Regulation of mRNA translation and cellular signaling by hepatitis C virus nonstructural protein NS5A.

The NS5A nonstructural protein of hepatitis C virus (HCV) has been shown to inhibit the cellular interferon (IFN)-induced protein kinase R (PKR). PKR mediates the host IFN-induced antiviral response at least in part by inhibiting mRNA translation initiation through phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). We thus examined the effect of NS5A inhibition of PKR on mRNA translation within the context of virus infection by using a recombinant vaccinia virus (VV)-based assay. The VV E3L protein is a potent inhibitor of PKR. Accordingly, infection of IFN-pretreated HeLa S3 cells with an E3L-deficient VV (VVDeltaE3L) resulted in increased phosphorylation levels of both PKR and eIF2alpha. IFN-pretreated cells infected with VV in which the E3L locus was replaced with the NS5A gene (VVNS5A) displayed diminished phosphorylation of PKR and eIF2alpha in a transient manner. We also observed an increase in activation of p38 mitogen-activated protein kinase in IFN-pretreated cells infected with VVDeltaE3L, consistent with reports that p38 lies downstream of the PKR pathway. Furthermore, these cells exhibited increased phosphorylation of the cap-binding initiation factor 4E (eIF4E), which is downstream of the p38 pathway. Importantly, these effects were reduced in cells infected with VVNS5A. NS5A was also found to inhibit activation of the p38-eIF4E pathway in epidermal growth factor-treated cells stably expressing NS5A. NS5A-induced inhibition of eIF2alpha and eIF4E phosphorylation may exert counteracting effects on mRNA translation. Indeed, IFN-pretreated cells infected with VVNS5A exhibited a partial and transient restoration of cellular and viral mRNA translation compared with IFN-pretreated cells infected with VVDeltaE3L. Taken together, these results support the role of NS5A as a PKR inhibitor and suggest a potential mechanism by which HCV might maintain global mRNA translation rate during early virus infection while favoring cap-independent translation of HCV mRNA during late infection.

Nuclear factor-90 of activated T-cells: A double-stranded RNA-binding protein and substrate for the double-stranded RNA-dependent protein kinase, PKR.

NFAT transcription factors play a central role in initiating T-cell activation through the induction of immediate-early T-cell specific genes including interleukin-2 (IL-2). NFAT transcription factors bind to a sequence in the IL-2 enhancer known as the antigen receptor response element 2 (ARRE-2). Multiple proteins exhibiting ARRE-2 binding activity have been isolated, including a heterodimer from stimulated T-cell nuclear extracts consisting of Mr = 90 000 (NF90) and Mr = 45 000 (NF45) subunits. The subunits of this heterodimer have been cloned, and NF90 was found to encode a protein containing two domains that are predicted to form motifs capable of binding to double-stranded RNA. Using in vitro translated polypeptides, we have demonstrated that NF90 specifically binds to double-stranded RNA. Furthermore, NF90 was phosphorylated in a double-stranded RNA-dependent manner likely by the interferon-induced, double-stranded RNA-dependent protein kinase, PKR. The NF90 protein was found to be expressed not only in T-cells, but also in nonimmune HeLa cells. In HeLa cells, the protein was almost exclusively localized to the ribosome salt wash fraction of cell lysates.

Characterization of viral double-stranded RNA-binding proteins.

Several viruses have been shown to code for proteins that specifically bind to double-stranded RNA or RNA with large amounts of secondary structure. These proteins have been implicated in providing interferon resistance to viruses and in inhibiting induction of apoptosis by viruses, and have been suggested to be involved in regulation of viral and cellular protein synthesis in infected cells. This article describes methods for detecting and analyzing viral double-stranded RNA-binding proteins.

Activation of antiviral protein kinase leads to immunoglobulin E class switching in human B cells.

An epidemiologic association between viral infections and the onset of asthma and allergy has been documented. Also, evidence from animal and human studies has suggested an increase in antigen-specific immunoglobulin E (IgE) production during viral infections, and elevated levels of IgE are characteristic of human asthma and allergy. Here, we provide molecular evidence for the roles of viral infection and of activation of the antiviral protein kinase (PKR) (double-stranded-RNA [dsRNA]-activated protein kinase) in the induction of IgE class switching. The presence of dsRNA, a known component of viral infection and an activator of PKR, induced IgE class switching as detected by the expression of germ line epsilon in the human Ramos B-cell line. Furthermore, dsRNA treatment of Ramos cells resulted in the activation of PKR and in vivo activation of the NF-kappaB complex. Interestingly, infection of Ramos cells with rhinovirus (common cold virus) serotypes 14 and 16 resulted in the induction of germ line epsilon expression. To further evaluate the role of PKR in the viral induction of IgE class switching, we infected Ramos cells with two different vaccinia virus (cowpox virus) strains. Infection with wild-type vaccinia virus failed to induce germ line epsilon expression; however, a deletion mutant of vaccinia virus (VP1080) lacking the PKR-inhibitory polypeptide E3L induced the expression of germ line epsilon. Collectively, the results of our study define a common molecular mechanism underlying the role of viral infections in IgE class switching and subsequent induction of IgE-mediated disorders such as allergy and asthma.

Developmental regulation of a plant encoded inhibitor of eukaryotic initiation factor 2 alpha phosphorylation.

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.

Double-stranded RNA is a trigger for apoptosis in vaccinia virus-infected cells.

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.

When two strands are better than one: the mediators and modulators of the cellular responses to double-stranded RNA.

Double-stranded RNA is a potent inducer of interferon, a modulator of the expression of a number of other genes involved in the response of cells to virus infection, an activator of the interferon-induced antiviral state, and may be involved in differentiation, induction of apoptosis, and control of oncogenic transformation. This review will attempt to summarize what is known about the cellular proteins that act to mediate the response of cells to double-stranded RNA and the viral and cellular macromolecules that may be able to modulate these responses.

Phosphorylation of plant eukaryotic initiation factor-2 by the plant-encoded double-stranded RNA-dependent protein kinase, pPKR, and inhibition of protein synthesis in vitro.

Regulation of protein synthesis by eukaryotic initiation factor-2alpha (eIF-2alpha) phosphorylation is a highly conserved phenomenon in eukaryotes that occurs in response to various stress conditions. Protein kinases capable of phosphorylating eIF-2alpha have been characterized from mammals and yeast. However, the phenomenon of eIF2-alpha-mediated regulation of protein synthesis and the presence of an eIF-2alpha kinase has not been demonstrated in higher plants. We show that plant eIF-2alpha (peIF-2alpha) and mammalian eIF-2alpha (meIF-2alpha) are phosphorylated similarly by both the double-stranded RNA-binding kinase, pPKR, present in plant ribosome salt wash fractions and the meIF-2alpha kinase, PKR. By several criteria, phosphorylation of peIF-2alpha is directly correlated with pPKR protein and autophosphorylation levels. Significantly, pPKR is capable of specifically phosphorylating Ser51 in a synthetic eIF-2alpha peptide, a key characteristic of the eIF-2alpha kinase family. Taken together, these data support the concept that pPKR is a member of the eIF-2alpha kinase family. In addition, the inhibition of brome mosaic virus RNA in vitro translation in wheat germ lysates by the addition of double-stranded RNA, phosphorylated peIF-2alpha, meIF-2alpha, or activated human PKR suggests that plant protein synthesis may be regulated via phosphorylation of eIF-2alpha.

Identification of a plant-encoded analog of PKR, the mammalian double-stranded RNA-dependent protein kinase.

Plant virus or viroid infection stimulates the phosphorylation of a plant-encoded protein of M(r) 68,000 to 70,000 (now termed pPKR) that is associated with double-stranded RNA-stimulated protein kinase activity. Using various biochemical and immunological comparisons, we have demonstrated that this plant protein is an analog of the mammalian PKR enzymes. pPKR is both cytosolic and ribosome associated, similar to mammalian PKR, and appears to be capable of phosphorylating exogenous histones. Monoclonal anti-serum to the human PKR as well as antiserum to a conserved double-stranded RNA-binding domain present on mammalian PKR demonstrated cross-reactivity with pPKR. Likewise, polyclonal antiserum to the pPKR detected the mouse and human PKR in western blot analysis. Northern blot analysis of a mammalian PKR cDNA detected a specific 2.5-kb transcript present in plant poly (A)+ RNA.

Nucleic acid affinity chromatography: preparation and characterization of double-stranded RNA agarose.

An improved method for the preparation of a double-stranded RNA-coupled resin is described. Periodate-oxidized double-stranded RNA, in the form of either purified reovirus double-stranded RNA or poly(rl). poly(rC), was covalently attached to agarose-adipic acid hydrazide. The resulting affinity resin had increased specificity for known double-stranded RNA binding proteins, as compared to a similar commercially available resin. The application of this resin in the purification and characterization of double-stranded RNA binding proteins is described.

Products of the porcine group C rotavirus NSP3 gene bind specifically to double-stranded RNA and inhibit activation of the interferon-induced protein kinase PKR.

The porcine group C rotavirus (Cowden strain) NSP3 protein (the group C equivalent of the group A gene 7 product, formerly called NS34) shares homology with known double-stranded RNA-binding proteins, such as the interferon-induced, double-stranded RNA-dependent protein kinase PKR. A clone of NSP3, expressed both in vitro and in COS-1 cells, led to the synthesis of minor amounts of a product with an M(r) of 45,000 (the expected full-length M(r) of NSP3) and major amounts of products with M(r)s of 38,000 and 8,000. Restriction enzyme digestion analysis prior to expression in vitro and amino-terminal sequence analysis suggest that the products with M(r)s of 38,000 and 8,000 are cleavage products of the protein with an M(r) of 45,000. The full-length protein and the product with an M(r) of 8,000, both of which contain the motif present in double-stranded RNA-binding proteins, bound specifically to double-stranded RNA. The products with M(r)s of 45,000 and 8,000 were also detected in Cowden strain-infected MA104 cells. NSP3 products expressed in COS-1 cells were capable of inhibiting activation of the double-stranded RNA-dependent protein kinase similar to other double-stranded RNA-binding proteins, and NSP3 products expressed in HeLa cells were capable of rescuing the replication of an interferon-sensitive deletion mutant of vaccinia virus.

TAR RNA-binding protein is an inhibitor of the interferon-induced protein kinase PKR.

A cDNA encoding a double-stranded-RNA (dsRNA)-binding protein was isolated by screening a HeLa cell cDNA expression library for proteins that bind the HIV-1 Rev-responsive-element RNA. The cDNA encoded a protein that was identical to TRBP, the previously reported cellular protein that binds the transactivation response element (TAR) RNA of human immunodeficiency virus type 1. TRBP inhibited phosphorylation of the interferon-induced ribosome-associated protein kinase PKR and of the eukaryotic translation initiation factor eIF-2 alpha in a transient-expression system in which the translation of a reporter gene was inhibited by the localized activation of PKR. TRBP expression in HeLa cells complemented the growth and protein-synthesis defect of a vaccinia virus mutant lacking the expression of the dsRNA-binding protein E3L. These results implicate TRBP as a cellular regulatory protein that binds RNAs containing specific secondary structure(s) to mediate the inhibition of PKR activation and stimulate translation in a localized manner.

Current activities within the National Biomonitoring Specimen Bank.

The National Institute of Standards and Technology (NIST) has been involved in biological environmental specimen banking activities since 1979. These activities, which are known collectively as the National Biomonitoring Specimen Bank (NBSB), include the banking of a variety of specimens (human liver, sediment, mussels/oysters, fish tissue and marine mammal tissues) from several different projects supported by different government agencies. The two most recent projects, the Alaska Marine Mammal Tissue Archival Project (AMMTAP) and the National Marine Mammal Tissue Bank (NMMTB), focus on the collection, banking and analysis of marine mammal tissues and they are part of a comprehensive plan to address marine mammal monitoring, specimen banking and quality assurance of analytical measurements associated with contaminant analyses in marine mammals.

Neutron Depth Profiling: Overview and Description of NIST Facilities.

The Cold Neutron Depth Profiling (CNDP) instrument at the NIST Cold Neutron Research Facility (CNRF) is now operational. The neutron beam originates from a 16 L D2O ice cold source and passes through a filter of 135 mm of single crystal sapphire. The neutron energy spectrum may be described by a 65 K Maxwellian distribution. The sample chamber configuration allows for remote controlled scanning of 150 × 150 mm sample areas including the varying of both sample and detector angle. The improved sensitivity over the current thermal depth profiling instrument has permitted the first nondestructive measurements of 17O profiles. This paper describes the CNDP instrument, illustrates the neutron depth profiling (NDP) technique with examples, and gives a separate bibliography of NDP publications.

Cytosolic double-stranded RNA-dependent protein kinase is likely a dimer of partially phosphorylated Mr = 66,000 subunits.

The work described in this report suggests the existence of two biochemically distinguishable forms of the interferon-inducible, double-stranded RNA-dependent protein kinase. Kinase isolated from the cytosolic fraction (S-100) and the ribosome salt wash fraction of interferon-treated cells differed in their chromatographic properties. S-100 kinase eluted from a gel filtration column with M(r) = 140,000-160,000 and was predominantly anionic in nature, whereas ribosomal kinase eluted with M(r) = 66,000 and was predominantly cationic in nature. Purified preparations of S-100 kinase contained the M(r) = 66,000 subunit, P1, as the only polypeptide present in stoichiometric amounts, and thus the S-100 kinase appears to be a dimer of P1 subunits. Dimerization of the S-100 kinase was dependent on the phosphorylation state of the enzyme. Kinase isolated from S-100 was partially phosphorylated. Dephosphorylation of the S-100 kinase by treatment with alkaline phosphatase resulted in a monomeric form of the enzyme with biochemical characteristics similar to that of the ribosome salt wash kinase.