Pharmac. Ther.Vol.54, pp. 307-317, 1992 Printed in Great Britain. All rightsreserved

0163-7258/92$15.00 © 1992PergamonPressLtd

Associate Editor: S. PESTKA

ROLE OF THE RNA-DEPENDENT PROTEIN KINASE IN THE REGULATED EXPRESSION OF GENES IN TRANSFECTED CELLS CHARLES E. SAMUEL

Department of Biological Sciences, Division of Molecular and Cellular Biology and the Graduate Program of Biochemistry and Molecular Biology, University of California, Santa Barbara, CA 93106, U.S.A. Abstract--The RNA-dependent P1/elF-2a protein kinase is a highly specific protein-serine/ threonine kinase that catalyzes the phosphorylation of the alpha subunit of protein synthesis initiation factor elF-2. The kinase plays a central role in translational control. The activity of the kinase is regulated by a variety of naturally occurring effector RNAs which bind to the regulatory domain of the enzyme. Certain RNAs are able to activate the elF-2~t kinase activity inherent within protein P1, a process which involves an autophosphorylation of protein P1, whereas other RNAs are able to antagonize the activation process. Translational repression mediated by the kinase may also be disrupted by RNA binding proteins that sequester activator double-stranded RNAs and by site-directed mutants and homologs of the elF-2a translation factor substrate. The P1/elF-2ct protein kinase is an important regulator of the translation of plasmid-derived mRNAs in transfected eukaryotic cells.

CONTENTS 1. 2. 3. 4.

Introduction The Enzyme The Substrate Regulated Expression of Genes by the Kinase in Transfected Cells 4.1. RNA effectors of kinase activity 4.1.1. Activators 4.1.2. Inhibitors 4.2. Purine effectors of kinase activity 4.3. Substrate analogs and protein phosphorylation 4.3,1. Point mutants of initiation factor elF-2~ 4.3.2. Homologs of initiation factor elF-2a 4.4. Protein effectors of kinase activity 4.4.1. Reovirus $4 4.4.2. Vaccinia virus E3L 5. Conclusion Acknowledgements References

307 3O8 309 309 310 310 310 311 312 312 312 313 313 313 314 314 314

1. I N T R O D U C T I O N A m o n g the processes in eukaryotic cells regulated by changes in protein phosphorylation is the initiation of messenger R N A (mRNA) translation. One of the key factors required for m R N A translation initiation whose activity is modulated by phosphorylation--dephosphorylation is elF-2. The phosphorylation state of the ~ subunit of elF-2 is often increased and m R N A translation

Abbreviations--P1 kinase, the RNA-dependent P1/elF-2ct protein-serine/threonine kinase; IFN, interferon; elF-2ct, the ct subunit of eukaryotic protein synthesis initiation factor 2; dsRNA, double-stranded RNA; CAT, chloramphenicol acetyltransferase; DHFR, dihydrofolate reductase. 307

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C.E. SAMUEL

PI/eIF-2~ Protein-Serine/Threonine Kinase (Inactive)

l

Autophosphorylation RNA-dependent

P1/eIF-2tx Protein-Serine/rhreonine Kinase.(~)(Active) (Ribosome-associated)

Initiation~ PhosphorylatedelF-2c¢-(~ ~ InitiationFactor i, Pi Phosphatase (Soluble)

elF-2c¢ Factor

~

Inhibition of Translation

FIG. 1. The inactive P1/elF-2ct protein-serine/threoninekinase is activated by an RNA-dependent autophosphorylation of protein PI, thereby yielding an active protein kinase capable of catalyzing the phosphorylation of serine residue 51 of the alpha subunit of protein synthesis initiation factor elF-2. Phosphorylation of elF-2~ leads to an inhibition of translation. decreased under a variety of conditions including interferon treatment and virus infection as well as transfection of cells with plasmid-based cDNA expression vectors. The RNA-dependent protein kinase responsible for the phosphorylation of the ct subunit of eukaryotic protein synthesis initiation factor 2 (elF-2~) is highly specific and finely regulated. By contrast, the type 2A protein phosphatase responsible for the elF-2c~P dephosphorylation exhibits broad specificity and is not known to be regulated in a manner comparable to the kinase. The phosphorylation of elF-2~ by the RNA-dependent P1/elF-2a protein kinase is summarized in Fig. 1. This review provides a brief background about the P1/elF-2ct kinase and its substrate and then summarizes in detail our understanding of the impact of the RNA-dependent kinase on the efficiency of synthesis of proteins from plasmid-derived mRNAs in transfected cells.

2. THE ENZYME The RNA-dependent elF-2ct protein kinase is a cyclic AMP-independent, protein-serine/ threonine kinase (Pestka et al., 1987; Samuel, 1991). This kinase is designated herein as the P1/elF-2~ protein kinase, but it is also known as the P1 kinase, p68 kinase, DAI and dsI (Samuel, 1979, 1991; Pestka et al., 1987). The P1/elF-2~ protein kinase is induced by type I interferons (IFN-~ and IFN-fl) in most kinds of animal ceils (Pestka et al., 1987; Samuel, 1991; Thomis et al., 1992). However, a basal level of PI/elF-2~ protein kinase activity is typically observed in untreated cells grown in culture. Catalytic activity of the P1/elF-2~ protein kinase is dependent upon RNA for activation, a process which involves an autophosphorylation of the interferoninducible protein PI (Samuel et al., 1984; Pestka et al., 1987; Barber et al., 1991; McCormack et al., 1992; Thomis et al., 1992). Certain RNA species selectively activate the kinase, whereas others selectively block activation (Samuel, 1991). Activated P1 kinase catalyzes the phosphorylation of the ~ subunit of elF-2 (Samuel, 1979; Samuel et al., 1984; Pathak et al., 1988). The P1/elF-2~ protein kinase from human cells is a 551 amino acid protein, as deduced from the cDNA nucleotide sequence (Thomis et al., 1992). The apparent size of the P1/elF-2~ kinase estimated from NaDodSO4-PAGE is about 67 kDa, somewhat larger than the 62 kDa size predicted from the cDNA clones of the human enzyme. The enzyme from murine cells appears to be somewhat smaller; the longest open reading frame of the murine cDNA would encode a 518 amino acid protein (Thomis et al., 1992; Feng et al., 1992; Icely et aL, 1991). The RNA-binding domain and the catalytic subdomains of the PI/elF-2~ protein kinase map to different regions of the P1 protein primary sequence (Thomis et al., 1992; McCormack et al., 1992). The C-terminal portion of the P1 protein includes the various catalytic subdomains of the P1/elF-2~ protein kinase which are conserved among all protein-serine/threonine kinases (Hanks et al., 1988; Meurs et al., 1990; Thomis et al., 1992). The N-terminal region of the PI protein

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309

constitutes the regulatory domain that includes the region of the P1/elF-2ct protein kinase that possesses the RNA-binding activity (McCormack et al., 1992; Katze et al., 1991; Feng et al., 1992; Patel and Sen, 1992). 3. THE SUBSTRATE The substrate of the activated RNA-dependent P1 protein kinase is the e subunit of eukaryotic protein synthesis initiation factor elF-2 (Samuel, 1979; Pathak et al., 1988). The RNA-dependent P1/elF-2e protein-serine/threonine kinase catalyzes the phosphorylation of the ct subunit of elF-2 on serine residue 51 (Samuel, 1979; Pathak et al., 1988). GTP, elF-2 and methionyl-tRNAmet together form a ternary complex which is involved in the binding of initiator transfer RNA to ribosomes during the initiation stage of translation (Hershey, 1989; Moldave, 1985). The elF-2 factor itself is composed of three nonidentical polypeptide subunits, e, fl and 7. Phosphorylation of the ct subunit, the smallest subunit at 36 kDa, correlates with an inhibition of protein synthesis (Hershey, 1989; Moldave, 1985). Protein synthesis is inhibited upon phosphorylation of elF-2ct because the recycling of elF-2 is effectively prevented as a consequence of sequestering elF-2B by elF-2eP:GDP, elF-2B, the factor that catalyzes the GDP:GTP guanine nucleotide exchange, is typically not as abundant as elF-2 in cells (Hershey, 1989; Moldave, 1985; Safer, 1983). Because elF-2B is present in relatively low levels, a localized phosphorylation of less than 100% of elF-2e can severely inhibit protein synthesis. 4. REGULATED EXPRESSION OF GENES BY THE KINASE IN TRANSFECTED CELLS The RNA-dependent P1/elF-2~ protein kinase plays a key role in the regulation of translation in eukaryotic cells under a variety of conditions, including interferon treatment, virus infection, heat shock and other physiologic conditions (Pestka et al., 1987; Schneider and Shenk, 1987; Hershey, 1989; Samuel, 1991). Important in pharmacology and molecular biology and potentially also in therapeutics, are the observations that the kinase can have a major effect on the efficiency of synthesis of proteins from plasmid-derived mRNAs in animal cells transfected with cDNA expression vectors. Some of the most direct evidence for the involvement of the RNA-dependent P1/elF-2~ protein kinase in the regulation of gene expression in transfected cells emerges from the analysis of effectors of kinase activity. These activators and inhibitors of the kinase represent many TABLE1. Effectors o f P l /eIF-2ct Protein Kinase Activity Effector

Reference

A. Activators RNA

Reovirus sl mRNA HIV TAR RNA Reovirus genome RNA

1 1-4 1, 5

RNA

Adenovirus VA RNA Epstein-Barr virus EBER RNA HIV TAR RNA

6, 7 8 9

Protein

Reovirus RNA binding protein a3 Vaccinia virus RNA binding protein E3L Vaccinia virus elF-2ct homoiogue K3L 2-Aminopurine

B. lnhibitors

Purine

10 11 12 13, 14

(1) Bischoffand Samuel (1989); (2) Edery et al. (1989); (3) SenGupta and Silverman (1989); (4) Roy et al. (1991); (5) Samuel (1979); (6) Kitajewski et al. (1986); (7) O'Malley et al. (1986); (8) Clarke et al. (1990); (9) Gunnery et al. (1990); (10) Imani and Jacobs (1988); (11) Chang et al. (1992); (12) Beattie et al. (1991); (13) Farrell et al. (1977); (14) DeBenedett and Baglioni (1984).

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classes of molecules which act by different mechanisms. They include virus-encoded RNAs and proteins, 2-aminopurine and substrate analogs of elF-2ct (Table 1). 4.1. RNA EFFECTORSOF KINASEACTIVITY The study of virus-specific RNAs which modulate kinase activity has provided some of the most direct evidence for the importance of the P1/elF-2~ protein kinase in the regulation of protein synthesis. Among these RNAs are some which selectively activate autophosphorylation of the RNA-dependent kinase and some which selectively inhibit the autophosphorylation and associated activation of the elF-2~t kinase activity. 4.1.1. Activators

The autophosphorylation of protein P1 is dependent upon double-stranded RNA (dsRNA), or single-stranded RNA that is able to assume the required structure (Lengyel, 1982; Pestka et al., 1987; Samuel, 1991). Attempts have been made to identify the structural requirements of RNA for the in vitro activation of the kinase autophosphorylation reaction. Both synthetic (e.g. poly rI~-poly rCn) and natural (e.g. reovirus genome RNA) dsRNAs are able to activate the Pl/elF-2a protein kinase. With synthetic homopolymer dsRNA, about 30 to 50 base pairs is the minimum required for activation. Synthetic polymers with an average of one mismatch every 45 nucleotides and triple-stranded RNA complexes are both good kinase activators. However, the P1/elF-2a kinase is not activated by either single-stranded or double-stranded DNA, RNA:DNA heteroduplexes, or synthetic homopolymer single-stranded R N A (Hunter et al., 1975; Minks et al., 1979; Samuel, 1979; Baglioni et al., 1981; Torrence et al., 1981; Lasky et al., 1982). In addition to dsRNA, certain natural single-stranded R N A s are potent activators of the RNA-dependent P1/elF-2ct kinase (Bischoff and Samuel, 1989; SenGupta and Silverman, 1989; Edery et al., 1989). For example, reovirus sl mRNA and human immunodeficiency virus RNAs containing the TAR region are good kinase activators. The ability of single-stranded RNA to activate the P1/elF-2ct kinase is quite selective and appears to require an appropriate secondary or higher-ordered structure of the RNA. For example, the reovirus sl mRNA is a potent activator of the kinase, but the reovirus s4 mRNA, globin mRNA, transfer RNA and ribosomal 5S RNA are not activators or are very poor activators of the kinase (Bischoff and Samuel, 1989). In transfected COS cells, plasmid-derived reovirus s4 mRNA, which does not efficiently activate the kinase, is translated about 5 times more efficiently than plasmid-derived sl mRNA which does activate the kinase (Munemitsu and Samuel, 1988). A 161 nucleotide region of the sl mRNA has been identified by mutational analysis which is sufficient for kinase activation, either alone or when fused to the otherwise inactive s4 RNA (Bischoff and Samuel, 1989). In the case of HIV, RNAs that contain the TAR region including the relatively short 60-nucleotide cytoplasmic RNAs function as HIV-specific kinase activators (SenGupta and Silverman, 1989; SenGupta et al., 1990; Edery et al., 1989). The integrity of the stem structure of the HIV TAR RNA is required for interaction with the kinase (Roy et al., 1991). These results indicate that the differential translational efficiency of mRNAs in vivo may be attributed in part to their differential ability to activate the RNA-dependent P1/elF-2a protein kinase. 4.1.2. Inhibitors

The analysis of virus-specific RNA inhibitors of the autophosphorylation of protein P1 has also provided direct evidence for the involvement of the RNA-dependent P1/elF-2~ protein kinase in the translational control of gene expression in transfected cells. Three animal viruses that produce small, virus-specific RNA inhibitors of kinase activation are adenovirus (VAI RNA), Epstein-Barr virus (EBERI RNA) and HIV (TAR RNA). The adenovirus VAI RNA is a small, 160-nucleotide RNA product transcribed by RNA polymerase III (Mathews and Shenk, 1991). VAI RNA antagonizes the activation of the P1/elF-2~ protein kinase (Kitajewski et al., 1986; O'Malley et al., 1986). Adenovirus VAI RNA binds to the N-terminal regulatory region of the P1 protein (McCormack et al., 1992) and antagonizes the activation of the P1/elF-2~ protein kinase by blocking the autophosphorylation of P1 (Ghadge

RNA-dependent protein kinase

311

et al., 1991; Kitajewski et al., 1986; Kostura and Mathews, 1989; Mellits et al., 1990; O'Malley et al., 1986). The Epstein-Barr virus EBER-1 RNA, like the adenovirus VAI RNA, binds to the

PI/elF-2~ protein kinase and prevents activation and subsequent translation inhibition (Clarke et al., 1990, 1991). And, the HIV TAR RNA may serve as an inhibitor as well as an activator of the kinase (Gunnery et al., 1990).

The translation of plasmid-derived mRNAs in transfected cells is increased by the presence of adenovirus VAI RNA, an antagonist of the P1/elF-2~ protein kinase (Kaufman, 1985; Kaufman et al., 1989; Svensson and Akusjarvi, 1985). The VAI RNA-mediated increase in translational efficiency results from blocking the RNA-dependent autophosphorylation activation of the kinase (Akusjarvi et al., 1987; Kaufman and Murtha, 1987). Adenovirus VAI RNA, a kinase inhibitor, and reovirus sl mRNA and synthetic poly rI:poly rC, kinase activators, all bind to the same RNA binding site on the the RNA-dependent P1/elF-2~ protein kinase (McCormack et al., 1992). However, the VAI RNA appears to contain two domains, one that promotes binding to kinase and the other which interferes with P1 autophosphorylation and kinase activation (Mellits et al., 1990). The involvement of VAI RNA in translation control is seemingly direct because of the following two results. First, a mutant VAI RNA which fails to function in vivo in virus replication also fails to inhibit the activation of the RNA-dependent P1/elF-2~ protein kinase (Furtado et al., 1989; Kitajewski et al., 1986). Second, the expression in cell lines of a ser51ala mutant of elF-2~ which cannot be phosphorylated by the P1/elF-2~ protein kinase complements adenovirus VAI gene deletion mutants and permits growth of the mutant virus (Davies et al., 1989). 4.2. PURINEEFFECTORSOF KINASEACTIVITY 2-Aminopurine, an adenine isomer, is a potent inhibitor of the P1/elF-2a protein kinase (DeBenedetti and Baglioni, 1983; Farrell et al., 1977). The effect of 2-aminopurine on the RNA-dependent P1/elF-2~ protein kinase is selective. Cellular protein kinases in general are not affected by treatment with the purine analog as determined from the analysis of protein synthesis and phosphorylation patterns of cells in culture (Zinn et al., 1988; Samuel and Brody, 1990). The translation of plasmid-derived mRNA is enhanced by treatment of transfected cells with 2-aminopurine (Kaufman and Murtha, 1987; Samuel and Brody, 1990; Kalvakolanu et al., 1991). The effects of 2-aminopurine are observed in both transiently transfected cells and permanently transfected cell lines. The translational efficiency of adenosine deaminase and dihydrofolate reductase (DHFR) mRNAs measured in transiently transfected cells is increased by treatment with 2-aminopurine (Kaufman and Murtha, 1987). Likewise, the translational efficiency of the reovirus sl mRNA, a potent activator of the RNA-dependent protein kinase, is increased about 5-fold in transiently transfected COS cells treated with 2-aminopurine. However, the translational efficiency of s4 mRNA, an RNA which does not activate the kinase, is not affected by 2-aminopurine treatment (Samuel and Brody, 1990). The improved synthesis of proteins mediated by 2-aminopurine observed in these transient transfection assays is selective. The 2-aminopurine treatment does not affect global mRNA translation in transfected cells (Kaufman and Murtha, 1987; Samuel and Brody, 1990). Possibly the localized activation and the subsequent localized inhibition of activation by the purine analog, may account for the selectivity (DeBenedetti and Baglioni, 1984). The chloramphenicol acetyltransferase and neomycin phosphotransferase enzymic activities are increased up to 50-fold by 2-aminopurine treatment of cells permanently transfected with the genes encoding these reporter proteins (Kalvakolanu et al., 1991). The increase in chloramphenicol acetyltransferase (CAT) activity is due to an increase in translation of the CAT mRNA and is observed in various cell lines stably transfected with various transcriptional regulatory elements (Kalvakolanu et al., 1991). In these permanently transfected cells, the transfected reporter genes are integrated into the cellular chromosomes (Kalvakolanu et al., 1991) and thus differ from the episomal location of the reporter cellular or viral genes in the transiently transfected cells (Kaufman and Murth, 1987; Samuel and Brody, 1990). The mechanism responsible for the selective effect of 2-aminopurine on the enhanced translation of mRNA of exogenous genes in stable transformants has not yet been fully elucidated, but presumably again involves the impairment of a selective and localized activation of the kinase.

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C.E. SAMUEL 4.3. SUBSTRATEANALOGSAND PROTEINPHOSPHORYLATION

4.3.1. Point M u t a n t s o f Initiation Factor eIF-2ct Protein synthesis initiation factor eIF-2 is a heterotrimeric complex composed of three subunits. Activated RNA-dependent P1/eIF-2ct protein kinase phosphorylates the ~ subunit of eIF-2 on the serine residue (Samuel, 1979) at position 51 (Pathak et al., 1988) of the eIF-2~ subunit. The role of phosphorylation of the eIF-2~ subunit in translational control has been clearly demonstrated by studying the synthesis of reporter proteins within whole cells that overexpress mutant forms of eIF-2~ (Kaufman et al., 1989). When cDNAs encoding mutant subunits of human eIF-2~ are expressed in transfected COS cells, the mutant ~ subunit readily exchanges with the endogenous wild-type ~ subunit in the trimeric factor. Two types of eIF-2~ mutants have been especially useful in the analysis of the importance of eIF-2ct phosphorylation in translational control. The ser51ala mutant of eIF-2~ possesses an alanine residue in place of serine at the site of phosphorylation, serine 51, and thus cannot be phosphorylated by the RNA-dependent protein kinase (Pathak et al., 1988; Choi et al, 1992). By contrast, the ser51asp mutant of eIF-2~ possesses an aspartic acid residue at position 51; this substitution appears to functionally mimic a phosphorylated serine residue at position 51 (Pathak et al., 1988; Choi et al., 1992). Translation of DHFR mRNA in COS cells transfected with a DHFR cDNA expression vector is inefficient due to activation of the RNA-dependent P1/eIF-2ct kinase in the transfected cells (Kaufman and Murtha, 1987). Cotransfection of COS cells with the ser51asp mutant of eIF-2ct, where an aspartic acid residue replaces the naturally occurring serine residue at position 51 of the wild-type human eIF-2~, causes further inhibition of DHFR protein synthesis (Kaufman et al., 1989; Choi et al., 1992). By contrast, the ser51ala mutant of eIF-2c~ does not cause an inhibition of protein synthesis, but rather stimulates the synthesis of DHFR reporter protein in transfected COS cells (Kaufman et al., 1989; Choi et al., 1992). When the extent of eIF-2~ phosphorylation is quantitated in COS cells by two-dimensional polyacrylamide gel electrophoresis, the stimulation observed in reporter protein synthesis by the ser51ala variant is readily explained by the failure of eIF-2~ to be phosphorylated (Choi et al., 1992). Furthermore, expression of the ser51ala mutant of eIF-2~ which is resistant to phosphorylation by the RNA-dependent P1/eIF-2~ protein kinase complements deletions in the adenovirus VA genes. That is, the adenovirus d/720 mutant which lacks both the VAI and the VAII RNA genes is severely defective for virus growth in human 293 cells that express wild-type eIF-27. However, d/720 virus growth is restored in 293 cells expressing the serine to alanine mutant of eIF-2~ (Davies et al., 1989). These results show that the primary function of the adenovirus VAI RNA is to block the activation of the RNA-dependent protein kinase and identify eIF-2~ as the primary substrate that mediates the effects of the activated kinase. 4.3.2. H o m o l o g s o f Initiation Factor elF-2ct Vaccinia virus (VV) encodes a homolog of the ~ subunit of eIF-2, K3L. The VV K3L gene includes an open reading frame predicted to encode an 88 amino acid product which has 28% identity with the 315 amino acid ~ subunit of eIF-2 (Beattie et al., 1991; Ernst et al., 1987; Goebel et al., 1990). The region of identity between VV K3L and eIF-2~ includes the region surrounding the site of phosphorylation on c~ subunit, serine 51; however, the serine residue is not conserved in K3L (Beattie et al., 1991; Pathak et al., 1988). Expression of the VV K3L gene in transfected COS cells acts to inhibit eIF-2~ phosphorylation. Expression of the VV K3L gene in transfected cells also potentiates the translation of plasmid-derived reporter mRNAs, for example dihydrofolate reductase and adenosine deaminase. The translation stimulation of reporter mRNAs correlates with a reduced level of eIF-2~ phosphorylation in the transfected cells (Davies et al., 1992). The K3L gene product appears to act as a decoy for eIF-2~ (Davis et al., 1992). The K3L protein, which inhibits eIF-2~ phosphorylation, is not phosphorylated. Furthermore, the K3L protein does not reverse the inhibition of protein synthesis mediated by the expression of the ser51asp mutant of eIF-2c~, a mutant designed to mimic phosphorylated eIF-2~P. Thus K3L cannot circumvent an inhibition of expression imposed by eIF-2c~ phosphorylation (Davies et al., 1992).

RNA-dependent protein kinase

313

Vaccinia virus is resistant to IFN treatment in some types of animal cells (Samuel, 1988, 1991). However, VV with a deletion of the K3L open reading frame displays a significantly increased sensitivity toward IFN (Beattie et al., 1991). The IFN-sensitivity of the VV-K3L deletion presumably derives, in part, from RNA-dependent P1 kinase mediated phosphorylation of elF-2ct. 4.4. PROTEINEFFECTORSOF KINASEACTIVITY The reovirus $4 gene and the vaccinia virus E3L gene each encode an inhibitor of the RNA-dependent Pl/elF-2ct protein kinase. The protein products of these two viral genes share a common property; they are both dsRNA binding proteins. The reovirus $4 and vaccinia virus E3L gene products likely prevent the activation of the RNA-dependent protein kinase by binding to the available dsRNA, thereby preventing the RNA from binding to and subsequently activating the P1/elF-2~ protein kinase. 4.4.1. Reovirus $ 4 Reovirus is a segmented, dsRNA virus (Joklik, 1984). The reovirus $4 genome segment encodes a 365 amino acid protein designated a 3 (Atwater et al., 1986). Sigma 3 binds dsRNA (Huismans and Joklik, 1976). The dsRNA binding activity is associated with an 85 amino acid domain within the C-terminal protein of the a 3 protein, a domain which possesses a repeated basic motif (Schiff et al., 1988; Miller and Samuel, 1992). Cotransfection of COS ceils with the reovirus $4 gene stimulates the expression of a reporter gene, CAT, at the level of CAT mRNA translation (Giantini and Shatkin, 1989). This effect likely reflects the capacity of S4-encoded tr3 to bind dsRNA (Huismans and Joklik, 1976), thereby blocking the RNA-dependent activation of the P1/elF-2~ protein kinase (Imani and Jacobs, 1988). The $4 genes of reovirus serotypes 2 and 3 are considerably more stimulatory than the $4 gene of serotype 1 in enhancing the expression of the CAT reporter gene in cotransfected COS cells (Seliger et al., 1992). 2-Aminopurine, an inhibitor of the RNA-dependent P1/elF-2~ kinase, does not enhance the expression of the reovirus $4 cDNA but does greatly stimulate the expression of the reovirus S1 cDNA in transfected COS cells (Samuel and Brody, 1990). This differential effect of 2-aminopurine can be explained by the fact that the $4 mRNA does not activate the kinase (Bischoff and Samuel, 1989), but rather the S4-encoded tr 3 protein antagonizes kinase activation by binding the available activator dsRNA (Imani and Jacobs, 1988). By contrast, the 1.5 kb S1 mRNA includes a 160 nucleotide region which efficiently activates the kinase (Bischoff and Samuel, 1989) and the Sl-encoded tr 1 and a Ins encoded proteins are not RNA binding proteins (Fields, 1990; Belli and Samuel, 1991). 4.4.2. Vaccinia Virus E 3 L Vaccinia virus is a large, double-stranded DNA virus (Fields, 1990). The VV E3L gene encodes a 190 amino acid protein (Ahn et al., 1990; Goebel et al., 1990) that binds dsRNA and is designated p25 (Chang et al., 1992; Watson et al., 1991). The p25 protein encoded by the VV E3L gene, when expressed in COS cells, inhibits activation of the RNA-dependent P1/elF-2~ protein kinase (Chang et al., 1992). The VV p25 E3L protein may function as a homolog of the regulatory region of the RNA-dependent P1/elF-2~ protein kinase. The RNA-binding activity of the 551 amino acid kinase has been mapped to the N-terminal region of the P1 protein (Katze et al., 1991; Feng et al., 1992; McCormack et al., 1992; Patel and Sen, 1992). The most conspicuous structural feature within the N-terminal 98 amino acids of the kinase, the region which possesses RNA binding activity, is a sequence which possesses striking homology with that of the VV E3L protein. The amino acid sequence of the RNA-dependent protein kinase from residues 58 to 75, which corresponds to the core of the RNA-binding domain, displays 67% identity (89% similarity) with residues 165 to 182 of the E3L protein (McCormack et al., 1992). Furthermore, this region of the kinase is both necessary and sufficient for RNA-binding activity (McCormack et al., 1992). The VV E3L gene product appears to act as a decoy of the RNA binding domain of the kinase regulatory region. The mechanism by which the VV E3L protein impairs activation of the P1/elF-2~ kinase is

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C.E. SAMUEL

probably similar to that mechanism used by the reovirus tr3 protein. Both E3L and tr3 bind dsRNA, thus lowering the effective concentration of activator R N A and subsequently eliminating RNA-dependent activation of the kinase and eIF-2g phosphorylation.

5. C O N C L U S I O N The RNA-dependent P1/eIF-2~ protein kinase regulates the expression of foreign genes in transfected cells. The translation of vector-derived mRNAs, both in transiently transfected cells and stably transfected cell lines, can be increased from 5- to 50-fold by impairing the action of the RNA-dependent protein kinase. The functional impact of the P1/elF-2~ protein kinase may be modulated by a variety of agents and approaches, including RNA inhibitors which bind to the kinase and block the RNA-dependent activation, protein inhibitors which sequester the activator RNA molecules, 2-aminopurine, mutant elF-20~ which is no longer a substrate for phosphorylation, or elF-2ct homologs which lack the phosphorylation site. The functional impact of the P1/elF-2~ protein kinase also may be modulated by R N A activators which bind to the kinase and mediate the RNA-dependent activation and by mutant elF-2ct which mimics phosphorylated elF-2ct. Modulation of the P1/elF-20~ protein kinase activity may be important for obtaining the optimal expression of foreign genes, whether in the setting of the biotechnology industry as applied to the efficient production of protein pharmaceuticals for diagnostic or therapeutic applications, or in the setting o f the medical community as applied to the regulated expression of protein products in gene therapy applications. Acknowledgments--Work from the author's laboratory was supported in part by research grants from the

National Institutes of Allergy and Infectious Diseases (AI-12520 and AI-20611).

REFERENCES AHN, B. Y., GERSHON,P. D., JONES,E. V. and Moss, B. (1990) Identification of rp30, a VV RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor. Molec. Cell Biol. 10: 5433-5441. AKUSJARVI, G., SEVENSSON,C. and NYGARD,O. 0987) A mechanism by which adenovirus virus-associated RNA I controls translation in a transient expression assay. Molec. Cell Biol. 7: 549-551. ATWATER,J. A., MUNEMITSU,S. M. and SAMUEL,C. E. (1986) Biosynthesis of reovirus-specified polypeptides. Molecular cloning and nucleotide sequence of the reovirus serotype 1 Lang strain s4 mRNA which encodes the major capsid surface polypeptide or3. Biochem. biophys. Res. Commun. 136: 183-192. BAGLIONI,C., MINKS, M. A. and DECLERCQ,E. (1981) Structural requirements of polynucleotides for the activation of 2,5A, polymerase and protein kinase. Nucleic Acids Res. 9: 4939-4950. BARBER,G. N., TOMITA,J., HOVANESSIAN,A. G., MEURS,E. and KATZE,M. G. (1991) Functional expression and characterization of the interferon-induced double-stranded RNA activated p68 protein kinase from Escherichia coli. Biochemistry 30: 10356-10361. BEATTIE,E., TARTAGLIA,J. and PAOLETTI,E. (1991) Vaccinia virus-encoded eIF-2~ homolog abrogates the antiviral effect of interferon. Virology 183: 419-422. BELLI,B. A. and SAMUEL,C. E. (1991) Biosynthesis of reovirus-specified polypeptides. Expression of reovirus Sl-encoded tr lns protein in transfected and infected cells as measured with serotype specific polyclonal antibody. Virology 185: 698-709. BISCHOFF,J, R. and SAMUEL,C. E. (1989) Mechanism of interferon action. Activation of the human P1/elF-2ct protein kinase by individual reovirus s-class mRNAs: sl mRNA is a potent activator relative to s4 mRNA. Virology 172:106-115. CHANG, H.-W., WATSON,J. C. and JACOBS,B. L. (1992) The E3L gene of VV encodes an inhibitor of the interferon-induced, double-stranded RNA-dependent protein kinase. Proc. natn. Acad. Sci. U.S.A. 89: 4825-4829. CHOI, S.-Y., SCHERER,B. J., SCHNIER, J., DAVIES, M. V., KAUFMAN, R. J. and HERSHEY,J. W. B. (1992) Stimulation of protein synthesis in COS cells transfected with variants of the ~-subunit of initiation factor elF-2. J. biol. Chem. 267: 286-293. CLARKE,P. A., SHARP,N. A. and CLEMENS,M. J. (1990) Translation control by Epstein-Barr virus small RNA EBER-1. Reversal of the double-stranded RNA-induced inhibition of protein synthesis in reticulocyte lysates. Eur. J. Biochem. 193: 635-641. CLARKE, P. g., SCHWEMMLE,M., SCHICKINGER, J., HILSE, I. K. and CLEMENS, M. J. (1991) Binding of

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