The vaccinia virus E3L gene codes for double-stranded RNA (dsRNA) binding proteins which can prevent activation of the dsRNA-dependent, interferon-induced protein kinase PKR. Activated PKR has been shown to induce apoptosis in HeLa cells. HeLa cells infected with vaccinia virus with the E3L gene deleted have also been shown to undergo apoptosis, whereas HeLa cells infected with wild-type vaccinia virus do not. In this report, using virus recombinants expressing mutant E3L products or alternative dsRNA binding proteins, we show that suppression of induction of apoptosis correlates with functional binding of proteins to dsRNA. Infection of HeLa cells with ts23, which leads to synthesis of increased dsRNA at restrictive temperature, induced apoptosis at restrictive but not permissive temperatures. Treatment of cells with cytosine arabinoside, which blocks the late buildup of dsRNA in vaccinia virus-infected cells, prevented induction of apoptosis by vaccinia virus with E3L deleted. Cells transfected with dsRNA in the absence of virus infection also underwent apoptosis. These results suggest that dsRNA is a trigger that can initiate a suicide response in virus-infected and perhaps uninfected cells.

An inhibitor of eIF-2a phosphorylation was identified in various plant species. The plant protein (termed PKI) specifically cross-reacts with monoclonal antiserum that recognizes the glycosylated, active form of a M(r) 87 kD protein analog (p67) from reticulocytes. Northern blot analysis using a probe to the reticulocyte inhibitor cDNA further supports the presence of analogous transcripts in plant tissue. PKI specifically inhibits the phosphorylation of the plant encoded eIF-2 alpha kinase (pPKR) as well as plant and human eIF-2 alpha phosphorylation. The interaction between PKI and pPKR is indicated by their copurification on dsRNA agarose, despite evidence showing that PKI does not bind dsRNA. Further, wheat PKI inhibits human PKR phosphorylation but activity is recovered by immuno-depletion of PKI from wheat germ fractions. PKI is temporally regulated during plant growth and development. It is maximally present in extracts from dormant seeds, however, it is not detectable soon after leaf emergence at approximately 48 h post-imbibition. PKI levels are again detectable at the mid-milk stage in seed development. Protein levels of pPKR in ribosomal salt wash and cytosolic extracts from healthy plant tissue remain essentially constant throughout the life cycle. In contrast, pPKR activity levels based upon autophosphorylation vary significantly and are inversely correlated with PKI protein levels. Phosphorylation of eIF-2 alpha is a classical mechanism for the downregulation of protein synthesis suggesting that inhibition of pPKR activity by PKI may contribute to the dramatic and rapid increase in protein synthesis observed during seed germination.