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  • Vol. 32, No. 3JOURNAL OF VIROLOGY, Dec. 1979, p. 8388440022-538X/79/12-0838/07$02.00/0

    Inactivation of Influenza and Vesicular Stomatitis Virion RNAPolymerase Activities by Photoreaction with 4'-Substituted

    PsoralensKUNIO NAKASHIMA, PRANAB K. CHANDA, VICTOR DEUTSCH,t AMIYA K. BANERJEE, AND

    AARON J. SHATKIN*Roche Institute ofMolecular Biology, Nutley, New Jersey 07110

    Received for publication 17 July 1979

    Irradiation of purified influenza virus and vesicular stomatitis virus (VSV) withlong-wavelength UV light in the presence of 4'-substituted psoralens inactivatedthe virion-associated RNA polymerase activity. Inactivation was apparently dueto psoralen modification of the viral genome RNAs, since cations that decreasepsoralen binding to nucleic acids had a protective effect, and reconstitution ofVSV RNA polymerase activity was inhibited by photoreaction of nucleoproteincores but not by pretreatment of soluble fraction from dissociated virions. Par-tially inactivated viral particles synthesized reduced amounts of full-length RNAproducts in vitro without an increase in prematurely terminated transcripts. VSVleader RNA formation was relatively resistant to psoralen photoinactivation, andsequential transcription was maintained by photoreacted VSV. The all-or-nonepsoralen effect on virion-associated RNA polymerase activities may be due to adifferential photosensitivity of promoter sites or to structural changes in modifiedviral genome RNAs that prevent formation of new mRNA chains.

    Psoralens photoreact with nucleic acids uponirradiation with long-wavelength UV light, form-ing covalent monoadducts with pyrimidines andalso cross-links in base-paired regions of poly-nucleotides (7, 14, 16, 18). As a consequence,both DNA and RNA viruses are inactivated bypsoralen photoreaction (11, 12, 19-21). The po-tential importance of this experimental ap-proach for studying viral genome structures insitu, as well as its practical application for vac-cine production, is evident.

    Previously it was shown that the inactivationof reovirus type 3 plaque-forming ability by UVirradiation with 4'-substituted psoralens was ac-companied by, and possibly due to, loss of thevirion-associated RNA polymerase activity (21).This enzyme transcribes one strand of each ofthe 10 double-stranded genome RNA segmentsof the virus to form the viral mRNA's (15).Reovirus particles that were partially photoinac-tivated synthesized reduced amounts of full-length transcripts corresponding to the large,medium, and small size classes of mRNA. Sur-prisingly, no prematurely terminated mRNAproducts were observed, and the same relativeproportions of the three size classes of mnRNAwere produced by particles that were either

    t Present address: Institut de Microbiologie, Universite deParis-Sud, 91405, Orsay, France.

    slightly or highly modified (20a). The resultssuggested that the presence ofa single covalentlybound psoralen molecule prevented initiation ofmRNA synthesis on the altered genome seg-ment, whereas unmodified segments continuedto function as templates for the reovirion RNApolymerase.RNA polymerase activities are also present in

    purified influenza virus (6) and vesicular sto-matitis virus (VSV) (3). The single-stranded ge-nome RNA of influenza virus, like that of reo-virus, consists of multiple segments that areindependently transcribed to form the comple-mentary RNAs (24, 27, 28). By contrast, theVSV genome is a single high-molecular-weightRNA molecule that is transcribed in a polarfashion by the particle-associated polymerase (1,2). Transcription starts preferentially at the 3'-end of the VSV genome with the formation of ashort leader RNA, followed by the sequentialsynthesis of the five viral mRNA's. These strik-ingly different mechanisms of influenza (24) andVSV transcription (1, 2) were deduced from UVinactivation studies. The findings made withreovirus suggested that psoralen photoreactionwould be an effective alternative method tostudy mRNA synthesis by the influenza andVSV RNA polymerase activities. We have usedthis approach to analyze the different patternsof in vitro mRNA synthesis by the virion-asso-

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  • PSORALEN PHOTOREACTION OF VSV AND INFLUENZA 839

    ciated RNA polymerase activities of these twoviruses.

    MATERLALS AND METHODSViruses. The Indiana strain of VSV was purified

    from infected BHK-21/13 cells as described (4). Puri-fied WSN influenza A virus from infected MDBK cells(23) was kindly provided by Robert Krug of Sloan-Kettering Institute, New York.

    Photoreaction with psoralen. Virus samples ata concentration of 250 itg of protein per ml in 10 mMTris-hydrochloride (pH 8) containing 1 mM EDTAand the indicated 4'-substituted psoralen were irradi-ated at 00C with UV light of 352-nm wavelength asdescribed previously (21). Triton N-101 was present atconcentrations of 0.2 and 0.05% for influenza virus andVSV, respectively.

    Transcription reactions. Influenza virus comple-mentary RNA (cRNA) was synthesized in incubationmixtures of 0.3 to 0.5 ml containing 30 to 50 jg ofpurified virus, 50 mM Tris-hydrochloride (pH 8), 0.1M KCI, 5 mM magnesium acetate, 2 mM dithiothrei-tol, 0.2% Triton N-101, 0.4 mM adenylyl-(3'--5')-gua-nosine dinucleoside monophosphate as primer (23), 0.1mM [a-3P]CTP or [a-3P]UTP (final specific activity,0.65 Ci/mmol), and 1 mM each of the other threeribonucleoside triphosphates. Incubation was at 310Cfor 1 h. For VSV mRNA synthesis, reaction mixturesof 0.3 ml containing 40 ug of purified virus, 50 mMTris-hydrochloride (pH 8), 0.1 M NaCl, 5 mM MgC12,4 mM dithiothreitol, 0.05% Triton N-101, 0.05 mM[a-3P]UTP (final specific activity, 0.25 Ci/mmol), and1 mM each of the other ribonucleoside triphosphateswere incubated at 300C for 2 h. Incorporation of radio-activity into acid-precipitable products was assayed asdescribed (21).

    Transcription product analysis. The influenzavirus single-stranded cRNA products were analyzedby electrophoresis in 3% polyacrylamide gels contain-ing 6 M urea (23). After hybridization of the cRNA tothe virion RNA and RNase T2 treatment, the double-stranded RNAs were resolved in 3% polyacrylamidegels without urea (23). VSV leader RNA, separatedfrom mRNA's by sucrose gradient centrifugation, wasquantitated by polyacrylamide gel electrophoresis, fol-lowed by autoradiography and tracing of the resultingfilm with an Ortec 4310 densitometer (8). The viralmRNA's were quantitated by hybridization to virionRNA, followed by electrophoresis in a 5% polyacryl-amide gel after the single-stranded regions weretrimmed by digestion with ribonuclease T2 (10, 25).Tracings of autoradiograms were used to determinethe relative amounts of the viral RNA products.

    Materials. 4'-Aminomethyl-4,5',8-trimethylpsora-len (AMT) was obtained from Calbiochem-BehringCorp., La Jolla, Calif. 4'-Hydroxymethyl-4,5',8-tri-methylpsoralen (HMT) was kindly provided by J. E.Hearst, University of California, Berkeley. Adenylyl-(3'- 5')-guanosine dinucleoside monophosphate wasobtained from P-L Biochemicals, Inc., Milwaukee,Wis., and proteinase K came from E. Merck, Darms-tadt, W. Germany. [a-3P]-labeled CTP and UTP (10to 30 Ci/mmol) were obtained from New EnglandNuclear Corp., Boston, Mass.

    RESULTSEffect ofpsoralen photoreaction on influ-

    enza virion RNA polymerase activity. Influ-enza virus contains an RNA polymerase thatbecomes active upon detergent treatment of pu-rified virions in vitro. The enzyme activity tran-scribes each of the eight genome segments intothe cRNA's and is markedly stimulated by thepresence of a primer dinucleotide such as aden-ylyl-(3'--+5')-guanosine dinucleoside monophos-phate (17, 22). The reaction is also stimulatedby capped globin mRNA (22) and reovirusmRNA's with the incorporation of the 5' capand 10 to 25 additional nucleotides from theprimer into the cRNA products (5a). Irradiationof detergent-treated virions in the presence ofAMT rapidly inactivated the adenylyl-(3',5')-guanosine dinucleoside monophosphate-primedinfluenza virus RNA polymerase activity (Fig.1). Irradiation or psoralen treatment alone hadlittle effect on cRNA synthesis. The extent ofinactivation was directly related to drug concen-tration and time of irradiation. Consistent withthe reported inhibition of psoralen binding tonucleic acids by cations (13), the presence of 0.1M KCI partially protected against inactivation.The same level of protection was observed inthe presence of 5 mM MgCl2 (data not shown).Photoreaction with another 4'-substituted deriv-ative, HMT, was about 10-fold less effective in

    80~60

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    0 2 3 4 5UV IRRADIATION (min)

    FIG. 1. Psoralen photoinactivation of influenzavirion RNA polymerase activity. Influenza virus wasirradiqted as described in the text in the absence(0) or presence of 2.8 x 10-5M HMT (C), 2.8 x 10-6MAMT (0), 2.8 x 10-5 MAMT (A), and 2.8 x 10-5MAMT plus 0.1 M KCI (A). At the times indicated,samples were taken for assay ofcRNA synthesis. The100% value for a 50-,ul incubation mixture containing9 pg of virus was 37,598 cpm or 26pmol.

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  • 840 NAKASHIMA ET AL.

    inhibiting influenza RNA polymerase activitythan with the same concentration ofAMT (Fig.1).As observed previously for reovirus-associated

    RNA polymerase activity (20a), AMT photo-inactivation of influenza cRNA synthesis appar-ently occurs at the level of initiation of polynu-cleotide chain formation. Total cRNA productsmade by the influenza virus RNA polymerasecomplex during a 60-min incubation were ana-lyzed by polyacrylamide gel electrophoresis.Short running times were used to detect rapidlymigrating, low-molecular-weight products thatmight be present as a result of premature ter-mination of transcription on modified templates(Fig. 2A). To avoid or at least to minimize losses,3'-polyadenylic acid was not removed from theproducts before electrophoresis. This resulted inlow resolution (lanes 1 to 4) as compared to apreparation of cRNA that had been deadeny-lated by hybridization with polydeoxythymi-dylic acid followed by RNase H treatment (lane5). Nevertheless, the gel patterns obtained forcRNA products, made by preparations of virion-associated RNA polymerase that had been in-activated to the extents of 0, 19, 29, and 47% byAMT photoreaction (Fig. 2, lanes 1 to 4), did notshow noticeable increases in short RNA prod-ucts. In agreement with the apparent lack ofincreased amounts of prematurely terminatedtranscripts, the annealed cRNA formed com-plete, RNase T2-resistant duplexes with the vir-ion RNA genome segments. Gel patterns of theduplexes obtained with products of both controland partially inactivated polymerase prepara-tions included the three large, three medium,and two small influenza RNAs (Fig. 2B). Asobserved for reovirus, the synthesis of each ofthe size classes of influenza cRNA was affectedby psoralen photoinactivation, the large seg-ments being relatively more sensitive (Fig. 2B,compare lanes 1 and 4). Thus AMT photoinac-tivation of influenza RNA polymerase appar-ently resulted in the formation of reduced yieldsof full-size products without a correspondingaccumulation of incomplete transcripts.Photoinactivation of VSV RNA polymer-

    ase activity. Irradiation of detergent-treated,purified VSV with long-wavelength UV lighthad no effect on the particle-associated RNApolymerase activity (Fig. 3). The addition ofAMT without irradiation diminished RNA syn-thesis by the virion polymerase by 20% or less.However, irradiation for 2 min in the presenceof 2.8 x 10'- M AMT resulted in >98% loss ofenzyme activity. Smaller decreases occurred atlower concentrations of AMT. As observed forthe reovirus and influenza virus-associated po-

    FIG. 2. Polyacrylamide gel electrophoresis ofcRNA synthesized by partially photoinactivated in-fluenza virus. Purified virus (50 jig) was irradiated in2.8 x 10-5MAMT for 2.5 s (lane 2), 5 s (lane 3), and10 s (lane 4). These samples and an untreated controlvirus suspension (lane 1) were incubated in the darkunder conditions of cRNA synthesis in 0.33-ml incu-bation volumes. The radioactive products were ex-tracted with phenol after digestion by proteinase K(0.5 mg/ml) in 0.5% sodium dodecyl sulfate for 1 h at37C. Unincorporated precursors were removed bygel filtration in Sephadex G-50 and ethanol precipi-tation. RNAs were analyzed (A) by electrophoresisfor 3 h at 75 V in a 3%o polyacrylamide slab gelcontaining 6M urea or (B) for 17 h at 30 mA in a 3%opolyacrylamide gel after hybridization to virion RNAand RNase T2 treatment (23). Gels were dried, andautoradiographs were prepared. Dye, Bromophenolblue. Lane 5 in (A) shows the profile ofdeadenylated,single-stranded cRNA. 1, m, and s, large, medium,and small species of influenza RNAs.

    lymerases, the activity was partially protectedby the presence of mono- or divalent cationswhich inhibit dark (noncovalent) binding ofpsoralens to nucleic acids.

    4'-Substituted psoralens modify both RNAand DNA, but photocatalyzed covalent attach-ment to proteins has not been observed. Theinactivation of the VSV RNA polymerase byAMT presumably results from alteration of thegenome RNA template. VSV RNA polymeraseactivity was reconstituted from AMT-treatedand untreated soluble and core fractions pre-pared by dissociating purified virions with deter-gent at high ionic strength (9). As previouslyshown (9), the high-salt-soluble fraction, which

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  • PSORALEN PHOTOREACTION OF VSV AND INFLUENZA 841

    contains the L, NS, M, and G proteins releasedfrom the genome RNA, had little RNA-synthe-sizing activity (Table 1). The cores derived fromvirions by high-salt treatment contained the ge-nome RNA and N protein and retained a lowlevel of RNA polymerase activity. Addition ofhigh-salt-soluble fraction, either untreated orafter photoreaction with AMT, increased theresidual polymerase activity of untreated high-salt cores by two- to threefold. However, theresidual RNA polymerase activity was not in-creased when soluble fraction was added to theAMT-reacted cores. These results are consistentwith inactivation of the VSV RNA polymeraseactivity by psoralen alteration of the genometemplate RNA.Analysis of the VSV RNA products. Pre-

    vious studies have shown that transcription ofthe VSV genome RNA in vitro by the virion-associated RNA polymerase is a sequential proc-ess (1, 2). Transcription begins at the 3' end ofthe template with the synthesis of a 48-nucleo-tide leader sequence (8) that is followed by read-ing of the N, NS, M, G, and L genes. Theproducts can be partially resolved by sucrosegradient centrifugation into the low-molecular-weight leader RNA, which remains at the top of

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    UV IRRADIATION (min)FIG. 3. Effect of AMT and UV irradiation on

    VSV-associated RNA polymerase activity. PurifiedVSVwas irradiated in the absence (0) orpresence of2.8 x 10-5MAMT (A), 2.8 x 10-5 MAMTplus 0.1MNaCl (), 2.8 x 10-5 M AMT plus 0.002 M MgC12(A), or 2.8 x 10- MAMT (-). At the times indicated,samples were taken for assay of RNA synthesis asdescribed in the text. The 100% value for a 200-ILIreaction mixture containing 40 pg of virus was 52,368cpm or 95pmol.

    TABLE 1. Effect ofAMTphotoreaction onreconstitution of VSVRNA polymerase activitya

    RNA synthesisViral components (cpm incorpo-

    rated)Untreated soluble fraction ......... 790Untreated core fraction ................ 3,980Untreated soluble + untreated core. 10,450Treated soluble + untreated core ....... 10,200Untreated soluble + treated core ...... 2,960

    aSoluble and core fractions were prepared by treat-ing 100 tig of purified VSV with 0.8 M NaCl and 1.88%Triton X-100 as described (9). Samples of each wereirradiated for 1 min in the presence of 0.1 M NaCl and2.8 x 10-4 M AMT. Soluble and core fractions or thereconstituted mixtures were incubated in 10 mM Tris-hydrochloride (pH 8), 0.1 M NaCl, 1 mM dithiothrei-tol, and 10% glycerol for 30 min at 300C. Ribonucleo-side triphosphates, including [3H]UTP (final specificactivity, 660 cpm/pmol) and 5 mM MgCl2, were thenadded as described in the text. After further incubationfor 1 h at 30C, the mixtures were assayed for acid-precipitable radioactivity.

    the gradient, a 12S peak that includes theM andNS messages, a 14.5-17S peak comprising the Nand G mRNA's, and the 31S mRNA for the Lprotein (5). Photoreaction with AMT under con-ditions that diminished overall VSV RNA syn-thesis by 47, 69, and 83% decreased the yields ofeach of the peaks ofmRNA, but synthesis of thelow-molecular-weight leader RNA that re-mained at the top of the gradient appeared tobe relatively unaffected (Fig. 4). Furthermore,as in the case of influenza and reovirus in vitroRNA synthesis, there was no indication of abor-tive transcript formation even in the most exten-sively inactivated VSV preparation. To test di-rectly for the apparent absence of prematuretermination and the relative resistance of leaderRNA synthesis to AMT photoinactivation, thelow-molecular-weight products corresponding tofractions 23 to 27 in Fig. 4 were analyzed bypolyacrylamide gel electrophoresis. As shown inFig. 5 (lane A), the small products synthesizedby untreated VSV included the leader RNA(arrow), mRNA products that were incompletebut larger than leader, and at least two otherlow-molecular-weight RNAs migrating fasterthan the 48-nucleotide leader RNA (see Discus-sion). Partially inactivated particles synthesizeda diminished yield of the same products, but thepredominant material made by the most exten-sively photoinactivated virus migrated in theposition of leader RNA (Fig. 5, lane D). Analysisof tracings of the autoradiographs confirmedthat synthesis of the leader RNA was differen-tially resistant to AMT photoreaction, i.e.,treated samples that retained 53, 31, and 17% of

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  • 842 NAKASHIMA ET AL.

    904x

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    FRACTION NUMBER

    FIG. 4. Sedimentation analysis of RNA productssynthesized by partially photoinactivated VSV. Pu-rified VSV (40 pg) was irradiated in the presence of2.8 x 10-5 M AMT for 15 s (-), 30 s (A), and 60 s(A) and then incubated together with an untreatedcontrol virus sample (0) in the dark under conditionsofRNA synthesis. Products were extracted byphenolin the presence of 0.5% sodium dodecyl sulfate andanalyzed by centrifugation (SW40, 33,000 rpm, 17 h,230C) in 15 to 30% sucrose gradients over a cushionof 0.6 ml of 60%o sucrose, containing 0.5% sodiumdodecyl sulfate, 0.1 M NaCl, 10 mM Tris-hydrochlo-ride (pH 7.4), and 1 mM EDTA. Fractions of 0.48 mlwere collected, and samples were assayed for acid-precipitable radioactivity.

    the total initial RNA synthesis activity synthe-sized 93, 70, and 69%, respectively, of the amountof leader RNA made by the untreated prepara-tion under the same conditions.

    Irradiation of VSV at a wavelength of 254 nminhibited differentially the in vitro synthesis ofthe five VSV mRNA's, and only the 3'-proximalN gene had a target size consistent with themolecular weight of its transcript (1, 2). Theother genes had considerably larger target sizesthat were cumulative, indicative of a compulsoryorder of gene transcription (1, 2). To determinethe relative sensitivity of the VSV genes toAMTphotoinactivation, the mRNA products (corre-sponding to fractions 1 to 22 in Fig. 4) wereannealed with viral genome RNA. The hybridswere treated with ribonuclease T2 to trim thesingle-stranded portions (10, 25), and the du-plexes were resolved by polyacrylamide gel elec-trophoresis. Gene N transcription was the leastaffected by AMT photoinactivation (Fig. 6).These results are similar to those obtained pre-viously with UV-inactivated VSV (1, 2).

    DISCUSSIONThe recently synthesized 4'-substituted psor-

    alens, AMT and HMT, effectively inactivatedthe infectivity of VSV (12) and reovirus (21) byphotoreacting with the viral genome RNAs.Modification of the viral RNAs in situ also abol-

    _ _

    FIG. 5. Analysis of the leader RNAs synthesizedin vitro byphotoreacted VSV. The fractions contain-ing the leader RNAs (fractions 23 to 27 in Fig. 4) werepooled, and the ethanol-precipitated RNAs were an-alyzed by electrophoresis in a 20%,o polyacrylamideslab gel containing 7M urea as described previously(8). Electrophoresis was for 15 h at 140 V, followed byautoradiography. Lanes A, B, C, and D contain theleader RNAs synthesized by VSV that was photo-reacted with AMT for 0, 15, 30, and 60 s, respectively.The arrow indicates the leader RNA band.

    FIG. 6. Polyacrylamide gel electrophoresis of theVSVmRNA products synthesized in vitro bypartiallyphotoinactivated VSVand hybridized to virion RNA.Fractions 1 to 22 in Fig. 4 representing the 12-18Sregion and containing the N, NS, G, and M proteinmRNA's (5) were pooled, and the RNA was ethanolprecipitated. Portions of the RNA samples were thenannealed with VSVgenome RNA, followed by diges-tion with RNase T2 as described previously (25). Theresulting RNA duplexes were analyzed by electropho-resis in a 5%polyacrylamide slab gel. Electrophoresiswas for 18 h at 50 V, followed by autoradiography.The positions of the mRNA duplexes are shown byletters on the left. Lanes A to D represent mRNA 'ssynthesized by VSV that was photoreacted with AMTfor 0, 15,30, and 60s, respectively. Lane E is the sameas lane A but exposed for a shorter time.

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  • PSORALEN PHOTOREACTION OF VSV AND INFLUENZA 843

    ished their ability to function as templates forthe RNA polymerase activity associated withthese virions (21; Fig. 3). Similarly, influenzaRNA polymerase activity was lost after psoralenphotoreaction of purified virions (Fig. 1). Itseems clear that the primary lesion is in thevirion RNA (16, 18; Table 1), although directeffects on the proteins may also occur, especiallyat high AMT levels or long times of photoreac-tion. The main effect on the virion RNA polym-erase activities appeared to be at the initiationstep of transcription, since AMT photoinactiva-tion was not accompnied by an increased pro-duction of prematurely terminated transcripts.Their absence suggests that transcription initi-ation is diminished by structural effects on theRNA-protein complexes in psoralen-photo-reacted particles. This might occur, for example,by a twisting ofthe strands ofthe duplex genomeRNA of reovirus or a similar unwinding of thehelical nucleocapsids of influenza virus by psor-alen intercalation. Alternatively, the RNA po-lymerase promoter sites may be more suscepti-ble to psoralen adduct formation than otherregions of the template RNAs, or modificationsin the promoter region may block initiationwhile alteration at internal sites may allow con-tinued elongation at a reduced rate of nucleotideincorporation.The observations with psoralen-photoreacted

    purified reovirus and influenza virus contrastsharply with findings made with VSV. It hasbeen shown previously that partial inactivationof VSV by irradiation with UV light at a wave-length of 254 nm apparently induced pyrimidinedimers randomly in the viral genome RNA (1).Their presence caused termination of RNA syn-thesis by the virion polymerase at the modifiedsites, resulting in the formation of a largeamount of products that were heterogeneous insize and not 3'-polyadenylated. If psoralen ad-ducts, like pyrimidine dimers, form at randomsites on the viral genome RNAs, prematurelyterminated transcripts would be expected. How-ever, psoralen-reactive sites on the VSV genomeRNA in situ appear to be specific, since theproducts made by AMT-photoreacted VSV didnot include increased amounts of prematurelyterminated transcripts (Fig. 4). On the otherhand, sequential transcription was apparentlymaintained in AMT-reacted VSV, since leaderRNA synthesis was highly resistant to inactiva-tion (Fig. 5) and N gene transcription.was af-fected less than the other viral genes (Fig. 6), aswas also observed with UV-inactivated VSV (1,2).

    In addition to leader RNA, the products ofuntreated and psoralen-treated VSV includedrapidly migrating, distinct bands of low-molec-

    ular-weight RNA (Fig. 5). It was recently shownthat they are not incomplete leader RNA mole-cules but correspond to 5'-terminal sequences ofVSV mRNA species. The small RNAs containthe unblocked 5'-sequence (p)ppA-A-C-A-G, ascompared to the capped 5'-sequenceG(5')ppp(5')A-A-C-A-G, present in full-lengthVSV mRNA's (26). The results indicate thatVSVmRNA synthesis in vitro is in fact mediatedby multiple initiation processes (D. Testa, P. K.Chanda, and A. K. Banerjee, submitted for pub-lication). Thus, the fornation of VSV leaderRNA and mRNA apparently occurs by inde-pendent processes. From Fig. 5 it can be seenthat the synthesis of the low-molecular-weightRNAs, like leader RNA, is resistant to psoralenphotoinactivation. The findings suggest furtherthat a site which is highly sensitive to psoralenmodification may be located 5'-distal to theleader template sequence, beyond the promoterregion ofN gene transcription. Further work onthe precise location of psoralen binding sites inviral genomes in situ should help to elucidatethe mechanisms of gene regulation.

    ACKNOWLEDGMENTSWe thank Robert Krug for providing purified influenza

    virus and the gel analyses of the influenza RNA products andYasuhiro Furuichi for valuable discussion.

    ADDENDUM IN PROOFIndependent transcription of individual influenza

    mRNA species has also been reported by G. Abraham(Virology 79:177-182, 1979).

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    3. Baltimore, D., A. S. Huang, and M. Stampfer. 1970.Ribonucleic acid synthesis of vesicular stomatitis virus.II. An RNA polymerase in the virion. Proc. Nati. Acad.Sci. U.S.A. 66:572-576.

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