J. Biochem. 105, 537-546 (1989) Control of Gene Expre ssion: Quantitative Analysis of E ach Viral RNA Species in Infected Cells ErikoHatada,1 Masakazu Hasegawa RyujiF ukuda1, 4 ,2Jun Mukaigawa,3Kazufumi Shimizu,3 and DepartmentofMolecular Genetics , NationalInstitute ofGenetics, Mishima , Shizuoka411 Receivedforpublication , September26,1988

We established a quantitative hybridization system by which th ree types of influenza viru(vRNA RNAs , mRNA, and cRNA) for the 8 genome segments were measured individualli As the hybridization probes , 32P-labeled RNAs of both plus and minus polarity we; produced employing an SP-6 transcription system and used in a large molar excess sufficient to overcome complementary RNAs present in the viral RNA samples. Employinsystem the , we studied the control of the synthesis of each viral RNA species in MDCKcell infected with A/Udorn/72 (H3N2) . Our new observations were as follows. 1) Segmenl specifi c transcription was observed at the primary transcription . 2) Replication of the viru g enome began simultaneously for all segments. No delay was observed in the replication c th e segments carrying late genes. 3) In addition to control at the transcriptional levels nes was regulated at some post-transcriptional step(s) , th ofexpressionviral latege s are not compatible with the concepts reported previously . Thes result , and lead us to proposregul unique ations operating on the expression of the viral late t enes.

The genome of influenza type A consists of eight It is followed by predominant synthesis of two mRNAs , th segment of single-strand RNAs having negative polarity ose coding for the NP and NS1 (early phase of (1-3). In virus-infected cells, three different types of viral transcription). Subsequently, the rate of synthesis of the RNAs are synthesized, the virion RNA (vRNA) and two Ml, HA, and NA mRNAs greatly increases (late phase of types of vRNA transcripts, the messenger RNA (mRNA) transcription) (4, 15). A good correlation was found and the complementry RNA (cRNA , the template for the between the relative synthesis rate of the individual vRNA vRNA replication) (3, 4). The mRNAs contain a 3•L poly(A) segments and the mRNAs transcribed from them, particu tail and a 5•L capped end of 10 to 13 nucleotides derived from larly at early phase of infection (15, 16). On this basis , it host cell heterogeneous nuclear RNAs, and are incomplete was postulated that the regulation of the transcription was copies of vRNA in that they lack a copy of the last about 16 not performed at the level of the mRNA synthesis itself , b to 22 nucleotides at the 5•L end of vRNA (5-8). The other ut through the control of the vRNA synthesis (15) . The type of transcript, cRNA, is a complete copy of vRNA, and hypothesis predicted that different relative proportions of possibly is initiated without a primer (4, 9). In addition, the eight vRNAs would be produced at various times , and th transcripts from segments 7 and 8 are processed to give two ese changes would be reflected in the relative synthesis or three, and two species of spliced mRNA, respectively rates of the respective mRNAs. That is, at early times, the (10, 11). Therefore at least 26 to 27 species of RNA in all NP and NS vRNAs may be synthesized preferentially , exist in infected cells, and the synthesis of each species is while those of Ml, HA, and NA are preferred at the later regulated temporally and in quantity during the infectious times (17). process (3). For detailed analysis of viral RNA synthesis of tem Generally accepted processes of viral RNA synthesis are perature-sensitive mutants as well as wild-type strains as follows. Immediately after infection, similar amounts of under various growth conditions, it is desirable to develop all eight viral mRNAs are synthesized by virion-associated a simple system for measuring the quantities of the viral RNA polymerase. This phase of the viral transcription is RNA species individually. For this purpose, we have termed the primary transcription, and is also observed in established a quantitative hybridization system in which cells infected in the presence of cycloheximide (4, 12-15). RNA probes intensely labeled with 32Pwere used in a molar excess sufficient to overpower complementary RNAs pres Present addresses: 1 Department of Biochemistry, Kanazawa Univer ent in the infected RNA. The probes were obtained by sity School of Medicine, Kanazawa, Ishikawa 920. 2 Chyugai Explor transcribing fragments of individual viral double-stranded atory Research Labs., Gotemba, Shizuoka 705-1. 3 Department of (ds)-cDNAs. In this report we measured the quantities of Microbiology, Nihon University, School of Medicine, Itabashi-ku, 24 RNA species in four test tubes. Tokyo 173.4 In the present study, we established the time courses of To whom correspondence should be addressed. the accumulation of individual viral RNAs in cells infected Abbreviations; vRNA, virion RNA; cRNA, complementary RNA to with wild-type virus (A/Udorn/72), and compared them vRNA; as, single-stranded; +ds, double-stranded; MDCK cells, Madin-Darby canine kidney cells; MEM, minimal essential medium; with the rates of viral synthesis. The following PBS, phosphate-buffered saline; pi, postinfection. observations do not agree with the concepts derived from

Vol. 105, No. 4, 1989 537 538 E. Hatada et al.

previous studies on the control mechanisms of the viral sis of hybridization probes. The reaction mixture for the RNA synthesis. For all genome segments , the vRNA SP6 transcription system was as described previously (21) synthesis rose simultaneously from 2 h postinfection (pi) , with the following modifications. Spermidine was omitted, and continued to increase beyond 5 h pi. We could not and the 100ƒÊl reaction mixture contained 0.75 u g of linear observe delay in the synthesis of those vRNA segments DNA template, 7.5 units of SP6 RNA polymerase, 50 units encoding the late genes. While the accumulation of the late of ribonuclease inhibitor, 50ƒÊM UTP and 20ƒÊCi of mRNAs was intimately coupled with that of vRNAs , the [ƒ¿-32P] UTP. After reaction for 2 hat 39•Ž, products were coordination was partial for the early genome segments extracted with phenol-chloroform, and precipitated with during 2 h pi. The accumulation of mRNA ceased to ethanol. The precipitates were dissolved in a denaturing increase or declined at 3 to 5 h pi for all segments, being loading buffer (80% formamide, 10mM Tris-HCI (pH 7.6), completely uncoupled from that of vRNAs. In the presence 1mM EDTA, 0.01% Xylene cyanol, and 0.01% bromo of inhibitors of protein synthesis, we could observe mRNAs phenol blue), heated for 3 min at 90•Ž, and applied to 5% for the early genes, but could hardly detect mRNAs for the polyacrylamide-8 M urea gel in 0.5xTBE (1xTBE; 0.1M late genes, indicating a control at the transcriptional level Tris-borate [pH 8.3], 2mM EDTA). After electrophoresis, during the primary transcription. 32P -RNA bands were cut out, and gel pieces were soaked in Results are also described, which indicate posttran- a gel elution buffer (22) containing 50mM ƒÀ-mercapto- scriptional regulation operating on the virus gene expres ethanol at 45•Ž overnight. The eluted 32P-RNA was extract sion. ed with phenol-chloroform, precipitated with ethanol and dissolved in 11,0. For those probes which overlapped with

MATERIALS AND METHODS template on gel electrophoresis, 1ƒÊg of DNase I was added to the reaction mixture for 10min at 39•Ž immedi Virus and Cell-SP626 is a wild-type strain of influenza ately after the transcription reaction. A/Udorn/72 (H3N2) virus, and was prepared in the Isolation of Cellular RNA from Infected Cells-Subcon allantoic cavity of 11-d-old embryonated chicken eggs fluent monolayers of MDCK cells in 85-mm plastic dishes which were incubated for 2 d at 34•Ž (18). MDCK cells were infected with approximately 1x108 PFU of virus were grown in Eagle's MEM supplemented with 10% fetal (moi; 10PFU/cell). After adsorption for 60 min at 4•Ž, the calf serum. cells were rinsed twice with PBS, overlaid with 5ml of Cloning of Virus cDNAs-Virion RNA was extracted as prewarmed MEM and incubated at 34•Ž. At the indicated described (19), and useduuused as thehe he template for the synthesis times, cells were chilled to 0•Ž, rinsed twice with cold PBS, of single-stranded (ss) cDNA, which was converted to ds and scraped from the dishes with a rubber policeman. From form and cloned in a pBR322-derived vector essentially as the washed cell pellet, total cellular RNA was isolated by

published previously (20). In brief, ss-DNA was synthe the guanidinium/hot phenol method as described previous- sized with avian myeloblastosis virus reverse tran ly (23). The concentration of RNA was measured photo- scriptase, using a synthetic dodeca-deoxynucleotide primer metrically. Usually 100ƒÊg of RNA was obtained from 1 x which was complementary to the common 3•L end of vRNA. 107 cells Der one 85 mm dish. The ss-DNA was converted to ds-DNA with the same The method used for preparation of poly(A) -containing enzyme, primed by a trideca-deoxynucleotide bearing the RNA was essentially as described by Aviv and Leder (24). 5•L end sequence of viral RNA. A XhoI linker was ligated to RNA-RNA Hybridization, RNase T1 Digestion and Gel the blunt-ended cDNAs except HA cDNA, for which Sail Electrophoresis-Two micrograms of total cellular RNA, linker was used. The cDNAs were then separated into each 100fmol of each probe, and 5ƒÊg of tRNA were mixed and segment by polyacrylamide gel electrophoresis, and each precipitated with ethanol. The precipitates were dried and cDNA was cloned either at the XhoI site or the SalI site of dissolved in 30ƒÊl of a hybridization buffer (50mM PIPES a pBR322-derived vector, in which a Xhol site was [pH 6.4], 0.4M NaCl, 1mM EDTA, 80% formamide) as introduced in the EcoRI site of pBR322, restoring two described previously (25). After heating of the hybridiza EcoRI sites at both sides of the Xhol site. cDNA clones of tion mixture for 5min at 90•Ž, RNA-RNA annealing was nearly full length were picked up, and their base sequences performed for 3 h at 37•Ž. At the end of incubation, 270,u l at both ends were determined. We have thus obtained of ice-cold T1 buffer (50mM Tris-HCl [pH 7.5], 5mM cDNA clones of full length for the 8 genome segments of the EDTA, 0.3M NaCl) was added together with 100 units of Udorn strain. RNase Ti, and the mixture was incubated for 60min at A restriction fragment containing the intact 5•L end 30•Ž to digest out single-stranded RNA. It was further sequence of each vRNA segment was isolated from the incubated for 30min at 30•Ž in the presence of 0.6% SDS cloned cDNA, and introduced into both of the SP-6 vectors, and 50ƒÊg of pronase K, and extracted with an equal volume of phenol-chloroform (1:1). The aqueous phase was mixed pSP64 and pSP65 (21). The cDNA fragments to the following 5•L termini of vRNAs were inserted between with 5ƒÊg of carrier tRNA, and RNA was precipitated with suitable restriction sites of both vectors; the 462 b ethanol for 60min at -80•Ž. The precipitates were washed PB2(HindIII), the 272 b PB1(EcoRI), the 527 b PA(StuI), with 70% ethanol, dried and dissolved in 5ƒÊ1 of denaturing the 155 b HA(BamHI), the 488 b NP(HindIII), the 353 b loading buffer. The RNA solution was heated for 4 min at NA(Sau3AI), the 287 b M(StuI), and the 172 b NS(TaqI). 95•Ž, chilled quickly at 0•Ž, and applied to a 0.35mm 6% In Vitro Synthesis of Hybridization Probes-Plasmid polyacrylamide-7M urea gel in 1xTBE. After electro DNAs carrying cDNA fragments were linearized by cutting phoresis, the gel was dried and exposed to Fuji RX X-ray at the unique PvuII site of the pSP vectors, which was films at -80•Ž. located at about 180 by from the promoter-distal end of the Quantitative Analysis of Autoradiograms-Autoradio multicloning site, and used as the templates for the synthe grams were exactly superimposed on the dried gels by the

RNA-RNA Hybridization, RNase TI Digestion J. Biochem.and Gel microgramsElectrophoresis-Twoof totalcellular RNA,fmol of 100 each probe, and5 ug of tRNAwere mixed and with precipitatedethanol. Theprecipitates were dried and in dissolved 30,ul of a hybridizationbuffer(50mM PIPES6.4],0.4 [pH M NaCl,1 mM EDTA,80% formamide)as describedpreviously(25).After heating of the hybridiza-tion mixturefor 5 min at 90°C,RNA-RNA annealingwasfor performed3 h at 37°C. At the end of incubation,270,ul of ice-cold Tl buffer (50 mM Tris-HCI[pH 7.5], 5 mM0.3 EDTA,M NaCl) wasadded together with100 units of RNaseTi,and the mixturewasincubated for 60 minat 30°Cto digestout single-strandedRNA.It wasfurther for 30 incubated minat 30°C in thepresence of 0.6% SDS 50,u andg of pronaseK,and extracted withan equalvolumephenol-chloroform (1 of : 1).The aqueous phase wasmixed 5 pgwith of carriertRNA,and RNA was precipitated withfor 60 ethanol min at -80°C.Theprecipitates were washed 70% ethanol, withdriedand dissolved in 5 p 1 of denaturingbuffer.TheRNA loadingsolution was heated for 4 minat chilled95°C,quickly at 0'C, and applied to a 0.35mm 6%M polyacrylamide-7 ureagel in 1 x TBE.After electro-phoresis, the gelwas dried and Quantitative exposed Analysis to of Fuji Autoradiograms-Autoradio. RX were X-rayexactly at superimposed -80°C. filmson .J. Ri.nehon, the dried gels by thegrams Control of Influenza Virus Gene Expression 539

use of several colored radioactive markers, and gel pieces densitometrically. corresponding to the radioactive bands on the film were cut Materials-SP6 RNA polymerase, pSP64, and pSP65 out and counted with a liquid scintillation counter. From the were kindly provided by Dr. Y. Nakanishi. SP6 RNA specific activity of [ƒ¿-32P]UTP and the content of U in the polymerase was also purchased from Takara Syuzo, Kyoto. complementary region of a probe, we could estimate the Sources of materials used for cDNA cloning were described

number of RNA-RNA hybrid molecules recovered in gel previously (20). Enzymes for DNA manipulations, phos bands. In some experiments, radioactivity of the hybrids phorylated linkers, and ribonuclease inhibitor were ob was estimated from the densitometric intensity of the tained from Takara Syuzo. Ribonucleases TI and A were autoradiograms, using a standard curve for the relationship from Sigma Chemical, U.S.A., while nuclease S1 and between the radioactivity and its density determined on the ribonuclease T2 were from Pharmacia P-L Biochemical, same autoradiogram. The recovery of RNA-RNA hybrids U.S.A. Cycloheximide, anisomycin, and puromycin were in the gel was corrected based on the recovery of the control obtained from Sigma Chemical, and actinomycin D was 32P -hybrid , which was formed between a pair of the comple from Calbiochem, U.S.A. [ƒ¿-32P]UTP (400 Ci/mmol) and mentary RNA transcripts added in advance to the total [35S]methionine (>800 Ci/mmol) were purchased from cellular RNA. The control RNAs were synthesized in the the Radiochemical Centre, Amersham, U.K. same way as the hybridization probes, using as the tem-

plate two SP6 vector DNAs with a 589 by fragment of E. RESULTS coli genome inserted in opposite directions. Analysis of Viral Polypeptides-Subconfluent mono The Hybridization System-In the quantitative hybridi layers of MDCK cells in 35-mm plastic dishes were infected zation system for influenza virus-specific RNAs, it is with virus, overlaid with 1.2ml of prewarmed MEM, and necessary to compete out the complementary RNA present incubated at 34•Ž. For radiolabeling, the medium was in test samples, by using an excess amount of probes. For discarded and replaced with 0.5ml of prewarmed methio the purpose, it is preferable to use an RNA probe. To get nine-free MEM containing 10ƒÊCi/ml [35S]methionine for such RNA probes, 155-500 by fragments of individual viral 30min. After pulse-labeling, the cells were rinsed with ds-cDNA containing the intact 5•L end sequence of the vRNA sense were cloned in two SP-6 vectors, pSP64 and pSP65. PBS, lysed directly in the dishes with 100ƒÊl of a lysis buffer (26), scraped from the dishes, and frozen-and- The plasmid DNAs were cut at the PvuII site of the vector, thawed several times. The cell lysates were sheared by and transcribed by SP-6 RNA polymerase. Usually 6 to 15 pmol of probe was obtained, containing 600 to 800cpm of passing through a hypodermic needle. Ten microliters of 32P per fmol . The 16 probes for the 8 genome segments are the lysates was heated for 5min at 95•Ž, and subjected to summarized in Table I. The probes for detection of mRNA electrophoresis on a 17.5% polyacrylamide gel (acrylamide and cRNA (minus-sense probes) were obtained by tran to bis-acrylamide ratio of 218.5:1) containing 4M urea scribing the SP-6 recombinant DNAs, which gave the intact (27), or in the absence of urea. A 10% gel containing 4M 5•L end sequence of vRNA. As mRNA lacks a copy of the last urea was also used. Fluorography was performed in 1 M sequence of about 16 nucleotides at the 5•L end of vRNA, the sodium salicylate according to Chamberlain (28). The hybrid formed between mRNA and the probe was shorter intensity of protein bands on the fluorogram was measured

TABLE I. The hybridization probes.

previously (10, 33-36). U content for segments 1 to 3 was deduced from the a The nucleotide sequences of segments 4 to 8 were published previously - of the fraements (our unpublished results). 'See text. csequencesThesignal offor PRSPBl strainvRNA (37),(also which known. is highly homologousdEstimatedfrom theto Udorn chainrlength strain inof thethe regionshybrids offormed, the fragmentswhichonger was(ourinversely unpublished related results). to theculated. bSeerecovery text. reason for the discrepancy values (see text).

Vol. 105, No. 4, 1989 540 E . Hatada et al.

Fig. 1. Autoradiogram of the signals for the virus-specific RNAs accumulated during the virus infection in MDCK cells. Total cellular RNA was isolated from the virus-infected cells har vested at the times (h pi) indicated at the top of the lanes. On the right, the left, and middle autoradiograms were overexposed to show signal bands exhibited at the early stage of infection. In lane m, mock-infected cellular RNA was used. The probe mixtures used are shown under the autoradiograms. PV and PMC indi cate the mixtures of plus and minus strand probes, respectively, for PB2, PB1, PA, NA, and NS. MV and MMC indicate the mixtures of plus and minus strands probes, respectively, for NP, M, and HA. Arrows, open triangles, and closed triangles indicate signals for vRNA, cRNA, and mRNA, respectively.

Fig. 2. Time course of accumula tion of 24 virus-specific RNA species in the infected MDCK cells. The intensity of the signals or the radioactivity in the gel pieces corre sponding to the signals on the auto- radiograms shown in Fig. 1 were measured as described in "MATE RIALS AND METHODS" and in the text. The estimated amount of each virus-specific RNA at various hours after infection is exhibited as the number of RNA molecules contained in an infected cell. Filled circles, open circles, and open triangles indicate vRNA, mRNA, and cRNA, respective ly.

than that between cRNA and the probe, after these hybrids probes, respectively), included those for segments 1, 2, 3, were digested with RNase. We could thus discriminate and 6; and the other two groups, termed MV and MMC (the cRNA from mRNA on polyacrylamide gel electrophoresis. plus and minus probes, respectively), for segments 4, 5, 7, The M and NS minus-strand probes could not discriminate and 8. Based on saturation experiments for those probes, the spliced mRNAs from their unspliced counterparts. For 100fmol of each probe was usually used for 2ƒÊg of total the discrimination, full-length probes (Mf- and NSf-) cellular RNA, which contained at most 5fmol of a specific were prepared. Transcripts of the opposite strand of viral RNA species during the infectious process. For cDNAs (plus-strand probes) gave the probes to detect example, 2ƒÊg of RNA sample at 5 h pi contained 4fmol of vRNA. As the nucleotide lengths of hybrids for each M vRNA (the maximum level of a virus-specific RNA), segment differed from one another, the plus or minus which formed a saturating level of the hybrid with 60fmol probes for the eight segments were combined into 4 groups: of the probe in the presence of 2fmol of corm_??_ting M the two groups, termed PV and PMC (the plus and minus mRNA (data not presented). The hybrid forma_??_ pro-

J. Biochem Control of Influenza Virus Gene Expression 541

infected cellular RNA was chromatographed on an oligo- (dT)-cellulose column to separate poly(A)+ and poly(A)- RNAs. The flow-through and the retained fractions of the column chromatography were separately hybridized against the four groups of probe mixtures. The hybridiza tion signals for vRNA were easily identified by comparing them with the signals obtained using purified virion RNA (Fig. 1, lane M). The signals for mRNAs moved a little faster than the corresponding signals for vRNA or cRNA

(Fig. 1, PMC and MMC), and most of them were detected in the retained fractions of the oligo(dT) column (data not presented). The signals for cRNAs were identified by three criteria: they had almost the same mobility as correspond ing vRNAs on gel electrophoresis; they were mainly detected in the flow-through fraction of the oligo(dT) column; and they were absent when hybridization was performed using RNA which was prepared from cells infected in the presence of cycloheximide (data are not Fig. 3. Time course of accumulation of NSI and NS2 mRNAs. presented). Total cellular RNA was prepared from virus-infected cells as shown in Time Course of Accumulation of Virus-Specific RNAs Fig. 1, and hybridized to NSf(-) probe (Table I). The signals -Employing the hybridization system , we measured expected for NS1 and NS2 mRNAs are illustrated on the upper right virus-specific RNAs, which were accumulated at various (filled bars; a, b, and c). Thick lines in the mRNAs show the sequence complementary to the vRNA and the dotted line shows the intron times after virus infection. Analyzing the autoradiograms which is spliced out in NS2 mRNA. On the left is shown the shown in Fig. 1, the numbers of three types of virus RNA autoradiogram, the numbers on top of which indicate hour pi. M and molecules were obtained as shown in Fig. 2. MDCK cells M•L are 32P-markers of nucleotide length (M: 488, 353 and M•L: 1,631, were infected with A/Udorn/72 virus at 10pfu per cell at 517, 506, 396, 344, 298, 221/220 bp, respectively, from the top). 34•Ž. Immediately after infection, we could not detect any Bands a and b were cut out, and their radioactivity was counted to virus-specific RNAs except for vRNAs from the inoculated calculate the copy numbers (see text); the results are summarized on virus (103 copies/cell). At 1 h pi, early mRNAs (for PB2, the lower right. PB1, PA, NP, and NS) appeared, among which NS mRNA predominated, but we could not detect any late mRNAs (for ceeded rapidly under our hybridization conditions. During a HA, NA, and M). Neither cRNAs nor increase of vRNAs 1 h incubation at 37•Ž, most of the hybrids were formed. To was observed for any of the segments. At 2 h pi, the level of digest out single-stranded regions of hybridized RNAs, we cRNA and vRNA began to rise simultaneously for all tried nuclease S1, ribonucleases, A, T2, and T1. S1 was segments. The amount of cRNA did not increase thereafter required in large amounts to digest RNA, and often cuts the and remained at a plateau level of 1,000 to 2,000 copies/cell double-stranded regions; A and T2 were more delicate in for each segment. NP and NS mRNAs further increased, digestion, and caused nibblings which hampered the identi prior to the increase in their respective vRNAs. The fication of cRNA. Ribonuclease T1 was much better in these mRNAs for three polymerase proteins also increased, respects. Even an excess amount of the enzyme did not cut reaching a plateau value thereafter (3,000 molecules/cell). the double-stranded region. G-specific digestion did not The mRNAs for late proteins started to be synthesized in cause problems, because we knew the base sequences close coupling with the rise of vRNAs. From 2 to 3 h pi the flanking the hybrid regions. mRNAs for NP and NS as well as for late proteins increased The final recovery of virus-specific RNAs from the in correlation to the increase of their respective vRNAs. hybridization procedure might differ from probe to probe. These correlations were broken thereafter, and the levels To estimate the recovery value, the following model of mRNAs were retained at plateau values or decreased experiments were performed. Mixtures of 5fmol of each gradually, except for M mRNA, for which some increase was observed until 5 h pi (10,000 copies/cell for NP, HA, probe and 2ƒÊg of RNA extracted from mock-infected cells, were hybridized against 100fmol of the respective comple and NA, and 20,000 copies/cell for NS and M). From 3 to mentary probe. The recovery values were inversely related 5 h pi, a steep increase in vRNA synthesis continued for all to the chain length of the hybrids formed (Table I). To segments, reaching a similar level (25,000-30,000 copies/ correct manipulative fluctuations among test tubes, a cell) for all the segments except for M (50,000 copies/cell) constant amount of 32P-labeled transcripts from both at 5 h pi. Both M and NS minus-strand probes which were used in strands of a 589 by E. coli DNA fragment was added to the cellular RNAs (see "MATERIALS AND METHODS"). the above experiments could not distinguish the spliced In this way, we could estimate the number of each viral mRNAs from their unspliced counterparts. For the RNA species by measuring the radioactivity in the signal discrimination, full-length probes (Mf- and NSf-) were employed in the hybridization experiments (Table I). The band on a polyacrylamide gel (U content was corrected; see NSf minus-strand probe should provide four signal bands Table I), and from the recovery values and corrections as mentioned above. The values were expressed as numbers of (Fig. 3); a doublet corresponding to NS cRNA and NS1 mRNA of 895 and 874 bases, respectively, and the other RNA copies in an infected cell, assuming that a cell contains two corresponding to NS2 mRNA of 349 (the 3•L region) and lOƒÊg of RNA. To identify hybridization signals for each viral RNA, 66 bases (the 5•L region). As shown in Fig. 3, the signal bands

Vol. 105, No. 4, 1989 542 E . Hatada et al.

Fig. 4. The accumulation of the virus-specific RNAs in MDCK cells which were infected with the Udorn strain in the presence of cycloheximide. The drug was added at 100ƒÊg/ml during the virus infec tion, rinsing of the cells and the subse

quent virus growth in MEM. A: Autoradiograms of the signals for the virus-specific RNAs. Total cellular RNA was isolated from the virus- infected cells at 3, 5, 7, and 9 h pi in the absence (lanes 1 to 4, respectively) and in the presence (lanes 5 to 8, respectively) of cycloheximide. For other symbols, see the legend to Fig. 1. Doublet bands near the top are from control hybrids added to the hybridi zation mixtures to normalize bands recovered on different gel lanes (see "MATERIALS AND METHODS") . B: The intensity of the signals, or the radioactivity in the gel pieces corr esponding to the signals on the auto- radiograms shown in A were measured as described in "MATERIALS AND METHODS" and in the text, and are plotted as the number of RNA mole cules contained in an infected cell. Closed circles, triangles, and squares represent NP, NS, and PB2 mRNAs, respectively, synthesized in cells in fected in presence of cycloheximide, while open symbols are for those mRNAs synthesized in the absence of the drug. In the presence of the drug, the signals for other mRNA species, and for all species of vRNA and cRNA were hardly detected.

Fig. 5. Rate of the synthesis, and the viral mRNA accumulation at various times postinfection. A: Time course of viral protein syn thesis. The infected MDCK cells were labeled for 30 min with [35S]methionine 15 min before the time (h pi) indicated at the top of each lane, and the labeled proteins were electrophoresed on a polyacrylamide gel. The lower photograph exhibits the M and NS1 proteins which were exposed for a shorter time. B: Rate of the viral protein synthesis is compared with the amounts of respective mRNA accumulated at various times (h pi). Densitometry of appropriate exposures of the gel shown in A and that for RNA measurement was used to generate each point of B, which is expressed as a percentage of the maximal value of the particular mRNA or protein . Open circles and closed circles represent mRNA and protein, respectively.

were observed at these predicted positions. Using the 0.15 during 2 to 5 h pi (Fig. 3). We could not detect signals recovery values indicated in Table I, the molar percentage corresponding to M2 (or M3) mRNA. of NS2 mRNA to total NS mRNA (NS1+NS2) was Effect of Cycloheximide on mRNA Accumulation-The estimated at various times after the infection. It was 0.1- synthesis of virus-specific RNAs was investigated in the

J. Biochem. Control of Influenza Virus Gene Expression 543

HA, M1, NP, and NS1 proteins with the amounts of respective mRNAs at various times after infection. For this

purpose, two sets of infected cell cultures were prepared. A portion of the infected cells was labeled with [35S]meth ionine for 30min and analyzed for the virus-specific

proteins (Fig. 5A). Fifteen minutes after addition of the radiolabel, RNA was prepared from the other nonlabeled portion. Densitometry of autoradiograms for 32P-hybrid RNAs and 35S-proteins was performed, and results were expressed as a percentage of the maximal amount of the

particular mRNA or protein in Fig. 5B. The synthesis rate of the NP protein increased in proportion to the amount of the mRNA. In contrast, the synthesis of the stayed at much lower level than the amount of its mRNA until 3 h pi. It increased abruptly thereafter to a maximum level during the late phase of infection, indicating a post- transcriptional control for the M1 synthesis. A critical dose of actinomycin D inhibited the synthesis of influenza virus late proteins but permitted the synthesis of early proteins (12, 30). With our infection system, 0.2 or 0.4ƒÊg/ml actinomycin D severely reduced the synthesis of the M1, HA, and, to a lesser extent, NS1 proteins, but allowed the synthesis of the NP and possibly polymerase proteins at nearly the normal level (Fig. 6). We measured the amounts of virus-specific RNAs accumulated at 5 h pi in these cells. As shown in Fig. 7, almost normal levels of all 24 virus-specific RNA species including mRNAs were Fig. 6. The effect of actinomycin D concentration on the pro tein synthesis of influenza virus. Actinomycin D was added to two detected in the presence of 0.1 or 0.2ƒÊg/ml actinomycin D. series of dishes of MDCK cells 1 h before tthe virus infection and Even with 0.4ƒÊg/ml of the drug, amounts of more than afterwards through the infectious process. One series of cells were 30% of these RNA species were detected. With decreasing labeled with [35S]methionine for 60min at 5 h pi. The cell lysates dose of the drug, the synthesis of late proteins increased in were prepared and applied to 17.5% gel without urea as described in "MATERIALS AND METHODS" (A) parallel with the extent of shutoff of the host protein . The numbers above gel lanes synthesis (Fig. 6A). These observations indicate that the of A, 1 to 7, indicate the cells infected in the presence of 0, 0.01, 0.04, syntheses of early and late viral proteins are regulated 0.1, 0.2, 0.4, and 1.0ƒÊg/ml actinomycin D, respectively. mo shows mock-infected cells. Densitometry of the autoradiogram of A was differentially not only at the transcriptional step(s), but also at some posttranscriptional step(s). performed and the results are plotted as a percentage of the maximal value of the particular protein (B).

DISCUSSION

presence of 100ƒÊg/ml cycloheximide, which completely In this paper we describe methods to measure accurately inhibited the protein synthesis in the infected cells. Only the amounts of virus-specific RNAs accumulated in cells mRNAs were synthesized in such cells. However, in con infected with influenza virus. Using 2 to 7ƒÊg of total RNA, trast to the previous reports (12-14, 29), we observed a the absolute amounts of three types of virus RNAs, i.e., notable difference in the accumulation level of each mRNA mRNA, cRNA, and vRNA for the 8 genome segments (24 species. As shown in Fig. 4, mRNAs for early proteins were species in all) could be measured in only 4 test tubes. This detected at about 103 molecules/cell at 3 h pi, and ac method is applicable to pulse-labeled RNA, if the poly(A) cumulated very slowly over the period studied (until 9 h tail at the 3•L end of mRNA is digested out using oligo(dT) pi). For three polymerase proteins, the mRNA levels were and RNase H. However, it is hard to label the virus-specific as high as those produced in the absence of the drug, RNAs to an adequate level for quantitative studies in suggesting that most of the polymerase mRNAs were MDCK cells, on which temperature-sensitive mutants of synthesized during the primary transcription. The levels of influenza virus A/Udorn had to be grown (18, 19). NP and NS mRNA were near those produced at 1 h pi in the In this study, we investigated the time courses of accu normal infection. In contrast to the early mRNAs, we could mulation of 24 virus-specific RNA species on cells infected hardly detect the late mRNAs; there was no NA mRNA at with a wild-type Udorn strain (Figs. 1 and 2), and compared all even at 9 h pi, and only a trace amount of M mRNAs, but them with the rates of the virus protein synthesis (Fig. 5). sometimes HA mRNA was detected at a level of 20 to 30% During the first hour postinfection when the virus protein of the early mRNAs. The same results were obtained using synthesis was hardly detectable, we could observe mRNAs other inhibitors of protein synthesis, anisomycin and for the NS, NP, and three polymerase proteins in that order puromycin (data not presented). The observations indicat of copy number, in agreement with the previous studies (4, ed a control at the transcription level during the primary 12, 16). However, we could hardly detect mRNAs for the transcription. late proteins. Nearly the same pattern of viral transcripts Uncoupling between Viral Transcription and the Protein was observed in cells as late as 9 h pi in the presence of Synthesis-In Fig. 5, we compared the rates of synthesis of inhibitors of protein synthesis (Fig. 4). In agreement with

Vol. 105, No. 4, 1989 544 E . Hatada et al.

Fig. 7. The effect of actinomycin D concentration on RNA synthesis of influenza virus. Total cellular RNA was isolated from the other series of cells harvested at 5 h pi as described in Fig. 6, and subjected to quantitative analysis of the virus-specific RNAs as described in the legend to Fig. 1. (A: for symbols, see Fig. 1.) The numbers above gel lanes of A, 1 and 4 to 6 indicate the cells infected in the presence of 0, 0.1, 0.2, and 0.4ƒÊg/ml actinomycin D, respectively. M indicates the signals obtained by hybridization between the plus probes and virion RNA. Doublet bands near the top are from control hybrids for normalization. Densitometry of the autoradiograms of A was performed and the results are plotted as a percentage of the amount of each RNA species detected in the absence of actinomycin D. Closed circles, open circles, and open triangles represent mRNA, vRNA, and cRNA, respectively.

TABLE II. Summary of the controls operating on the expression of each genome segment of influenza virus.

aHour postinfection . b±, weak response. `No evidence. d++, strong response.

this transcription pattern, we could detect the synthesis of difference in the virus strain or the host cell, or the high moi the early proteins, but not of the Ml and HA proteins adopted by the previous authors (60 to 200) compared to immediately after lifting the protein-synthesis block (Ref. ours (10). We have so far observed the same tran 31 and our unpublished observations). This phase of scriptional selectivity at moi 50 and 100 (data not present transcription occurring in the absence of any detectable ed). No such selectivity was observed in the in vitro viral protein synthesis is called the primary transcription, reaction catalyzed by disrupted virions of the Udorn strain and it has been reported that an almost equal number of (our unpublished observations). It was thus suggested that mRNA copies is transcribed unselectively from each the transcriptional selectivity for the early genes was genome segment (12-14, 29). At present we could not find endowed in the infected cells, and led to the preferential the reason for these discrepancies; it may be because of the synthesis of early mRNAs during 2 h pi, at least for MDCK

J. Biochem. Control of Influenza Virus Gene Expression 545 cells infected with Udorn strain. For transcription of the tion indicates that the late mRNAs can not function late genes, it is conceivable that some early gene product(s) effectively during the early phase. Some virus- of the virus is required, which has to be synthesized de programmed process, possibly through the synthesis of novo, or is supplied by the infecting virions inoculated at a some virus protein(s), eliminates this barrier on the high moi. Another possibility is that the late gene transcrip protein-synthesizing system. The process may be sensitive tion occurs using solely newly synthesized vRNAs as the to a low dose of actinomycin D (Figs. 6 and 7). It has been templates (secondary transcription). suggested that the barrier is on the nuclear-cytoplasmic The synthesis of the late mRNAs commenced after 1 h pi, transport system of mRNA (12), or on the translation and their copy number as well as that of NP and NS machinery. proteins increased in close proportion to the copy number of It is also noteworthy that there was a correlation between the respective vRNAs until 3 h pi. On average, one the shutoff of host protein synthesis and the synthesis of the molecule of vRNA accumulated one molecule of mRNA for late proteins (Fig. 6). It is conceivable that NS1 partici the HA, NP, NA, and M genes, and two to three molecules pates in both of these processes (Hatada, E., et al., submit of mRNAs for the NS gene. These results are consistent ted for publication). Both NS1 and NP are classified as early with the previous notion that the control of mRNA synthe proteins, but the synthesis of NS1 protein was delayed sis is predominantly a direct consequence of the regulation compared to that of the NP protein (Fig. 5B), and a part of of the vRNA synthesis (4, 15). The increase in the mRNA its synthesis was under the posttranscriptional control accumulation ceased abruptly at 3 h pi for the HA, NP, NA, operating on the late protein synthesis. and NS genes or leveled off gradually until 5 h for the M In Table II, we summarize the regulations operating on gene, in agreement with Shapiro et al. (16), who measured the expression of each genome segment. It is indicated that the synthesis rate of virus-specific RNAs. In contrast with early expression of the genes for NP and polymerase these mRNAs, the mRNAs for three polymerase gennnesdid proteins is ensured because their inoculated vRNAs are not increase after 2 h pi, retaining a similar low level for preferentially transcribed to their mRNAs, which are each mRNA. As thehesame level of each polymerase mRNA immediately translated into the proteins. On the other was maintained in the presence of cycloheximide, it was hand, the posttranscriptional barrier has to be removed for indicated that most of these mRNAs were synthesized the late and the NS1 mRNAs to be efficiently translated. during the primary transcription. The synthesis of vRNAs as well as cRNAs began after 1 h pi simultaneously for all We wish to thank Dr. Akira Ishihama for helpful comments on this segments. We could not observe notable priority in the manuscript, and Dr. Masakazu Hatanaka for discussionsand encour synthesis of NS or NP vRNAs, in disagreement with the agement. We are very grateful to Miss Miyuki Ogino for her skillful assistance in preparing the manuscript. previous reports (4, 15-17). The copy number of cRNAs did not increase after 2 h, and remained at a similar level for all segments (4, 15), while the synthesis of vRNAs REFERENCES increased and continued beyond 5 h pi to attain approxi 1. McGeoch, D.J., Fellner, P., & Newton, C. (1979) Proc. Natl. mately the same copy number for all segments except for Acad. Sci. U.S. 73, 3045-3049 M, in agreement with the previous report (20). 2. Lamb, R.A. & Choppin, P.W. (1983) Annu. Rev. Biochem. 52, At 2 h pi, when replication of all genome segments 467-506 started, the synthesis rate of the NP protein attained a 3. MacCauley, J.W. & Mahy, B.W.J. (1983) Biochem. J. 211, 281- substantial level, but that of the NS1 protein still remained 294 4. Hay, A.J., Lomniezi, B., Bellamy, A.R., & Skehel, J.J. (1977) low (Fig. 5). Similar patterns were observed when infection Virology 83, 337-355 was performed in the presence of a critical dose of 5. Hay, A.J., Abraham, G., Skehel, J.J., Smith, J.C., & Fellner, P. actinomycin D (Fig. 6). The NP protein was synthesized at (1977) Nucleic Acids Res. 4, 4197-4209 almost the normal level, whereas the synthesis rate of NS1 6. Bouloy, M., Plotch, S.J., & Krug, R.M. (1978) Proc. Natl. Acad. and the late proteins was severely reduced. Under these Sci. U.S. 75, 4886-4890 conditions, however, all species of virus RNAs were 7. Beaton, A.R. & Krug, R.M. (1981) Nucleic Acids Res. 9, 4223- 4236 synthesized at nearly the normal level. If the NS1 protein 8. Robertson, J.S., Schubert, M., & Lazzarini, R.A. (1981) J. Virol. participates in virus replication along with the NP and 38, 157-163 polymerase proteins as was suggested previously (Ref. 32 9. Hay, A.J., Skehel, J.J., & McCauley, J. (1982) Virology 116, and Hatada, E., et al., submitted for publication), it may be 517-522 required in only a small amount. The remainder of the 10. Lamb, R.A. & Lai, C.-J. (1980) Cell 21, 475-485 11. Inglis, S.C. & Brown, C.M. (1981) Nucleic Acids Res. 9, 2727- protein may function at another step(s) of the infectious 2740 process. 12. Barrett, T., Wolstenholme, A.J., & Mahy, B.W.J. (1979) In addition to the control on the level of viral RNA Virology 98, 211-225 synthesis, it was indicated that the expression of the virus 13. Mark, G.E., Taylor, J.M., Broni, B.B., & Krug, R.M. (1979) J. genome was regulated at various steps after the transcrip Virol. 29, 744-752 tion (4, 16, 29). The experiments shown in Figs. 5 to 7 14. Inglis, S.C. & Mahy, B.W.J. (1979) Virology 116, 517-522 indicate such posttranscriptional regulation. The synthesis 15. Smith, G.L. & Hay, A.J. (1982) Virology 118, 96-108 of early proteins, the NP and polymerase proteins, 16. Shapiro, G.I., Gurney, T., Jr., & Krug, R.M. (1987) J. Virol. 61, 764-773 proceeded immediately after their mRNA production, 17. Varich, N.L. & Kaverin, N.V. (1987) J. Gen. Virol. 68, 2879- though the synthesis of NS1 protein was somewhat 2887 retarded (Fig. 5B). On the other hand, the efficient synthe 18. Shimizu, K., Mullinix, M.G., Chanock, R.M., & Murphy, B.R. sis of the late proteins (Ml and HA) was delayed for more (1982) Virology 117, 38-44 than one hour after their mRNA production. The observa 19. Shimizu, K., Mullinix, M.G., Chanock, R.M., & Murphy, B.R.

Vol. 105, No. 4, 1989Vol.105,No.4, 1989 546 E. Hatada et al.

(1982) Virology 117, 45-61 177-187 20. Enami, M., Fukuda, R., & Ishihama, A. (1985) Virology 142,68- 27. Lamb, R.A., Etkind, P.R., & Choppin, P.W. (1978) Virology 91, 77 60-78 21. Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, 28. Chamberlain, J.P. (1979) Anal. Biochem. 98, 132-135 K., & Green, M.R. (1984) Nucleic Acids Res. 12, 7035-7056 29. Lamb, R.A. & Choppin, P.W. (1976) Virology 74, 504-519 22. Maxam, A.M. & Gilbert, W. (1980) in Methods in Enzymology 30. Minor, P.D. & Dimmock, N.J. (1975) Virology 67, 114-123 (Grossman, L. & Moldave, K., eds.) Vol. 65, pp. 499-560, 31. Skehel, J.J. (1973) Virology 56, 394-399 Academic Press, New York 32. Wolstenholme, A.J., Barrett, T., Nichol, S.T., & Mahy, W.J. 23. Feramisco, J.R., Smart, J.E., Burridge, K., Helfman, D.M., & (1980) J. Viral. 35, 1-7 Thomas, G.P. (1982) J. Biol. Chem. 257,11024-11031 33. Sveda, M.M., Markoff, L.J., &Lai, C.-J. (1982) Cell 30,649-656 24. Aviv, H. & Leder, P. (1972) Proc. Natl. Acad. Sci. U.S. 69, 34. Buckler-White, A.J. & Murphy, B.R. (1986) Virology 155, 345- 1408-1412 355 25. Berk, A.J. & Sharp, P.A. (1978) Proc. Natl. Acad. Sci. U.S. 75, 35. Markoff, L. & Lai, C.-J. (1982) Virology 119, 288-297 1274-1278 36. Lamb, R.A. & Lai, C.-J. (1981) Virology 112, 746-751 26. Peluso, R.W., Lamb, R.A., & Choppin, P.W. (1977) J. Virol. 23, 37. Fields, S. & Winter, G. (1982) Cell 28, 303-313

J. Biochem.