaction (set to 1). Results for one representative experiment out of three, producing similar results, are shown.

FIG. 6.

MVMp permissiveness of rodent and human transformed cells negatively correlates with PKR activity and eIF2α phosphorylation. (A, B) Rodent (MEF, NIH 3T3, PKR^o/o, BHK21, and A9) and human transformed (NB324K, U373, and HepG2) cell lines, either mock (−) or MVMp infected, were analyzed for NS1 expression and eIF2α phosphorylation at the indicated time points (hpi) by Western blotting. Panels A and B show results for two representative experiments performed with parallel infections. Membranes were first probed with the anti-phospho eIF2α antibody, then stripped, and subsequently reprobed for total eIF2α accumulation. Note that substantial differences in the levels of total and phosphorylated eIF2α forms were found among the cell lines. Bands were quantified by densitometry, and the ratio between phosphorylated and total eIF2α is shown below. Since the basal level of phosphorylated eIF2α was undetectable in uninfected NIH 3T3 cells, its ratio was set to 0, and an arbitrary value of 1 was assigned to MVMp-infected 3T3 cells at 15 hpi. (C) The levels of PKR protein accumulation in the cell lines, compared to the level for the β-actin (upper) or the eIF2α (lower) housekeeping protein control, are shown. Equal amounts of total protein were probed with the indicated antisera.