RNA-Profein Interactions in the Regulation of Coronavirus RNA Replication and Transcription

Transcription

1 Biol. Chem., Vol. 378, pp , June 1997 Copyright by Walter de Gruyter & Co Berlin - New York Minireview RNA-Profein Interactions in the Regulation of Coronavirus RNA Replication and Transcription Michael M.C. Lai Howard Hughes Medical Institute, Department of Molecular Microbiology and Immunology, University of Southern California School of Medicine, Los Angeles, CA , USA Coronavirus, with a 31 -kb RNA genome, replicates its own RNA and transcribes subgenomic mrnas by complex mechanisms. Viral RNA synthesis is regulated by multiple RNA regions, which appear to interact either directly or indirectly. Multiple cellular proteins bind to these regions and may undergo additional protein-protein interactions. These findings suggest that coronavirus RNA synthesis is carried out on a ribonucleoprotein via a mechanism that involves both viral and cellular proteins associated with viral RNA, similar to DNA-dependent RNA transcription. This mode of RNA synthesis may be applicable to most RNA viruses. Key words: hnrnpal / Mouse hepatitis virus / Transcription complex / UV cross-linking. Introduction RNA viruses, in general, have small genomes relative to DNA viruses. Replication of the viral genome and expression of viral genes in RNA viruses are thought to be regulated by a much simpler mechanism than in DNA viruses. Very little temporal regulation of viral gene expression or genome replication has been observed. Usually, an RNA virus encodes its own RNA polymerase, which recognizes a specific viral RNA sequence to initiate RNA synthesis. For instance, the 3'-end of a (+)-strand RNA genome may provide a replication signal for (-)-strand RNA synthesis, while the 5'-end [corresponding to the 3'-end of the (-)- strand template] serves as the replication origin of (+)- strand RNA. An internal promoter may provide a signal for subgenomic RNA synthesis. It is assumed that viral polymerase, possibly with the help of other viral proteins, is responsible for much of the specificity of RNA synthesis. In this sense, RNA viruses seem to be more self-reliant than DNA viruses, which involve a large number of cellular factors and extensive genomic interaction (e.g. promoterenhancer interaction) to regulate their genomic replication and gene expression. However, recent studies suggest that many cellular of viral RNA functions involve direct or indirect RNA-RNA interactions between different regions of the same RNA. For example, the 3'-end sequence of an RNA can regulate translation, which usually start from the 5 ; -end (Danthinne ef a/., 1993; Kwon and Hecht, 1993; Leathers et a/., 1993), suggesting a cross-talk between both ends of the mrnas. Replication and transcription of several RNA viruses have also been shown to require interactions between the 5'- and 3'-ends of the viral RNA. These may be mediated by either direct RNA-RNA interaction or RNA-protein and protein-protein interaction. For example, complementarity between the 5'- and 3 ; -ends of influenza virus RNA segments is required for viral RNA synthesis (Luo eta/., 1991). In vesicular stomatitis virus, the extent of complementarity between the 5'- and 3'- ends of a defective-interfering (Dl) RNA regulates either replication or transcription (Wertz ef a/., 1994). Other examples, such as brome mosaic virus, also suggest the involvement of the 3'-end sequence of the viral RNA in regulating RNA synthesis that initiates from the other end of viral RNA (Lahseref a/., 1993). Thus, it appears that complex direct or indirect RNA-RNA interactions are involved in the regulation of viral RNA synthesis, contrary to the more simplistic models. Coronavirus, e.g. mouse hepatitis virus (MHV), provides a good paradigm for understanding this model of regulation of RNA synthesis as it contains a very large RNA genome (31 kb) and employs a complex mechanism of discontinuous transcription to generate multiple subgenomic mrnas. It involves multiple c/s- and trans-acting RNA elements in regulating RNA synthesis. This review will discuss various aspects of Coronavirus RNA replication and transcription to illustrate the general principles of regulation of RNA synthesis in RNA viruses. Structure of Coronavirus Genomic RNA and Subgenomic mrnas and Mechanism of Discontinuous Transcription The genomic RNA of MHV is a 31-kb, single-strand, (+)- sensed RNA, which is considerably longer than all other known viral RNAs (Lai, 1990). It contains eight or more genes, most of which are expressed from separate mrnas. Each mrna starts from a region termed 'intergehic sequence' (IG) at the beginning of the gene and extends to the 3'-end of the viral RNA: thus, mrnas have a 3'-coterminal, nested-set structure (Figure 1). Accordingly, the regulation of mrna synthesis appears to occur at the initiation step; the termination of mrna synthesis seems to lack specific regulation since it takes place at the very end of the genomic RNA, where the polymerase simply runs off the template. The 5'-end of each mrna also has

2 478 M.M.C. Lai Leader Leader r Π >Ν -(UCUAA)- 2-4 polymerase \\\ IG IG IG W V UCUAAAC- 1 v IG v IG v IG v IG -r * T 4 T 5 T 6 ' 7 ^ 3 genome mrna Fig. 1 Diagram of the Regulatory Sequences for Coronavirus RNA Synthesis. The regulatory sequences required for MHV RNA transcription are indicated in grey. These sequences are extrapolated from studies of MHV Dl RNAs (see text). The left-to-right orientation represents (+)-strand RNA synthesis; the opposite orientation represents (-)-strand RNA synthesis. The structure of the mrnas synthesized is shown. The thick arrows indicate the translatable regions of the RNAs. (Diagram is not to scale.) a nucleotide stretch of leader sequence, which is derived from the 5'-end of the genomic RNA. Therefore, each mrna consists of two discontiguous parts. Existing evidence clearly shows that these two parts very often are derived from two different RNA molecules, which are joined during transcription (Jeong and Makino, 1994; Zhang ef a/., 1994). Thus, coronavirus RNA synthesis represents one type of discontinuous transcription. There is a stretch of pentanucleotide repeats (UCUAA) at the 3'-end of the leader sequence, which is homologous to the consensus motif (UCUAAAC) in the IG sequences (Figure 1). Fusion between the leader sequence and the mrnas takes place at these homologous sequences. Mechanistically speaking, discontinuous transcription can occur during either (-)- or (+)-strand RNA synthesis. In the (-)-strand RNA model, the viral genomic RNA serves as the template for (-)-strand RNA synthesis, which terminates at the IG sites; the nascent RNA then jumps to the leader sequence to complete the synthesis of subgenomic (-)-strand RNA. In (+)-strand RNA model (termed 'leader-primed' transcription), a full-length (-)-strand RNA is synthesized first and serves as the template for leader RNA synthesis. The leader RNA then jumps to the IG sites on the (-)-strand template to complete subgenomic mrna synthesis. The UCUAA sequence acts as a guiding sequence for leadermrna fusion in either case. Existing data cannot unequivocally distinguish these two models. Conceivably, both mechanisms may operate at different time points during viral infection. This interpretation is consistent with some of the experimental data (e.g. differences in UV target size for mrna synthesis between early and late time points of viral infection) (den Boon et a/., 1995; Yokomori et a/., 1992). Regulation of RNA transcription by the leader and IG sequences is presumed to occur at the termination of subgenomic (-)-strand RNA in one model and at the initiation of subgenomic (+)-strand RNA in another (leaderprimed transcription) (Lai, 1990). Different mrnas are transcribed at different rates, but the relative ratios of these transcriptional rates remain constant throughout the viral life cycle. Thus, the fransacting factors affecting RNA transcription arecoordinately regulated. Interactions of RNA Regulatory Regions Using Dl RNAs of MHV as a model, the RNA sequences required for MHV RNA replication and mrna transcription have been determined (Figure 1). The first step of viral RNA synthesis is the synthesis of a (-)-strand RNA. Only the 55 nucleotides at the 3'-end of the RNA genome plus poly(a) sequences are required to provide the c/s-acting recognition signal for this step (Lin et a/., 1994); the remaining sequences on the viral RNA do not affect the initiation of Η-strand RNA synthesis. The (-)-strand RNA then serves as the template for (+)-strand RNA synthesis, including replication of genomic RNA and transcription of subgenomic mrnas. Surprisingly, more than 400 nucleotides at both the 5'- and 3'-ends of the RNA genome are required for RNA replication (Kim ef a/., 1993; Lin and Lai, 1993).

3 Regulation of Coronavirus RNA Replication and Transcription 479 This requirement is surprising, because only 55 nucleotides at the 3'-end of the viral RNA are required for (-)- strand RNA synthesis. Thus, the additional required nucleotides at the 3'-end are c/s-acting signals for (+)-strand but not (-)-strand RNA synthesis. Since (+)-strand RNA synthesis starts at the 5'-end of the genome, why is it regulated by sequences at the 3'-end of the viral RNA? A simple explanation is that there is a cross-talk between the 5'- and 3'-ends of the viral RNA during RNA replication. In some Dl RNAs, an internal sequence of viral RNA is also required for RNA replication (Kim ef a/., 1993; Lin and Lai, 1993). This additional sequence may be required for maintaining a specific conformation in the template RNA. For mrna transcription, at least four RNA regions serve as regulatory sequences (Figure 1): The IG sequence, which serves as the promoter for mrna synthesis (Makino ef a/., 1991); the 5'-end sequence, including the leader sequence and an additional sequence downstream of the leader sequence (Liao and Lai, 1994); a leader RNA derived from other RNA molecules, which serves as both a frans-acting leader sequence and the source of the leader in the mature mrnas (Zhang ef a/., 1994); and the 3'-end untranslated region (305 nucleotides) (Lin ef a/., 1996). As in RNA replication, the requirement of the 3'-end sequence for mrna transcription implies an interaction between the 5'- and 3'-ends of the RNA during RNA synthesis. The efficiency of mrna synthesis depends on the sequence context of the IG sequence and the leader sequence. Some combinations of leader and IG resulted in efficient mrna transcription from that particular IG site, while others did not. For example, an RNA containing two UCUAA repeats in the leader sequence can transcribe an mrna from the IG site for mrna 2-1, whereas an RNA with three UCUAA repeats cannot transcribe from the same site (Makino and Lai, 1989). This differential transcription is seen both when the critical leader RNA sequence is in the same molecule as the IG sequence (in cis) and when it is in a different molecule (in trans) (Zhang ef a/., 1994). Other IG sites are constitutively transcribed irrespective of the nature of the leader sequence. These findings imply that there is an intra- or intermolecular interaction between the leader and IG sequences. These studies of the c/s-acting and frans-acting sequence requirements for RNA replication and transcription thus suggest a complex interaction among various RNA regions during MHV RNA synthesis. Identification of Proteins at the RNA Regulatory Sites Since there is no obvious complementarity among the regulatory sequences mentioned above, their interactions are more likely mediated by protein-rna and protein-protein interactions than direct RNA-RNA interactions. The proteins involved may be either viral or cellular proteins; however, no viral proteins have been shown to bind to these regulatory regions of MHV RNA specifically. On the other hand, several cellular proteins have been shown by UVcrosslinking experiments to bind to some of these regulatory regions, such as the (+)- and (-)-strands of IG and leader sequences and the 3'-end of the viral genomic RNA (Furuya and Lai, 1993; Yu and Leibowitz, 1995b; Zhang and Lai, 1995). One of these proteins, p35, binds to the (-)- strand RNA of the leader and IG sites; the binding efficiency of this 35-kDa protein correlates with the efficiency of transcription from this particular IG site, suggesting that it is a transcription factor (Zhang and Lai, 1995). p35 has been identified as heterogeneous ribonucleoprotein A1 (hnrnp A1) (Li and Lai, unpublished observation), a nuclear protein normally involved in the transport of cellular RNA and in the regulation of alternative pre-mrna splicing (Dreyfuss ef a/., 1993). Thus, MHV, which replicates exclusively in the cytoplasm, appears to usurp a nuclear protein for its RNA synthesis. This raises an interesting question regarding the potential role of the nucleus in the viral infection. hnrnp A1 contains RNA-binding domains and protein-protein-interacting motifs. The consensus RNA motifs recognized by hnrnp A1 (as determined by the in vitro SELEX approach) (Burd and Dreyfuss, 1994) match the sequences of the (-)-strand leader and IG sites. Conceivably, hnrnp A1 binds to the (-)-strand sequence of the IG site and leader and interacts with itself and other proteins to bring together the regulatory RNA domains to form a transcription initiation complex. Since the protein-binding sites at the 3'-end of viral genomic RNA are also critical for RNA replication (Yu and Leibowitz, 1995a), the cellular proteins binding to these sites may also participate in forming this transcription complex. Mechanism of Formation of RNA Transcription and Replication Complexes The findings described above suggest that MHV RNA synthesis involves extensive protein-rna and protein-protein interactions. In MHV, the binding of hnrnp A1 to the (-)-strand leader and IG site may trigger a series of proteinprotein interactions, which recruit other cellular proteins, such as p55 and p70 identified by UV-crosslinking experiments (Furuya and Lai, 1993) (Figure 2). Conceivably, viral polymerase and other viral proteins (among these, the nucleocapsid protein N) bind to one of these proteins. As a result, the leader sequence on the template and the fransacting leader sequence on a separate RNA may interact directly or indirectly with the IG site to form a transcription complex. In this context, it is interesting to note that hnrnp A1 can promote the annealing of complementary DNA or RNA molecules (Kumar and Wilson, 1990), potentially facilitating the joining of the leader RNA to the template at the IG site, which is complementary to the leader sequence. The capacity of these RNA elements to form a ribonucleoprotein complex through hnrnp A1 has recently been demonstrated (Zhang, Li, and Lai, unpublished). Some of the proteins in this complex may be basal transcription factors; others may be transcription activa-

4 480 M.M.C. Lai S'(-) Fig. 2 The Proposed Mechanism for the Formation of the MHV Transcription Complex. A double-stranded RNA is proposed to be the template for RNA transcription. The identity of the proteins, except for hnrnp A1, is not known. The binding of the RNA polymerase is hypothetical. The possible participation of the 3'-end RNA in the formation of the transcription complex is not shown here. tors. Because of the ability of hnrnp A1 to bind directly to the(-)-strand leader and IG site, it may play a similar role to that of the TATA-binding protein (TBP) in DMA-dependent RNA transcription complexes. The cellular proteins that bind to the 3'-end of the viral genome (Yu and Leibowitz, 1995b) may also be components of the transcription or replication complexes. This transcription complex is similar in concept to the transcription complex in DNA-dependent RNA transcription, which consists of DNA enhancer and promoter elements and viral and cellular transcription factors. Additional studies will be needed to determine whether these cellular proteins indeed participate in viral RNA synthesis. Perspectives An increasing number of RNA viruses have been shown to involve cellular proteins in the replication of their RNAs. These cellular proteins can be part of the viral RNA polymerase, as evidenced by the inclusion of cellular factors in purified cucumber mosaic virus and brome mosaic virus RNA polymerase preparations (Haynes and Buck, 1990; Quadt ef a/., 1993) and the binding of the nuclear protein Sam68 to poliovirus RNA polymerase (McBride ef a/., 1996). Alternatively, they can bind directly to viral RNA, as is the case with cellular protein p36, which, together with viral 3CD protein (polymerase), binds to the 5'-end of poliovirus RNA to catalyze in trans the initiation of (+)-strand RNA synthesis (Andino ef a/., 1993). The protein-binding region on the poliovirus RNA appears to be required for viral RNA replication (Andino ef a/., 1990). Many other cellular proteins bind to different regions of other viral RNAs, e.g. La protein to Sindbis virus RNA (Pardigon and Strauss, 1996) and calreticulin to rubella virus RNA (Singh ef a/., 1994). However, direct proof that these proteins are involved in the RNA synthesis of these viruses or coronavirus presented here is still lacking. Most of the purified viral RNA polymerases from RNA viruses lack template specificity in in vitro RNA replication and transcription systems. It is tempting to suggest that the cellular proteins that bind to a viral RNA may provide the specificity needed for the RNA polymerase to replicate only viral, but not cellular, RNAs in the virus-infected cells. This possibility is reminiscent of cellular gene expression, in which RNA polymerases do not bind to the promoter region directly; rather, TBP binds to the promoter and serves to recruit RNA polymerases and other transcription factors to form a transcription initiation complex. The cellular proteins that bind to the regulatory regions of viral RNA genomes and their complementary strands may prove to be such transcription factors. Identification of the components of transcription complexes for RNA viruses and the elucidation of the formation mechanism of these complexes will provide novel insights into the mechanism of RNA synthesis for RNA viruses in general. Acknowledgement The work described here was supported by U.S. Public Health j Service Research Grant AI The editorial assistance by '

5 Regulation of Coronavirus RNA Replication and Transcription 481 Daphne Shimoda is greatly appreciated. The author is an Investigator of HHMI. References Andino, R., Rieckhof, G.E., and Baltimore, D. (1990). A functional ribonucleoprotein complex forms around the 5'-end of poliovirus RNA. Cell 63, Andino, R., Rieckhof, G.E., Achacoso, RL, and Baltimore, D. (1993). Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA. EMBO J. 72, Burd, G.G., and Dreyfuss, G. (1994). RNA binding specificity of hnrnp A1: significance of hnrnp A1 high-affinity binding sites in pre-mrna splicing. EMBO J. 73, Danthinne, X., Seurinck, J., Meulewaeter, R, van Montagu, M., and Cornelissen, M. (1993). The 3' untranslated region of satellite tobacco necrosis virus RNA stimulates translation in vitro. Mol. Cell. Biol. 73, den Boon, J.A., Spaan, W.J., and Snijder, E.J. (1995). Equine arteritis virus subgenomic RNA transcription: UVinactivation and translation inhibition studies. 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7 Biol. Chem., Vol. 378, pp , June 1997 Copyright by Walter de Gruyter & Co Berlin New York Minireview The Molecular Anatomy of Influenza Virus RNA Polymerase Ayae Honda 1 ' 2 and Akira Ishihama 1 '* 1 National Institute of Genetics, Department of Molecular Genetics, Mishima, Shizuoka 411, Japan 2 CREST, Japan Science and Technology Corporation (JST), Kawaguchi, Saitama332, Japan * Corresponding author The genome of influenza virus is composed of eight RNA segments of negative polarity. The RNA-dependent RNA polymerase is associated with each viral RNA (vrna) segment and in virus-infected cells, involved in both transcription, i.e. vrna-directed synthesis of viral mrna, and two step reactions of vrna replication, i.e. vrna-dependent synthesis of complementary RNA (crna) and crna-dependent synthesis of vrna. The RNA polymerase is composed of three viral proteins, PB1, PB2 and PA. PB1 is the core subunit for not only the RNA synthesis but also the assembly of PB2 and PA into this multifunctional enzyme complex. PB1 alone is able to catalyze vrna-dependent RNA synthesis, but PB2 is required for capped RNA-dependent transcription, both together forming the transcriptase. The third P protein, PA, and an as yet unidentified host factor(s) are involved in the conversion of RNA polymerase from transcriptase to replicase. The functional map is being made for both PB1 and PB2 proteins. Key words: Replication / RNA genome / RNA virus / Subunit function/transcription. Introduction The genome of influenza virus is composed of eight RNA segments of negative polarity. Each of the segments 1-6 encodes one viral protein while the segments 7 and 8 code for two proteins, altogether producing 9 structural proteins (PB1, PB2, PA, HA, NP, NA, M1, M2 and NS1) and 1 non-structural protein (NS2) (for a review see Lamb, 1989; Yasudaef a/., 1993a). The viral RNA-dependent RNA polymerase is present in virions, forming complexes with viral RNA (vrna), and after infection, functions as the transcriptase which catalyzes all steps of mrna synthesis, i.e. the generation of capped primers, primer-directed initiation of RNA synthesis, elongation of RNA chains, and addition of poly(a) tails (reviewed in Ishihama and Nagata, 1988; Krug ei a/., 1989; Ishihama and Barbier, 1994; Ishihama, 1996). In eukaryotic cells, these processes are catalyzed by three enzyme complexes: capping enzyme, RNA polymerase II and poly(a) polymerase, each consisting of a number of different subunits. Late after infection, the viral RNA polymerase catalyzes replication, which involves two step reactions, i.e. vrna-dependent synthesis of full-sized complementary RNA (crna) without modifications at 5' and 3' termini and crna-dependent synthesis of vrna. The RNA polymerase with such extremely multiple functions is, however, composed of only one molecule each of three viral proteins, PB1, ΡΒ2 and PA, which are encoded by three large RNA segments (Honda ef a/., 1990). Here we summarize our up-to-date knowledge of the molecular anatomy of this multifunctional RNA polymerase. Transcription of Viral RNA Transcription initiation of influenza viral RNA (vrna) takes place by utilizing capped RNA primers derived from host cell mrnas (Plotch ef a/., 1981). The virus-associated RNA polymerase possesses the capped RNA cleavage activity so as to generate capped oligonucleotides of 10- Ι 2 nucleotides in length. Krug and colleagues (1989) proposed that the RNA polymerase recognizes and cleaves the capped RNAs at the 3' proximal side of purine nucleotides located about 10 nucleotides apart from 5' termini. Using various synthetic RNA molecules with or without various cap structures, we found that the RNA polymerase recognizes three RNA signals, (i) thecap-1 structure, but neither the cap core nor cap-0 structures, (ii) a distance from the cap-1 structure to the cleavage sites, and (iii) the bases of either A or U located at the 3'-proximal side of the cleavage sites (Kawakami ef a/., 1984). To meet the observations by two groups, we proposed that the RNA polymerase-associated capped RNA endonuclease carries a unique recognition property of either A or U residues at the 5' proximal side of the cleavage site. Transcription is initiated by polymerizing GMP, which is complementary to C at the second position of vrnas' termini, to capped oligonucleotide primers. In transcription in vitro, this process can be bypassed by adding high concentrations of dinucleotide ApG, which is complementary to(3')upcofalleightvrnasegments(hondaefa/.,1987). In the presence of GTP alone at high concentrations, multiple G residues are polymerized, yielding 7m GpppAmN- (10-12)-(A/U)GG-G, but erroneously polymerized GMP are removed by the addition of CTP, the second substrate

8 484 A. Honda and A. Ishihama polymerized into 7m GpppAmN-(10-12)-(A/U)G (Ishihama et a/., 1986). Thus, the viral RNA polymerase appears to carry 3'- to 5'- exonuclease activity. Such an exonuclease activity has never been found for DMA-dependent RNA polymerases involved in transcription of DNA genomes. The biological role of this apparent proofreading activity, however, remains unsolved. Once RNA transcription is initiated using capped RNA primers, termination is followed by the addition of poly(a) tails to transcripts (Krug et a/., 1989). A polyadenylation signal, consisting of U clusters, is located near the 5' end of vrna immediately upstream of a barrier of the RNA duplex formed by the 3' and 5' terminal sequences (Robertson etal., 1981; Liz and Palese, 1994). Poly(A) is considered to be synthesized by a slippage of the RNA polymerase on this U cluster. All these activities associated with the virion-associated RNA polymerase ultimately lead to synthesis of viral mrnas with the cap-1 structure at 5' termini and the poly(a) tail at 3' ends. Structure and Function of the Viral RNA Polymerase The RNA polymerase was isolated from influenza virus A/PR/8 virions using a step-wise dissociation method by centrifugation in cesium chloride and cesium trifluoroacetate (Honda et a/., 1990). The purified RNA polymerase is composed of one molecule each of three subunits, i.e., two basic subunits, PB1 (757 amino acid residues in the case of A/PR8/34) and PB2 (759 residues), and one acidic subunit PA (716 residues). The RNA polymerase activity was detected for the isolated RNA polymerase only when model templates of 50 to 300 nucleotides long were used, which contained the terminal sequences at 5' and 3' ends conserved among eight vrna segments (Pan/in ef a/., 1989; Seong etal., 1992), indicating that the promoter for transcription by the influenza virus RNA polymerase is located within the RNA duplex consisting of nucleotides in length. RNAfootprinting experiments indicate the association of RNA polymerase at the stem region formed by 5'-and 3'-terminal conserved sequences (Honda ef a/., 1987). These findings led to the development of influenza vector which allows the viral RNA polymerase-dependent expression of foreign genes inserted between these terminal sequences (Luytjesef a/., 1989). Photochemical crosslinking studies indicate that the RNA polymerase is associated at residues 9 to 12 from the 3' terminus of vrna (Fodorefa/., 1993). Mutant studies of the putative promoter sequences also supported that the terminal sequences are essential for transcription and replication (Yamanaka etal., 1991; Picconeef a/., 1993). The RNA polymerase purified from virus particles requires primers for RNA synthesis in vitro (Honda ef a/., 1990). Solubilized RNA polymerase transcribes vrna-sense model templates more efficiently than crna-sense templates (Parvin etal., 1989; Seong etal., 1992). Since the replicase should recognize both vrna- and crna-sense templates, the RNA polymerase associated with virions was considered to carry the catalytic specificity of transcriptase. The same specificity of template recognition was found for the reconstituted RNA polymerase (Kobayashi etal., 1992). These findings suggested the involvement of an additional factor(s) for functional conversion of the RNA polymerase into the replicase (see below). RNA synthesis by the isolated RNA polymerase-vrna complex (P-RNA) is attenuated about nucleotides from the start point. However, concomitantly with the increase of viral nucleoprotein (NP) addition, the size of transcripts increases (Honda ef a/., 1988). In the case of RNP cores, in which vrna is fully covered with NP at an average interval of about 15 nucleotides in length, the size of in vitro transcription products is as large as that of vrnas, indicating that NP is required for anti-termination of RNA chain elongation (Honda ef a/., 1990). The domain on NP for vrna binding is located in the internal part from amino acid residues 91 to 188, of which the sequence is widely conserved among influenza A, B and C viruses (Kobayashi ef a/., 1994). On the other hand, membrane or matrix (M1) protein binds to RNP cores and interferes with the migration of RNA polymerase along the template (Hankinsetal., 1990). Two M proteins are encoded by RNA segment 7, but genetic studies of M gene reassortants indicated that M1 but not M2 is involved in this transcription inhibition in vivo (Yasuda etal., 1993b). The region of M1 involved in the RNA polymerase inhibition has been mapped (Yeef a/., 1987; 1989; Hankins ef a/., 1989) and peptides carrying the sequence for this region inhibit the virus growth when added in cell cultures (Nasser ef a/., 1996). Since the discovery of the subunit composition of viral RNA polymerase, several trials have been devoted to establishing a reconstitution system of the RNA polymerase. Szewczykef a/. (1988) succeeded in reconstituting the RNA polymerase from individual subunits separated on SDS-PAGE and renatured in the presence of thioredoxin, while Kobayashi etal. (1992) achieved the reconstitution from three P protein subunits isolated from insect cell culture expressing each subunit using recombinant baculoviruses. The reconstituted RNA polymerase is able to catalyze model RNA-directed RNA synthesis. Thus these three subunits are considered to be the essential subunits of RNA polymerase with transcriptase activity. The RNA polymerase from virus-infected cell extracts is able to initiate de novo RNA synthesis without primers and transcribe the model templates of both negative and positive polarity (Del Rio ef a/., 1985; Takeuchi ef a/., 1987; Shapiro and Krug, 1988; Nagata ef a/., 1989; Toyoda ef a/., 1994). Triphosphate is associated at 5' termini of the majority of vrna (Young and Content, 1971; Honda ef a/., in preparation), indicating that at least the second step of replication in vivo takes place de novo without using primers. Since both purified-and reconstituted RNA polymerases lack the activity of de novo initiation, we propose that in virus-infected cells, the viral RNA polymerase is converted from the transcriptase to the RNA replicase,

9 The Molecular Anatomy of Influenza Virus RNA Polymerase 485 supposedly by interaction with an as yet unidentified host factor(s). Functional Role of Each RNA Polymerase Subunit Until recently, our knowledge of the function(s) of each RNA polymerase subunit has been limited. Studies of temperature sensitive (ts) mutants have indicated that PB1 is involved in RNA polymerase, and PB2 recognizes and binds the cap-1 structure at 5'-termini of RNA (Krug ei a/., 1975; Mahy ef a/., 1981; Mowshowitz, 1981; Ulmanenef a/., 1983). UV-cross-linking experiments indicated that PB1 is cross-linked to both substrate GTP (Asano ef a/., 1995) and 3'-terminal nucleotides of elongating mrna chains (Braam et a/., 1983). These results support the concept that PB1 carries the catalytic site for RNA synthesis. On the other hand, both PB1 and PB2 are cross-linked to synthetic RNA with 3'-terminal sequence of vrna (Seong and Brownlee, 1992), suggesting that the RNA polymerase recognizes and binds the conserved sequences of viral RNA 3'-termini. RNA-footprinting experiments of the RNA polymerase-rna complexes showed that the RNA polymerase is associated with the stem region of panhandle-shaped vrna segments (Honda ef a/., 1987). One way to get detailed understanding about the function of each subunit is to establish a reconstitution assay system from isolated individual subunits. At first, we succeeded in the assembly in vitro of subunits individually isolated from insect cells after infection with recombinant baculoviruses carrying cdnafor P proteins (Kobayashi ef a/., 1992). All the isolated subunits were inactive in RNA synthesis in vitro when assayed separately, but the reconstituted mixture of all three subunits exhibited a low level activity of model vrna-directed RNA synthesis. The low yield of enzyme activity might be due to incorrect refolding of RNA polymerase subunits, which were isolated in the presence of protein denaturants. In order to test the function of each subunit, we then examined in vitro activities of nuclear extracts of cells that express one or combinations of the RNA polymerase subunits using recombinant baculo- or vaccinia viruses (Kobayashi et a/., 1996; Toyodaef a/., 1996b). Nuclear extracts containing all three subunits catalyzed RNA synthesis in vitro in the absence of primers. Furthermore, not only c-sense RNA but also v-sense RNA is produced using a v-sense model RNA template. These two characteristics together indicate that these nuclear extracts carry the activity to catalyze the complete cycle of replication. In addition, these observations suggest that the recombinant baculovirus-infected insect cells and recombinant vaccinia virus-infected HeLa cells both contain the putative host factor(s) for replication of the influenza virus genome. The nuclear extract of cells expressing PB1 alone was able to catalyze the model template-directed dinucleotide-primed RNA synthesis (Kobayashi ef a/., 1996; Toyodaef a/., 1996b). This result supports the concept that PB1 is the catalytic subunit of RNA polymerase. Since the isolated PB1 was unable to catalyze RNA synthesis in vitro (Kobayashi ef a/., 1992), PB1 may be correctly folded in vivo by itself or may form a complex with a cellular protein(s) in place of PB2 and/or PA so as to express the intrinsic activity of RNA polymerization. Shi ef a/. (1996) detected the capped RNA-binding activity for the PB2 protein expressed using a recombinant baculovirus. In parallel with the in vitro approach, Nakada and colleagues established cell lines expressing one or combinations of the influenza virus RNA polymerase subunits under the control of MMTV promoter and assayed the activities in vivo associated with each subunit and binary complexes between the three subunits (Kimura ef a/., 1992; Nakagawaefa/., 1995; Nakagawaefa/., 1996). For detection of the//? vivo activities of transcription and replication, a reporter gene encoding chloramphenicol acetyltransferase (CAT) was inserted between 5'- and 3'-noncoding sequences of the NS gene (segment 8) and transfected into these cell lines. The CAT activity was detected in clone 76, which expresses all three RNA polymerase subunits and NP only after the addition of dexamethasone (Kimura ef a/., 1992). The CAT activity was, however, not detected in clone 64 expressing PB1 and PA but lacking PB2 (Nakagawa ef a/., 1995). Clone 64, however, produced transcripts of the reporter gene. At least a population of the sense-strand RNA transcripts in clone 64 carried a poly(a) tail at their 3'-termini but lacked the cap structure at 5' termini. Such RNA becomes translatable after the addition of cap structures using the vaccinia virus capping enzyme. In clone 64, the v-sense model RNA was also reproduced after transfection of the v-sense RNA, indicating that both transcription and replication of the influenza virus genome can be supported by two subunits, PB1 and PA, but PB2 is required forthe addition of capped RNA. Recently, the model vrna template was found to be transcribed into the complementary RNA even in cell lines, clone 25 and 27, expressing only PB1 and NP (Nakagawa ef a/., 1996). Furthermore, we demonstrated v-sense model RNA-dependent activity of c-sense RNA synthesis in vitro using extracts of cells expressing only PB1 subunit (Toyodaef a/., 1996b). Taking the in vitro and in vivo observations together, we concluded that all three subunits are necessary for the complete cycle of transcription and replication; but PB1 alone is able to catalyze RNA synthesis. These biochemical studies also support the concept previously obtained from mutant studies that PB2 carries the function(s) required forthe utilization of capped RNA primers and PA is required forthe replication. Functional Map of each RNA Polymerase Subunit The availability of viral RNA polymerase sequences from a number of RNA viruses led to a recognition of four conserved motifs, motifs A to D, including the canonical GDD

10 486 A. Honda and A. Ishihama IPIB2 FBI CAP BINDING NUCLEAR SIGNAL \ NTP PA CONTACT NTP NUCLEAR SIGNAL A BCD ***».^ RNA-RPASE MOTIF PB2 CONTACT IPA NUCLEAR SIGNAL NTP BINDING. PB1 CONTACT Fig. 1 Functional Map of the Subunits of Influenza Virus RNA Polymerase. The sites for subunit-subunit contacts and of nucleoside 5'-triphosphate binding have been determined (Toyoda etal., 1996a; Asano and Ishihama, in preparation), while the cap binding site on PB2 and the NTP binding site on PA are estimated from the amino acid sequence similarity. The four conserved motifs, A to D, among RNA-dependent RNA polymerases are located within the central domain of the PB1 subunit. tripeptide hallmark of the RNA-dependent polymerases in motif D (Kamar and Argos, 1984; Delarue et a/., 1990). These RNA polymerase motifs exist in the central part of PB1, suggesting that PB1 is the catalytic subunit of influenza virus RNA polymerase (Figure 1). In fact, the modification of the conserved D445D446 in the motif D renders PB1 inactive in RNA synthesis as demonstrated by an RNP transfection assay (Biswas and Nayak, 1994). On the other hand, PB2 carries a sequence with weak homology with cellular cap-binding proteins, supporting that BP2 is the subunit involved in cap-rna recognition. Upon exposure of influenza virus RNP to UV in the presence of radioactive 8-azido (N 3 )GTR the BP1 was specifically cross-linked (Asano ef a/., 1995). After protease V8 digestion, two cross-linked fragments were identified (Figure 1). The upstream binding site is located near the motif A of RNA-dependent polymerase, while the downstream binding site is located in a fragment which contains the motif D including the D445-D446 sequence (Asano and Ishihama, in preparation). One possible explanation for the presence of two GTP-binding sites is that these two regions form a single GTP-binding site on the tertiary structure. Alternatively, two nucleotide-binding sites exist in the PB1, for instance, one for the substrate-binding and the other for the primer-binding. We also carried out the cross-linking experiments of capped RNA with radioactivity only at the cap structure. The radioactive cap-structure was cross-linked to the PB2 (Honda, A., unpublished). Isolation and identification of the cross-linked fragment is in progress. The subunit-subunit contact sites have been determined by analysis of binary complex formation in vivo between two different subunits, one intact subunit and the other truncated subunit, which are expressed in the same cells by co-transfection of the respective expression plasmids (Toyodaef a/., 1996a). All three RNA polymerase subunits carry their own nuclear localization signals and can be transported into nuclei even when they are expressed individually (Nath and Nayak, 1990; Mukaigawa and Nayak, 1991) (Figure 1). The results of co-expression experiments indicated that PB1 is the core subunit for assembly of both PB2 and PA, each binding to the aminoterminal and carboxy-terminal domain, respectively, of the PB1 subunit (Figure 1). The regions on PB1 carrying the functional sites are therefore sandwiched between two subunit-subunit contact domains. Proteolytic cleavage experiments support the three domain structure for PB1 (Asano ef a/., in preparation) and the two domain structure for PB2 protein (Honda ef a/., in preparation). Concluding Remarks The influenza virus RNA polymerase is a multi-functional and multi-subunit enzyme. The assembly of three subunits, PB1, ΡΒ2 and PA, is required to carry out all of the discrete reactions of both transcription and replication of the genome RNA, but under certain conditions, the individual subunits exposed at least parts of the intrinsic activities. The combination of PB1 and PB2 is necessary for transcription but PA and an as yet unidentified host protein(s) are needed for replication. Acknowledgements We thank the Ministry of Education, Science and Culture of Japan, the New Energy and Industrial Technology Development Organization (NEDO) of Japan, and the Japan Science and Technology Cooperation. References Asano, Y, Mizumoto, K., Maruyama, T., and Ishihama, A. (1995). Photoaffinity labeling of influenza virus RNA polymerase PB1 subunit with 8-azido GTRJ. Biochem. 777,

11 The Molecular Anatomy of Influenza Virus RNA Polymerase 487 Biswas, S.K., and Nayak, DP. (1994). Mutational analysis of the conserved motifs of influenza A virus polymerase. J. Virol. 68, Braam, J., Ulmanen, I., and Krug, R.M. (1983). Molecular model of a eukaryotic transcription complex: functions and movements of influenza P proteins during capped RNA-primed transcription. Cell 34, Del Rio, L., Martinez, C., Domingo, E., and Ortin, J. (1985). In vitro synthesis of full-length influenza virus complementary RNA. EMBOJ. 4, Delarue, M., Poch, P., Tordo, N., Moras, D., and Argos, P. (1990). An attempt to unify the structure of polymerases. Protein Engineering 3, Fodor, E., Seong, B.L., and Brownlee, G.G. (1993). Photochemical cross-linking of influenza virus polymerase to its virion RNA promoter defines a polymerase binding site at residues 9 to 12 of the promoter. J. Gen. Virol. 74, Hankins, W.R., Nagata, K., Bucher, D.J., Popple, S., and Ishihama, A. (1989). 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In: The Influenza Viruses, R.M. Krug, ed. (New York: Plenum Press), pp Liz, Z., and Palese, P. (1994). Characterization of the polyadenylation signal of influenza virus RNA. J. Virol. 68, Luytjes, W., Krystal, M., Enami, M., Pan/in, J.D., and Palese, P. (1989). Amplification, expression and packaging of a foreign gene by influenza virus. Cell 59, Mahy, B.W.J., Barrett, T, Nichol, ST., Penn, C.R., and Wolstenholme, AJ. (1981). Analysis of the functions of influenza virus genome RNA segments by use of temperature-sensitive mutants of fowl plaque virus. In: The Replication of Negative Stranded Viruses. D.H.L Bishop and R.W. Compans, eds. (New York: Elsevier/North-Holland), pp Mowshowitz, S.L. (1981). RNA synthesis of temperature-sensitive mutants of WSN influenza virus. In: The Replication of Negative Stranded Viruses. D.H.L. Bishop and R.W. Compans, eds. (New York: Elsevier/North-Holland), pp Mukaigawa, J., and Nayak, D.R (1991). Two signals mediate nuclear localization of influenza virus (A/WSN/33) polymerase basic protein 2. J. Virol. 65, Nagata, K., Takeuchi, K., and Ishihama, A. (1989). In vitro synthesis of influenza viral RNA: Biochemical complementation assay of factors required for influenza virus replication. J. Biochem. 706, Nakagawa, Y, Kimura, N., Toyoda, T, Mizumoto, K., Ishihama, A., Oda, K., and Nakada, S. (1995). RNA polymerase PB2 subunit is not required for replication of the influenza virus genome, but is involved in capped RNA synthesis. J. Virol. 69, Nakagawa, Y, Oda, K., and Nakada, S. (1996). The PB1 subunit alone can catalyze crn A synthesis, and the PA subunit in addition to the PB1 subunit is required for viral RNA synthesis in replication of the influenza virus genome. J. Virol. 70, Nasser, E.H., Judd, A.K., Sanchez, A., Anastasiou, D., and Bucher, D.J. (1996). Antiviral activity of influenza virus M1 zinc finger peptides. J. Virol. 70, Nath, ST., and Nayak, D.R (1990). Function of two discrite regions is required for nuclear localization of polymerase basic protein 1 of A/WSN/33 influenza virus (H1N1). Mol. Cell. Biol. 70, Parvin, J.D., Palese, P., Honda, A., Ishihama, A., and Krystal, M. (1989). Promoter analysis of influenza virus RNA polymerase. J. Virol. 63, Piccone, M.E., Fernandes-Sesma, A., and Palese, P. (1993). Mutational analysis of the influenza virus vrna promoter. Virus Res. 28, Plotch, S.J., Bouloy, M., Ulmanen, I., and Krug, R.M. (1981). A unique cap (m7gpppxm)-dependent influenza virion enconuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. Cell 23, Robertson, U.S., Schubert, M., and Lazzarini, R.A. (1981). Polyadenylation sites for influenza virus mrna. J. Virol. 38, Seong, B.L, and Brownlee, G.G. (1992). Nucleotides 9to 11 of the influenza A virus RNA promoter are crucial for activity in vitro. J. Gen. Virol. 73,

12 488 A. Honda and A. Ishihama Seong, B.L., Kobayashi, M., Nagata, K., Brownlee, G.G., and Ishihama, A. (1992). Comparison of two reconstituted systems for in vitro transcription and replication of influenza virus. J. Biochem. 777, Shapiro, G., and Krug, P.M. (1988). Influenza virus RNA replication in vitro: Synthesis of viral template RNAs and virion RNAs in the absence of an added primer. J. Virol. 62, Shi, L, Galarza, Ü.M., and Summers, D.F. (1996). Recombinantbaculovirus-expressed PB2 of the influenza A virus RNA polymerase binds cap groups as an isolated subunit. Virus Res. 42, 1-9. Szewczyk, B., Laver, W.G., and Summers, D.F. (1988). Purification, thioredoxin renaturation and reconstituted activity of the three subunits of the influenza A virus RNA polymerase. Proc. Natl. Acad. Sei. USA 85, Takeuchi, K., Nagata, K., and Ishihama, A. (1987). In vitro synthesis of influenza viral RNA: Characterization of an isolated nuclear system that supports transcription of influenza viral RNA. J.Biochem. 707, Toyoda, T, Adyshev, D., Kobayashi, M., Iwata, A., and Ishihama, A. (1996a). Molecular assembly of influenza virus RNA polymerase: Identification of the subunit-subunit contact sites on each P protein subunit. J. Gen. Virol. 77, Toyoda, T., Kobayashi, M., Nakada, S., and Ishihama, A. (1996b). Molecular dissection of influenza virus RNA polymerase: PB1 subunit alone is able to catalyze RNA synthesis. Virus Genes 72, Toyoda, T., Kobayashi, M., and Ishihama, A. (1994). Replication in vitro of the influenza virus genome: selective dissociation of RNA replicase from virus-infected cell ribonucleoprotein complexes. Arch. Virol. 736, Ulmanen, l., Broni, B.A., and Krug, R.M. (1983). Influenza virus temperature sensitive cap (m 7 GpppNm)-dependent endonuclease.j. Virol. 45, Yamanaka, K., Ogasawara, N., Yoshikawa, H., Ishihama, A., and Nagata, K. (1991). In vivo analysis of the promoter structure of the influenza virus RNA genome using a transfection system with an engineered RNA. Proc. Natl. Acad. Sei. USA 88, Yasuda, J., Nakada, S., Kato, A., Toyoda, T, and Ishihama, A. (1993a). Molecular assembly of influenza virus: Association of the NS2 protein with virion matrix. Virology 796, Yasuda, J., Toyoda, T, Nakayama, M., and Ishihama, A. (1993b). Regulatory effects of matrix protein variations on influenza virus growth. Arch. Virol. 733, Ye, Z., Pal, R., Fox, J.W., and Wagner, R.R. (1987). Functional and antigenic domains of the matrix (M1) protein of influenza A virus. J. Virol. 67, Ye, Z., Baylor, W., and Wagner, R.R. (1989). Transcription-inhibition and RNA-binding domains of influenza virus A matrix protein mapped with anti-idiotypic antibodies and synthetic peptides. J. Virol. 63, Young, R.J., and Content, J. (1971). 5'-Terminus of the influenza virus RNA. Nature New Biol. 230,

13 Biol. Chem., Vol. 378, pp , June 1997 Copyright by Walter de Gruyter & Co Berlin New York Minireview Role of Cellular Kinases in the Gene Expression of Nonsegmented Negative Strand RNA Viruses Bishnu P. De*, Tapas Das and Amiya K. Banerjee Department of Molecular Biology, Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, NC20, Cleveland, OH 44195, USA * Corresponding author Nonsegmented negative strand RNA viruses package an RNA-dependent RNA polymerase composed of two subunits, a large protein L and a phosphoprotein P, for transcription and replication of their genome RNAs. The RNA polymerase activity resides within the L protein, while the P protein acts as a transcription factor ortransactivator of the polymerase. Since P protein is heavily phosphorylated and phosphorylation is known to regulate function of many viral as well as cellular proteins, the role of phosphorylation of P protein in the gene expression of this group of RNA viruses has recently been investigated. Through expression in bacteria the P protein was produced in large quantity in the nonphosphorylated form and involvement of cellular kinase(s) in its phosphorylation was studied. Casein kinase II and/or protein kinase C have been shown to play a critical role in the activation of P protein in transcription. These findings have opened up a new avenue for studying an important regulatory step in virus gene expression that may lead to the development of an effective antiviral agent. Key words: Casein kinase II / Phosphoprotein / Protein kinase C / Replication / RNA virus / Transcription. Introduction The nonsegmented negative strand RNA viruses are widely distributed in nature and constitute a large group of animal pathogens infecting both vertebrates and invertebrates. They are classified into three families, namely Rhabdoviridae, Paramyxoviridae, and Filoviridae that contain a single linear RNA genome. A large body of data support the view that these viruses adopt a common strategy for their gene expression (Banerjee et a/., 1991; Banerjee and Barik, 1992; Wagner and Rose, 1996; Lamb and Kolakofsky, 1996). For example, the genome RNAs of these viruses are encapsidated by a nucleocapsid protein to form a ribonucleoprotein complex (RNP), which serves as template in transcription as well as in replication. In the transcription mode, the RNA polymerase synthesizes in a sequential fashion a short leader RNA (about 47nt to 55nt long) and mrnas encoding the virus structural proteins. The intracistronic sequences and extracistronic 5'-trailer region are not copied during this process. The mrnas, but not the leader RNA, contain the hallmark of typical eukaryotic mrnas, i.e., 5' cap structure, 3' poly(a) tail, and also methylated cap in the presence of methyl donor, S- adenosylmethionine. The replication occurs in two distinct steps: first synthesis of a full-length antigenome, followed by production of negative strand RNA, which is packaged in the virions. Thus, in the replication mode the same RNA polymerase that until then was engaged in mrna synthesis copies entire genome-length RNA with concomitant encapsidation of the nascent chain by the nucleocapsid protein. Both transcription and replication are mediated by avirion-associated RNA-dependent RNA polymerase consisting of two subunits, a large protein L (~250kDa) and a phosphoprotein P (23kDa to 68kDa). Thus, the RNA polymerase activity plays a central role in the gene expression of nonsegmented negative strand RNA viruses, and its structure/function analysis has become a major subject of study for the last two decades. Over the past several years our laboratory has been involved in understanding the role of phosphorylation of P protein in transcription using vesicular stomatitis virus (VSV) and human parainfluenza virus type 3 (HPIV3) as model systems for Rhabdoviridae and Paramyxoviridae, respectively. The present review summarizes our recent results on the role of phosphorylation in transcription of these two viruses and discusses some relevant findings in other nonsegmented negative strand RNA viruses. Role of Phosphorylation in P Protein Function The P protein (formerly known as NS) of Rhabdoviridae has long been known to exist in various phosphorylated forms and was one of the major subjects of study. Using VSV Indiana serotype, two phosphorylated forms of NS, designated NS1 and NS2, were identified where NS2 was the hyperphosphorylated form (Clinton et a/., 1979; and Hsu and Kingsbury, 1982). However, because of the difficulty in purifying these forms, the intriguing question as to what role they play in the virus genome RNA transcription/replication remained unknown. Also, the protein kinase(s) involved in phosphorylation of NS protein remained uncharacterized. Only recently, the P protein has

14 490 B.P.Deefa/. been expressed in bacteria in nonphosphorylated form (PO) and many fundamental questions in Rhabdoviridae gene expression have been resolved. Specific cellular kinase has been identified, role of phosphorylation in transcription/replication process has been demonstrated, and the phosphorylated states of the P protein in vitro and in vivo have been established. Using the P protein of VSV New Jersey, Barik and Banerjee (1992a) demonstrated that it exists in three different forms: (i) A bacterially expressed nonphosphorylated form, P 0 ; (ii) a reticulocyte lysate translated form, P^ ; and (iii) two virion associated forms, P! and P 2. Cellular kinase-mediated phosphorylation of P 0 is required to procude the P! form. However, cellular kinase failed to convert P 0 to P 2 indicating that P^ is the end product of cellular kinase-mediated phosphorylation of P 0. Further phosphorylation of P^ by a viral L protein-associated kinase (LAK) (Barik and Banerjee, 1992a) produced a fully phosphorylated form (P 2 ). Thus, a cascade phosphorylation pathway, P 0 > PI > P 2, was elucidated for the P protein of VSV New Jersey; seemingly two different kinases, viz., cellular kinase and LAK, were involved in this process. The cellular kinase was subsequently purified from BHK cell extract and was found to be indistinguishable from CK II, a ubiquitous cyclic AMP-independent protein kinase (Barik and Banerjee, 1992b). The role of CK II in the virus life cycle was suggested by the findings that CK II was involved in the phosphorylation of P protein in vivo (Chen ef a/., 1997), and CK II (Gupta ef a/., 1995) or CK ll-like (Beckes and Perrault, 1991) activity was also packaged in the progeny virion and remained tightly associated with the RNP. The LAK, on the other hand, could not be purified for biochemical characterization due to its presence in small amounts in the virus. Thus, the identity of this kinase still remains unknown. However, several biochemical properties clearly indicated that the VSV New Jersey LAK is a unique kinase and different from CK II (Barik and Banerjee, 1992a). That CK ll-mediated phosphorylation of P protein is essential for P protein function in transcription was demonstrated using a reconstitution assay with P 0, kinase-free L protein, and N-RNA template of VSV New Jersey serotype (Barik and Banerjee, 1992b). Subsequently, Takacs ef a/. (1992) showed that Ser-59 and Ser-61 together account for CK ll-mediated phosphorylation of the P protein. Phosphorylation defective mutant P proteins were then used to probe the role of phosphorylation in transcription. The double mutant S59/S61A was totally inactive while single mutants S59A or S61A were active by about 50% in in vitro transcription (Takacs ef a/., 1992). Interestingly, when both Ser residues were altered to acidic amino acids such as Glu, the mutant protein was active in transcription at the level of wild type P protein suggesting involvement of negative charge in the activation of the P protein (Das ef a/., unpublished). Furthermore, phosphorylation was found to increase α-helical content of the P protein that leads to the formation of P-P dimer, which appears to be directly involved in transcription activation (Das ef a/., 1995a). The sites of LAK-mediated phosphorylation were similarly mapped at Ser-236 and Ser-242 in the P protein of VSV New Jersey (Chattopadhyay and Banerjee, 1987). The double mutant S236/S242 was inactive in transcription indicating a role for LAK in VSV New Jersey transcription (Chattopadhyay and Banerjee, 1987). Because P protein is also involved as P-N complex in replication, the role of phosphorylation in this complex formation was also investigated using a two hybrid system (Takacs ef a/., 1993). Phosphorylation of P protein by CK II at Ser-59 and Ser-61 was found to have no effect on N-P interaction suggesting no role for CK II in encapsidation. In the case of VSV Indiana, again both CK II and LAK were involved in the phosphorylation of P protein suggesting a specific role for these kinases in the virus life cycle (Chen ef a/., 1997; Gaoand Lenard, 1995a, b). Gaoand Lenard (1995a, b), using an in vitro reconstitution system, demonstrated that CK ll-mediated phosphorylation of P protein was required in transcription. By mutational analysis, CK ll-mediated phosphorylation sites were mapped at Ser-60, Thr-62, and Ser-64 in the P protein (Chen ef a/., 1997). Furthermore, Jackson ef a/. (1995) observed an obligatory phosphorylation of Ser-64 for the phosphorylation of Thr-62 to occur. Transcription and replication studies with mutant P proteins of VSV Indiana, in contrast, provided complex and somewhat perplexing results. For example, a bacterially expressed doubly substituted S60A/S64A mutant was as active as the wild type, whereas the same double mutant, when expressed in COS cells, was active only by 10-15% of the wild type (Mathur ef a/., in preparation). Recently, Pattnaikef a/, (manuscript in preparation), using mini-antigenomic VSV construct in transcription and defective interfering (DI) RNA in replication in vivo demonstrated that the double mutant S60A/S64A is virtually inactive (10%) in transcription but fully active in replication. Thus, in the case of VSV Indiana P protein, it seems that the structure obtained by the phosphorylation defective mutant P protein in bacteria is similar to that of the wild type P protein rendering it active in transcription. The LAK-mediated phosphorylation sites in the P protein were mapped at positions Ser-226 and Ser-227 (Chen ef a/., 1997), and the mutant S226A/S227A was inactive in in vitro transcription, indicating a role for LAK in this process (Chen ef a/., unpublished). Additionally, a comparison of migration positions and tryptic peptide maps of the in vitro phosphorylated P protein of Indiana serotype with that of in vivo labeled NS forms (NS1 and NS 2) established that the P1 and P2 forms are identical to the NS 1 and NS 2 forms, respectively (Chen ef a/., 1997). Similar to VSV New Jersey, the role of phosphorylation in N-P interaction was investigated using a bacterial expression system where these proteins, when coexpressed in bacteria, formed a complex in the absence of phosphorylation suggesting again that phosphorylation of P protein may not be required for com- plex formation in vivo (Gupta ef a/., 1997). \

15 Role of Kinases in the Gene Expression of RNA Viruses 491 HPIV3 The phosphorylation of P protein of HPIV3 by cellular kinase(s) was similarly investigated. The P protein was expressed in bacteria and using the nonphosphorylated form (PO), phosphorylation by cellular kinase was carried out (De ef a/., 1995). Cellular kinase that phosphorylated the P protein was purified from CV-1 cell extract. In contrast to VSV, biochemical and immunological characterization revealed that the HPIV3 P phosphorylating kinase was protein kinase C isoform ζ (PKC ζ) of the atypical family. Interestingly, similar to CK II, the PKC ζ is also ubiquitous, and is present in the cytosol, which is in agreement with the broad host range of this class of viruses. The role of PKC ζ was further supported by the observation that purified RNP packaged a kinase activity that phosphorylated the Ρ protein, and the kinase displayed similar biochemical properties as PKC ζ. These results indicated that PKC ζ is packaged in HPIV3 virion and remains tightly associated with the RNP for phosphorylation of the Ρ protein. Metabolic labeling with 32 P-inorganic orthophosphate and protease digestion of the labeled Ρ protein also suggested that PKC ζ is involved in the phosphorylation of HPIV3 Ρ protein during virus replication in infected cells. By site-directed mutagenesis of potential PKC consensus sites (S/T-x-R/K), the primary site of phosphorylation in the Ρ protein was identified at S333 (Huntley et a/., 1995). The same site appeared to be modified when viral P protein was phosphorylated in vitro by RNP-associated PKC and in the P protein of progeny virion labeled in vivo. The role of PKC ζ for HPIV3 gene expression could not be determined in vitro using a reconstitution system, similar to that used for VSV, because such a system for HPIV3 has not yet been developed. Accordingly, this study took advantage of the fact that PKC ζ-specific pseudosubstrate (peptide Z) strongly inhibits PKC ζ activity in cells (Dominguezefa/., 1992). Delivery of peptide Ζ abrogated HPIV3 replication in a dose dependent fashion while a conventional PKC-specific pseudosubstrate peptide inhibitor (peptide A) had no effect (De ei a/., 1995). This inhibition was specific and not due to a toxic effect of peptide Z to the cells because peptide Z did not inhibit VSV replication. These results thus demonstrated that PKC ζ-mediated phosphorylation of P protein is required in the gene expression of HPIV3. Development of an in vitro transcription reconstitution system would certainly help determine the effect of PKC ζ-mediated phosphorylation in HPIV3 transcription in greater detail. Cellular Kinases in the Phosphorylation of P Proteins of Other Viruses The findings on VSV and HPIV3 stimulated considerable interest in understanding the role of P protein phosphorylation of different nonsegmented negative strand RNA viruses in the virus gene expression. Table 1 summarizes the results of several studies where cellular kinases for different viruses have been characterized and in some cases the role of phosphorylation in virus gene expression has been elucidated. A brief description of those studies are given below. In the rhabdoviridae family, cellular kinases that phosphorylate the P protein of rabies virus were purified from cell extract using PO in the in vitro phosphorylation assay (Gupta ef a/., in preparation). Two cellular kinases, a major kinase different from both CK II and PKC, which remains uncharacterized (referred to as novel kinase) and a minor kinase of the PKC family, the PKC ζ, were identified. Both the novel kinase and PKC ζ were packaged in purified virions suggesting their specific role in the transcription/replication process. In the paramyxoviridae family, cellular kinase for the Ρ protein of respiratory syncytial virus has been characterized as CK II (Mazumderef a/., 1994). Phosphorylation sites have been mapped at S232 (major) and S237 (minor) in the Ρ protein (Sanchez-Seco ef a/., 1995). In vitro reconstitution of tran- Table 1 Cellular Kinases Involved in the Phosphorylation of Ρ Proteins of Non-segmented Negative Strand RNA Viruses. RNA viruses Ρ protein (Total amino acid) Cellular kinases Phosphorylation sites Rhabdoviruses VSV Rabies Chandipura 271 (NJ) 265 (IND) CKII ΡΚΟ Ύ >ζ>α,νκ CKII S59, S61 (NJ) S60,T62,S64(IND) ND S62 Paramyxovi ruses RSV HPIV3 Sendai Measles CDV CKII PKC ζ PKC ζ, PDK CKII, PKC ζ PKC ζ, CKII S232.S237 S333 S249(PDK) S86.S1 51,8180 (CKII) ND VSV, vesicular stomatitis virus; RSV, respiratory syncytial virus; HPIV3, human parainfluenza virus; CDV, canine distemper virus; NK, novel kinase; PDK, proline-directed kinase; ND, not determined.

16 492 B.P.Deefa/. scription using mutant P proteins demonstrated that phosphorylation of S232, but not S237, is directly involved in the regulation of transcriptional activity of the P protein, whereas S237 was suggested to be involved in a modulatory role. Byrappa et a/. (1996) investigated phosphorylation of Sendai virus P protein in mammalian cells and reported the involvement of a proline-directed kinase (PDK), although detailed characterization of the kinase was not performed. By mutational analysis, S249 was identified as the potential phosphorylation site. More recently, Huntley et a/. (1997), using an in vitro phosphorylation and purification approach, demonstrated involvement of PKC ζ in the phosphorylation of Sendai virus Ρ protein and also in the virus replication in vivo. Thus it seems likely that Sendai virus utilizes more than one cellular kinase for modification of its Ρ protein. In measles virus and canine distemper virus (CDV), both CK II and PKC ζ were found to be involved in the phosphorylation of Ρ proteins (Das et a/., 1995b; Liu ef a/., 1997). The sites of CK ll-mediated phosphorylation in the MV P protein were identified by site-directed mutagenesis and located at the Ser residues at positions 86, 151, and 180 (Das ef a/., 1995b). Major phosphorylation was found to occur at S86 and S151, while S180 appears to play a regulatory role in phosphorylation. However, the site of PKC ζ-mediated phosphorylation has yet to be determined. In the case of CDV, the sites of phosphorylation on the P protein for both PKC ζ and CK II currently remain unidentified. As demonstrated for HPIV3 and Sendai virus, replication of CDV was also inhibited by peptide Ζ indicating a role of PKC ζ-mediated phosphorylation of Ρ protein in the virus life cycle (Liu etal., 1997). 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