Cap-recognizing protein of influenza virus

VIROLOGY

116, 339-348 (1982)

Cap-Recognizing DIETER Institute

BLAAS,

of Biochemistry,

ERIK

University

Received

Jul?~

Protein

of Influenza

PATZELT,

AND ERNST

of Vienna,

A-1090

Vienna,

28, 1981; accepted September

Virus KUECHLER’

WCihringerstraJe

17, Austria

10, 1981

Globin mRNA-primed in vitro transcription of influenza virus is inhibited by m7GpppGm, m7GpppG, and m7GTP indicating that the cap analogs block the specific binding site involved in the cap transfer reaction. y-[4-(Benzoylphenyl)methylamido]-7-methylguanosine-5’-triphosphate, a photoreactive derivative of m7GTP, was used as an affinity label for the cap-binding protein. Evidence for the specificity of the labeling reaction is provided by competition with various cap analogs. At low concentrations inhibition of affinity labeling was observed only with guanosine derivatives which also exert a strong inhibitory effect on in vitro transcription. The cap-binding protein was tentatively identified as P2 by electrophoresis on polyacrylamide gels.

tion complex is clearly caused by interactions other than base pairing with the viral template RNA. This leaves the cap structure as the only common signal of recognition of the primer RNA. Therefore it seems likely that some cap recognition site must be present within the polymerase complex. In this respect the initiation of influenza virus RNA transcription resembles the initiation of protein synthesis on cellular messenger RNAs in eukaryotic systems. Affinity labeling and affinity chromatography experiments (Sonenberg et al., 1978; Sonenberg et al., 1979) have demonstrated that a polypeptide of M; 24,000, a component of eukaryotic initiation factors 3 and 4B, is involved in cap binding. This approach appears to be particularly well suited for the study of factors involved in cap binding. More recently it was shown that in the presence of ATP and Mg ions oxidized reovirus mRNA is crosslinked to two proteins of M, 28,000 and 50,000, which may be structurally related to the 24,000 polypeptide (Sonenberg, 1981). Studies with monoclonal antibodies indicate that the 24,000 cap-binding polypeptide may be contained in even larger protein precursors (Sonenberg et al., 1981). It has also been demonstrated that translation of capped mRNAs can be competitively inhibited by various cap analogs (Both et al.,

INTRODUCTION

Influenza virus, a negative strand RNA virus with a segmented genome has the unique feature to use host cell-coded capped RNAs as primers for the synthesis of its own mRNA. In the priming reaction the cap together with some 10 to 15 nucleotides is cleaved from host RNA by a viral endonuclease and is subsequently used to initiate viral RNA transcription (Plotch et al., 1981). As a result a host RNA-derived capped sequence is found to be linked to the 5’-end of the viral mRNA (Krug et ah, 1979; Caton and Robertson, 1980; Dhar et ab, 1980). This cap transfer reaction can also be performed in vitro using a variety of mRNAs such as globin mRNA or reovirus mRNAs as primers (Bouloy et ah, 19’78; Bouloy et ah, 19’79; Plotch et ab, 1979; Robertson et al., 1980). The structural requirements for an RNA to be active as a primer have been investigated (Bouloy et aZ., 1980). Only RNAs containing a capped 5’-terminus can efficiently initiate viral mRNA synthesis. In addition to natural mRNAs, synthetic polynucleotides such as capped poly(A) and capped poly(AU) can prime viral transcription (Krug et ab, 1980). Thus binding of the capped RNA to the viral transcrip1 To whom reprint

requests should be addressed. 339

0042-6822/82/010339-10.$02.00/0 Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

340

BLAAS,

PATZELT,

1976; Canaani et al., 1976; Groner et al, 1976; Hickey et al., 1976; Roman et al, 1976). The same methylated guanosine derivatives were employed as inhibitors of the affinity reaction to demonstrate the specificity of labeling in the translation system (Sonenberg and Shatkin, 1977; Sonenberg et ah, 1978; Sonenberg, 1981). For the influenza virus system a protein involved in cap binding has not been characterized so far. The present study describes a photoaffinity label for the identification of the cap-binding protein in the influenza virus transcriptase complex. Evidence for the specificity of the labeling reaction is provided by competition using cap analogs.

MATERIALS

AND

METHODS

Materials. [32P]Orthophosphate (carrier free) and [5-3H]UTP (2 Ci/mmol) were obtained from the Radiochemical Centre, Amersham, United Kingdom. m’GTP, m7GpppG, m7GpppGm, and GpppG were obtained from P-L Biochemicals (Milwaukee, Wise.). 4-(Aminomethyl)benzophenone was synthesized by Dr. A. Haslinger in our laboratory. Globin mRNA was prepared from rabbit reticulocytes by proteinase K digestion, phenol extraction, and affinity chromatography on oligo(dT)-cellulose following published procedures (Lockard and RajBhandary, 1976). Influenza virus, human strain PR 8, was kindly provided by Dr. H. Bachmayer (Sandoz Forschungsinstitut, Vienna) and was purified by rate-zonal centrifugation on 10 to 40% sucrose density gradients containing 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA for 1 hr at 25,000 rpm using the Beckman SW 27 rotor. Transcriptuse activity assay. The assay mixture contained in a final volume of 50 ~1: 100 mM KCl, 15 mM NaCl, 5 mM MgClz, 50 mM HEPES-K salt (pH 7.8), 1 mM dithiothreitol, 0.2% Nonidet NP 40, 1 m&Z each of ATP, GTP, and CTP, 0.2 mM E3H]UTP (0.5 Ci/mmol), 1.5 pg globin mRNA, influenza virus PR 8 corresponding to 30 pg of viral protein, and guanosine derivatives as indicated in the figures. In-

AND

KUECHLER

cubation was for 30 min at 30”. Reaction mixtures were spotted onto Whatman No. 1 filter paper disks pretreated with 10% trichloroacetic acid containing 20 mM sodium pyrophosphate. Filter disks were dried, washed 15 times with cold 10% trichloroacetic acid containing 10 mM sodium pyrophosphate and once with ethanol, acetone, and ether. Dried paper disks were counted in a toluene-based scintillator. Synthesis of r-[.l-(benxoylphenyl)methylamido] - 7 - methyl - guanosine - 5’ - triphos phate (y-[82P]-BP-m7GTP). GTP was labeled with [32P]phosphate in y position (Glynn and Chappell, 1964) and subsequently methylated with dimethylsulfate (Leng et aZ., 1968). 4-(Aminomethyl)benzophenone was coupled to -y-[“PIm7GTP according to Girshovitch et al. (1977). The specific activity of r-[32P]-BPm7 GTP was 50 Ci/mmol. The synthetic procedure will be described in full detail elsewhere (D. Blaas, E. Patzelt, and E. Kuechler, in preparation). PhotoafJinity labeling and electrophoretie separation of viral proteins, The incubation mixture for photoaffinity labeling contained in a final volume of 25 ~1: 100 mM KCl, 15 mM NaCl, 5 mM MgC12, 50 mM HEPES-K salt (pH 7.8), 1 mM dithiothreitol, 0.2% Nonidet NP 40, influenza virus PR 8 corresponding to 15 pg of viral protein, 2.5 PM y-[32P]-BP-m7GTP, and guanosine derivatives as indicated in the figures. The purity of the various cap analogs was checked by thin-layer chromatography, the concentrations were determined by measuring the A250 using the specifications given by the manufacturer. Incubation was for 5 min at 30”. The samples were irradiated in quartz tubes under argon atmosphere for 5 min at 4’. Irradiation was carried out using a 500-W super high-pressure mercury lamp (Philips, Eindhoven, Netherlands) and a WG 320 filter (Schott & Gen., Mainz, GFR) at 98 mW/cm2. Subsequently the proteins were precipitated with 5 vol of cold acetone for 1 hr at -2O”, washed with ethanol, ethanol:ether (l:l, v/v) and ether, and dried. Electrophoresis on 10% polyacrylamide gel was performed using the

CAP-RECOGNIZING

PROTEIN

OF INFLUENZA

341

VIRUS

Laemmli system (Laemmli, 1970), except that the running buffer contained 0.05 M Tris base, 0.38 M glycine, and 0.1% SDS. Gels (140 X 210 X 1.5 mm) were run at 5 W constant power until the bromophenol blue marker entered the separation gel. Power setting was then increased to 10 W. Running time was 4.5 hr. Gels were fixed in 50% methanol, 7% acetic acid, dried, and exposed to X-ray film (Kodak XR-5) at -70” using Cronex Lightning Plus intensifying screens (Du Pont). Autoradiographs were scanned in a Beckman DU-8. RESULTS

In order to devise a suitable photoaffinity label the minimal structural requirements for specific binding were tested in a competition experiment. In vitro transcription of detergent-disrupted influenza virus, human strain PR 8, primed with globin mRNA was tested in the presence of different cap analogs (Fig. 1). m7GpppGm and m’GpppG inhibited the polymerase reaction strongly already at low concentrations (Fig. 1A). An almost complete inhibition of incorporation was observed at high concentrations. In contrast, the unmethylated analog GpppG inhibited the transcription reaction only at high concentrations to a significant extent (Fig. 1A). This indicates that the m7G is required for maximal inhibition. Figure 1B presents the inhibition by m7GTP, m7GDP, and m7GMP. Comparison of the curves shows that m7GTP efficiently inhibits influenza virus transcription whereas m7GMP, m7G, and unmethylated guanosine derivatives such as GMP and GDP had no inhibitory effect (GTP is required for the polymerization and is present in the incubation mixture). An effect of m7GDP is clearly detectable but it is less than that of m7GTP. Apparently the presence of the m7G moiety alone does not suffice for competition with the mRNA. m7GTP was therefore chosen as starting point for the synthesis of the photoaffinity label. Aromatic ketones, in particular deriv-

INHIBITOR

CONCENTRATION

bM)

FIG. 1. Inhibition of globin mRNA-primed influenza transcription by cap analogs. The conditions of the transcriptase assay using [3H]UTP were as described under Methods. Cap analogs were added to the incubation mixtures at the concentrations indicated. The incorporation in the absence of cap analogs (100% value) was 970 pmol rH]UTP/mg of influenza virus protein in 1 hr. (A) m”GpppGm (A); m7GpppG (0); GpppG (v). (B) m7GTP (D); m7GDP (k) m7GMP (a); m7G (A); GMP (0); GDP (0); BP-m7GTP (V). atives of benzophenone, have been used previously as photoaffinity labels (Galardy et al., 1974; Barta et al., 1975; Barta and Kuechler, 1977). The photoactivation of the carbonyl group occurs at 320 nm which is known not to affect ribonucleoproteins (Barta et ab, 1975). 4-(Aminomethyl)benzophenone was attached to the y-phosphate of m’GTP because the resulting structure is expected to resemble most closely the cap analogs which inhibit priming of the viral transcription. Attachment of the benzophenone derivative did not significantly alter the inhibitory properties of m’GTP (Fig. 1B). The results of the photoaffinity labeling with r-[““PIBP-m7GTP are presented in Fig. 2A. Radioactivity was incorporated into bands corresponding to proteins P2, NP, HAl, M,

342

BLAAS,

PATZELT,

AND

KUECHLER

P2, NP, HA1 F

ABCa

bc

B

ab

c

d

e

FIG. 2. Gel electrophoresis of photoaffinity-labeled influenza proteins. (A) Influenza viral proteins were incubated and irradiated in presence of [32P]-BP-m7GTP as indicated under Methods. Before electrophoresis on a 10% polyacrylamide gel 150 pg of unlabeled viral protein was added to each sample to allow subsequent identification of the proteins by Coomassie brillant blue staining. Exposure to X-ray film was for 48 hr. Lanes A, B, and C present the staining pattern, lanes a, b, c the corresponding autoradiographs. Inhibitors present during incubation: Lane a, none; lane b, 0.5 mM m7GTP; lane c, 0.5 mM m7GpppGm. (B) Effect of preincubation with SDS and test of photoreactivity. Autoradiographs: Lane a, virus heated for 5 min in 2% SDS to 95” before addition to the incubation mixture; lanes b and c, controls; lane d, no irradiation; lane e, y-[32P]-m7GTP replacing y-[32P]-BP-m7GTP in the incubation mixture.

and HA2 (lane a). When the photoaffinity labeling reaction was performed in the presence of 0.5 mM m7GTP (lane b) or m’GpppGm (lane c) labeling of P2 was drastically reduced, but the incorporation into NP, HAl, M, and HA2 was not affected. This is taken to indicate that m7GTP and m?GpppGm compete with y-

[32P]-BP-m7GTP only at the specific cap recognition site of P2 but do not influence the nonspecific labeling of the other proteins. Considering that influenza virus contains about 15 copies each of Pl, P2, andP3, about 1000copies each of NP,HAl, and HA2, and about 2400 copies of M (Inglis et al, 1976) the preferential labeling

CAP-RECOGNIZING

PROTEIN

of the band corresponding to P2 becomes apparent. There was no difference between the samples in the Coomassie blue staining pattern (lanes A, B, C). If photoaffinity labeling of P2 is specific, the reaction should depend on the integrity of the viral transcriptase complex. To clarify this point the virus was briefly preincubated in 2% SDS at 95” before addition to the reaction mixture in order to destroy its native structure (Fig. 2B, lane a). Comparison to the control (lane b) clearly demonstrates that specific labeling occurs only with the intact protein complex. In contrast, nonspecific labeling (particularly of HA1 and HA2) is even increased presumably because of a better accessibility of the unfolded proteins. Experiments to check the photoreactivity of y-[32P]-BP-m7GTP are given in lanes c through e (Fig. 2B). The control is in lane c. No incorporation is obtained either when irradiation is omitted (lane d) or when the viral transcriptase complex is irradiated in an incubation mixture containing y-[32P]-m7GTP instead of Y-[~‘P]BP-m7GTP (lane e). This proves that the labeling occurs via the photoreaction of the benzophenone moiety. If the photoaffinity labeling reaction indeed occurs at the cap recognition site of the viral transcriptase complex the various cap analogs should be expected to inhibit both the affinity reaction and the priming of the RNA synthesis to a similar extent. Nevertheless it must be emphasized that the transcriptase assay does not allow discrimination between an inhibition of the initiation and an inhibition of the elongation of RNA chains. Measurement of total incorporation would truly reflect the initiation only if the event of initiation is the rate-limiting step in the transcription. This, however, may be a reasonable assumption to make in the light of the known complexity of the initiation of the influenza virus transcription (Plotch et aZ., 1981). In order to compare the effects on transcription and on the photoaffinity labeling the concentration dependence of the inhibition of the photoaffinity reaction by m’GpppGm and m7GpppG was determined. As a con-

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trol for the specificity of the inhibitory effect parallel experiments were performed using GpppG. The results are shown in Fig. 3. Lane A presents the pattern obtained in the absence of a competing guanosine derivative. Lanes B and C show the effect of different concentrations of m7GpppG and m’GpppGm, respectively. It can be clearly seen that the labeling of P2 is strongly suppressed even at low concentrations (0.1 mM, lanes 4), of the inhibitor. As expected, the labeling of the other proteins is not affected (the high background in lane C3 is presumably due to an incomplete washing of the sample resulting in an insufficient removal of adsorbed radioactive reagent). The control experiments with GpppG are presented in lanes D. In agreement with the data on the inhibition of the viral transcriptase reaction (Fig. 1) GpppG shows a pronounced decrease of labeling only at high concentration (1 mM) whereas no inhibition is observed at the lowest concentration tested (0.1 m&Y). Thus, as expected, the specificity of the inhibitors is increased if low concentrations are employed. The concentration range in which the inhibitory effect is observed is similar both in the transcriptase assay and in the photoaffinity reaction. This is taken to support the idea that the labeling indeed takes place at the domain of the viral transcriptase involved in binding of the capped messenger RNA. The comparison of the inhibitory effects of the guanosine derivatives tested previously in the transcriptase assay was then performed at a concentration of 0.1 mM. To demonstrate the influence of the various cap analogs on the photoaffinity labeling, autoradiographs were scanned as shown in Fig. 4. In comparison to the control (containing GTP, scan 1) no significant inhibitory effect was found for GDP (scan 2), GMP (scan 3), m7G (scan 4), m7GMP (scan 5), and GpppG (scan 6), whereas a strong inhibition of incorporation into P2 was observed with the ‘7-methylated guanosine derivatives containing three phosphate residues, i.e. m7GTP (scan 8), m’GpppG (scan 9), and m7GpppGm (scan 10). The importance of

344

BLAAS,

PATZELT,

AND

KUECHLER

4P2

1 FIG. 3. Inhibition conditions were as teins. Addition to trations employed

234

12341234

of specific labeling by different concentrations of cap analogs. The experimental in Fig. 2A. Autoradiography following electrophoretic separation of viral proincubation mixture: A, None; B, m7GpppG; C, m7GpppGm, D, GpppG. Coneenwere in samples numbered 1, 1 mM, 2, 0.5 mu, 3, 0.25, mM; 4, 0.1 mM.

the 7-methyl group is clearly demonstrated when comparing GpppG (scan 6) with m7GpppG (scan 9) and GTP (scan 1) with m7GTP (scan 8). m7GDP (scan 7) shows only a small effect. In order to facilitate the quantitative comparison the areas under the peak of P2 were computed. The results are expressed as percentage of inhibition in Table 1. The pattern of inhibition is in reasonable agreement with the data on the inhibition by the various cap analogs of the transcriptase reaction (Fig. 1). The strongest inhibitory effect is observed with m7GpppGm, the analog which resembles most closely the 5’-capped end of eukaryotic mRNAs. This is consistent with the previous findings by Bouloy et al. (1980) that mRNAs containing a m’GpppGm (cap I structure) are better primers for the transcriptase than mRNAs bearing a m’GpppG (cap 0 structure). In addition, Table 1 also shows the effect of 0.1 mM ApG on the photoaffinity labeling (not included in Fig. 4). As expected no signifi-

cant inhibition served.

by the dinucleotide

is ob-

DISCUSSION

Translation of capped mRNAs in eukaryotic systems can be inhibited by a variety of cap analogs such as m?GpppGm, m’GpppAm, m’GTP, m’GDP, and m7GMP (Both et al., 1976; Canaani et ah, 1976; Groner et aL, 1976; Hickey et al, 1976; Roman et ab, 1976). Initiation of transcription in the influenza system using capped mRNAs as primers is similarly inhibited by the various cap analogs but the structural requirements for inhibition are more stringent. m7GMP shows no significant effect at the concentrations employed whereas m7GDP inhibits only to a moderate extent. This finding is in line with the idea that influenza uses a presumably virus-coded protein for cap binding which is different from the known cap binding protein involved in the initiation of translation. It appears to be of significance that

CAP-RECOGNIZING

PROTEIN

NP .

HA2 .

I-L

1,

2 d-

L--

1

3

d

4

-LA-

5

d

6

-LA

7d

TABLE

-w-h-

1

INHIBITION OF THE PHOTOAFFINITY-LABELING VARIOUS GUANOSINE DERIVATIVES

Q-LA-

Compound (0.1 w

10 ,-LA0 DISTANCE

345

VIRUS

both the priming activity of globin mRNA and the affinity labeling are not only inhibited by the same cap analogs but also that the range of concentrations in which the inhibition is observed, is similar for both systems. This can be taken to indicate that the photoaffinity probe is bound to the site which normally recognizes the cap of the mRNAs. Furthermore Bouloy et al. (1980) have demonstrated that at high concentrations methylated cap structures are able to prime viral transcription, although not as efficiently as ApG (510%). A similar effect was observed with GpppG. This is apparently due to a binding of the methylated or unmethylated cap structure to the same site as ApG. On the other hand, the transcription reaction primed with ApG is stimulated by 7-methylated cap analogs indicating that the two binding sites are different (Bouloy et al., 1980). The lack of inhibition of the photoaffinity reaction by ApG (Table 1) also indicates that binding of BP-m7GTP occurs at a site different from the ApG binding site. For the photoaffinity reaction [32P]-BP-m7GTP was applied at the very low concentration of 2.5 piI4 The strategy of using labeling probes at very low concentrations is generally utilized to ensure preferential bind-

M .

P2 .

OF INFLUENZA

10 OF

MIGRATION

(cm)

FIG. 4. Inhibitory effect of different guanosine derivatives on specific labeling. The experimental conditions were as in Fig. 2A. All guanosine derivatives were employed at a concentration of 0.1 m&f. Autoradiographs of photoaffinity-labeled viral proteins were scanned. The densitometric tracings were normalized to the peak of NP. Sean 1, control in the presence of GTP, 2, GDP, 3, GMP; 4, m”G; 5, m7GMP, 6, GpppG; 7, m7GDP; 8, m7GTP; 9, m7GpppG; 10, m7GpppGm.

GTP GDP GMP m7G m7GMP GPPPG m7GDP m7GTP m7QwG m7GpppGm APG

BY

Percentage inhibition 0 10 13 6 0 17 29 58 66 85 2

Note. Data are obtained from scans of autoradiographs as in Fig. 4 except for ApG. Areas under the peak of P2 were determined. The value measured in the presence of GTP was taken as a reference.

346

BLAAS,

PATZELT,

ing to high affinity sites. This was of particular importance in the case of influenza, where the molar ratios of individual proteins differ by as much as two orders of magnitude. The fact that under proper conditions only ‘7-methylated cap analogs compete with BP-m7GTP in the photoaffinity labeling proves that the reaction occurs at a binding site specific for a 7methylated cap structure. An important advantage of the technique of photoaffinity labeling is the ease of control of the reaction. Since the reactive product is generated at the site to which the probe is bound, side reactions occurring during intermediate stages of binding are minimized. Nevertheless, certain precautions have to be taken to prevent undesirable artefacts such as structural changes due to binding of more than one molecule of reagent per virion and damage due to prolonged irradiation. For this reason the conditions of the irradiation were adjusted such as to incorporate less than one molecule of the photoaffinity probe per virion. The actual ratio determined was about one molecule of BPm7GTP incorporated per three virions. Previous experiments by Bouloy et al. (1980) have demonstrated that the lack of the 2’-O-methyl-residue at the 5’-terminus (cap 0 structure) decreases the priming efficiency of capped mRNAs. Qualitatively this is also observed when comparing the inhibitory effect of m7GpppGm and m’GpppG on both the transcriptase assay and the photoaffinity labeling. However, the difference is much smaller with these low-molecular compounds. This is to be expected because of the well-known influence of a 2’-O-methyl group on the stacking to the neighboring base within the context of a polynucleotide (Drake et ah, 1974). The essential control of specificity of the photoaffinity reaction is therefore the comparison of the inhibitory effect of the m7GpppG(m) with that of GpppG at low concentrations. As shown in Figs. 3 and 4 the inhibitory effect is linked to the presence of a 7-methyl group. Since the 7methyl group results in a positive charge on the purine ring system the characteristic properties of this methyl substitution

AND

KUECHLER

should be maintained in the small molecular weight cap analogs. The identification of the labeled influenza protein is based so far only on the comigration of the radioactivity with the stained band of P2 in SDS-gel electrophoresis. Comparison of the electrophoretic mobility of the unlabeled M protein (Mr 25,000) as indicated in the staining pattern (Fig. 2A, lanes A-C) with that of the nonspecifically labeled, radioactive M protein (Fig. 2A, lanes A-C) indicates that the addition of the y-[32P]-BP-m7GTP (Mr 750) slightly decreases the electrophoretic migration of the labeled protein due to the increase in the Mr. As expected this shift is most pronounced for the smallest protein and decreases with increasing M, of the protein. The shift should therefore be negligible for P2 (MT 87,000). Nevertheless, a few cases have been reported in the literature in which the addition of small molecules has resulted in an increase of the apparent M, exceeding that expected from the size of the substituent (Tung et al., 1971; Higgins et ok, 1977). It can therefore not be excluded with absolute certainty that labeling of the closely migrating protein P3 results in an electrophoretic shift which leads to a coincidence with the band of unlabeled P2. Experiments to unambiguously identify the labeled protein are presently underway in our laboratory. ACKNOWLEDGMENTS This work would not have been possible without the generous help of Dr. H. Bachmayer (Sandoz Institute, Vienna) who contributed in discussions and suggestions and who supplied the influenza virus. We thank Dr. A. Haslinger for a gift of 4-(aminomethyl)benzophenone. We also thank Dr. P. Palese (Mount Sinai Hospital, New York) for advice on the gel system and Mrs. B. Gamperl for typing the manuscript. This work was supported by a grant from the Austrian “Fonds zur Fijrderung der wissenschaftlichen Forschung.” REFERENCES BARTA, A., KUECHLER, E., BRANLANT, C., SRI WIDADA, J., KROL, A., and EBEL, J. P. (1975). Photoaffinity labelling of 23s RNA at the donor-site of the Escherichia coli ribosome. FEBS I&t. 56, 170-174.

CAP-RECOGNIZING

PROTEIN

BARTA, A., and KUECHLER, E. (1977). Aromatic ketone derivatives of aminoaeyl-tRNA as photoaffinity labels for ribosomes. In “Methods in Enzymology” (W. B. Jakoby and M. Wilchek, eds.), Vol. 46, pp. 676-683. Academic Press, New York. BOTH, G. W., FURUICHI, Y., MUTHUKRISHNAN, S., and SHATKIN, A. J. (1976). Effect of 5’-terminal structure and base composition on polyribonucleotidebinding to ribosomes. J. Mol. BioZ. 104, 637-658. BOULOY, M., PLOTCH, S. J., and KRUG, R. M. (1978). Globin mRNAs are primers for the transcription of influenza viral RNA in vitro. Proc. Nat, Acad. Sci. USA 75,4886-4890.

BOULOY, M., MORGAN, M. A., SHATKIN, A. J., and KRUG, R. M. (1979). Cap and internal nucleotides of reovirus mRNA primers are incorporated into influenza viral complementary RNA during transcription in vitro. J Viral. 32, 895-904. BOULOY, M., PLOTCH, S. J., and KRUG, R. M. (1980). Both the 7-methyl and the 2’-O-methyl groups in the cap of mRNA strongly influence its ability to act as primer for influenza virus RNA transcription. Proc. Nat. Acad Sci. USA 77,3952-3956. CANAANI, D., REVEL, M., and GRONER, Y. (1976). Translational discrimination of “capped” and “noncapped” mRNAs: Inhibition by a series of chemical analogs of m7GpppX. FEBS L&t. 64,326-331. CATON, A. J., and ROBERTSON, J.S. (1980). Structure of the host-derived sequences present at the 5’-ends of influenza virus mRNA. Nucleic Acids Res. 8, 2591-2603. DHAR, R., CHANOCK, R. M., and LAI, CH.-J. (1980). Nonviral oligonucleotides at the 5’-terminus of cytoplasmic influenza viral mRNA deduced from cloned complete genomic sequences. Cell 21, 495 500. DRAKE, A. F., MASON, S. F., and TRIM, A. R. (1974). Optical studies of the basestacking properties of the 2’-0-methylated dinucleoside monophosphates. J. Mel

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GALARDY, R. E., GRAIG, L. C., JAMIESON, J. D., and PRINTZ, M. P. (1974). Photoaffinity labeling of peptide hormone binding sites. J. Biol. Chem. 249, 3510-3517. GIRSHOVICH, A. S., POZDNYAKOV, V. A., and OVCHINNIKOV, Y. A. (1977). Photoactivated GTP analogs. In “Methods of Enzymology” (W. B. Jakoby and M. Wilchek, eds.), Vol. 46, pp. 656-660. Academic Press, New York. GLYNN, I. M., and CHAPPELL, J. B. (1964). A simple method of the preparation of 32P-labelled adenosine triphosphate of high specific activity. B&hem J. 90, 147-149. GRONER, Y., GROSFELD, H., and LITIAUER, U. Z. (1976). B-capping structures of artemia salina mRNA and the translational inhibition by cap analogs. Eur. J. Biochem. 71,281-293. HICKEY, K. D., WEBER, L. A., and BAGLIONI,

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C. (1976). Inhibition of initiation of protein synthesis by 7-methylguanosine-5’-monophosphate. Proc.

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