Incomplete splicing and deficient accumulation of the fiber messenger RNA in monkey cells infected by human adenovirus type 2

J. Mol. Biol. (1980)

139, 221-242

Incomplete Splicing and Deficient Accumulation of the Fiber Messenger RNA in Monkey Cells Infected by Human Adenovirus Type 2 DANIEL

F. KLESSIG

AND LOUISE

T. CHOW

Cold Spring Harbor Laboratory P.O. Box 100 Cold S$ng Harbor, N. Y. 11724, U.S.A. (Received 8 October 1979, and in revised form

19 December 1979)

Monkey cells are non-permissive hosts for human adenoviruses due to a block in the synthesis of certain late viral proteins. Analysis of the RNA produced in abortively infected monkey cells indicated that the depressed synthesis of many of the late proteins can be ascribed to the reduced concentration of their corresponding mRNAs (Klessig & Anderson, 1975). An exception is the capsid polypeptide, fiber. Fiber RNA concentration is reduced by five- to ZO-fold in monkey cells, whereas fiber protein synthesis is depressed by 100 to lOOO-fold. Experiments designed to detect defects in the cytoplasmic fiber RNA which would account for its poor translatability showed that the RNA had a normal, capped tripartite leader at its 5’ terminus. However, a large proportion of these molecules contained long sequences between the tripartite leader and the main body which failed to be spliced out of the initial transcript during maturation of the fiber mRNA. Similarly, region 3 RNAs at late times also contained the same long upstream sequences in abortive infections. In contrast, few cytoplasmic fiber or region 3 RNAs from a productive infection with an adenovirus type 2 mutant, which grows lytically in monkey cells, retained such long upstream sequences. The other late RNAs from abortively or productively infected monkey cells had splicing patterns identical to those seen in permissive human cells. These observations suggest a role for RNA splicing in the control of gene expression and may represent an example of limited host range of a virus due to deficient RNA splicing.

1. Introduction The use of phage to study bacterial host functions is well-documented. Similarly, viruses can serve as probes to understand the intricate controls governing the molecular machinery of a eukaryotic cell. Viruses offer the advantages of small, well-defined genomes and the availability of viral mutations that affect their replication, transcription, or translation processes. In addition, by placing a virus in alternative host environments the normal processes occurring during the lytic cycle can be perturbed. The complex processes taking place within the cell can then be dissected to aid in our understanding of these events, much as cellular mutants have helped define many biosynthetic and degradative pathways. Growth of human adenoviruses is suppressed in monkey cells, and results in a lOOO-fold reduced yield compared to infections in human cells (Rabson et al., 1964). 221 0022-2836/80/140221-22.

$02.00/O

0 1980 Academic

Press Inc. (London)

Ltd.

222

I).

F.

KLESSIG

AND

1,. T.

CHOW

This provides a convenient system to study the mechanisms employed by eukaryotic systems to control the expression of viral as well as host genes. The ability to manipulate this system t,o allow efficient multiplication of the virus in monkey cells permit,s the study of the molecular events within the same cell type under both permissive and non-permissive conditions. Specifically, the block to viral multiplication can bc overcome by providing a function which is encoded by a small segment, of the SV4Ot genome located at t,he distal end of the early region. This information can be supplied by coinfection with SV40 (Rabson et al., 1964) or by SV40 DNA which is integrated either into the host’ genome, as in SV40-transformed monkey cells (Hashimoto et ul., 1973). or into the infecting human adenovirus genomc, as in several Ad2-SV40 hybrid viruses such as Ad2+NDl (Lewis et al., 1969; Grodzicker et al., 1976; Fey et uZ., 1979; Lukanidin, Sambrook, Lewis & Grodzicker, unpublished results). Alternatively, a mutation in a segment of the Ad2 genome which encodes the DXAbinding protein results in the efficient growth of the mutant (Ad2hr400, abbreviated hr400) in monkey cells (Klessig, 1977a; Klessig & Hassell, 1978: Klessig & Grodzicker, 1979). The nature of the block to the growth of human adenovirus in monkey cells has not been understood clearly. Adsorption and entry appear normal (Feldman et aZ., 1966), and both the time of onset and the rate of viral DNA synthesis are similar t,o those seen in productive infections (Friedman et al.: 1970; Hashimoto et al., 1973; Reich et al., 1966). The early viral proteins (T-antigen and the 72,000 M,, (72K) DNBbinding protein) that have been analyzed are found in normal amounts (.Feldman et al., 1966; van der Vliet & Levine, 1973). In contrast, the synthesis of many viral late proteins is variably reduced (Baum et al., 1972; Friedman et aZ., 1970; Klessig & Anderson, 1975). The depression in synthesis of most’ of t’hese late proteins can be attributed to the decreased concentrations of their corresponding mRNAs (Klessig & Anderson, 1975; Farber & Baum, 1978). However, the five- to 20-fold lowe concentration of the fiber gene transcripts cannot account for the 100 to lOOO-fold reduction in the synthesis of the fiber protein (Klessig & Anderson, 1975). We now report our studies to examine the cause for the anomalously low level of fiber protein in abortively infected monkey cells.

2. Materials and Methods (a) Cells

ad

viruses

CVl cells, an established line of African green monkey kidney cells, were obtaIned from J. Mertz. The human cell line HeLa was obtained from J.. F. Williams. CVl ceh were cultivated in Dulbecco modified Eagle minimal essential medium (DMEM) (cat,alog no. 11305; Microbiological Associates) supplemented with 5 y0 fetal bovine serum (Reheis Chemical Co.), 100 pg of streptomycin/ml (Sigma Chemical Co.), and 100 pg of penicillin/ml (Sigma). Suspension cultures of HeLa cells were grown in Eagle F13 medium (Grand Island Biological Co.) supplemented with 50/b horse serum (Reheis). Ad2 was originally obtained from U. Pettersson, and Ad2+NDl and 140 were provided a host range mutant of Ad2 by T. Grodzicker. These 3 viruses, as well as Ad2hr400, (Klessig, 1977a), were propagated in suspension cultures of HeLa cells. SV40 strain 776 was obtained from J. Sambrook and prepared as previously described (Sharp et al., 1973). t Abbreviations units.

used: SV40, simian virus

40; Ad2, adenovirus

type 2; p.f.u., plaque-forming

INCOMPLETE

SPLICING (b) RNA

OF FIBER

mRNA

223

preparation

The preparation of total cytoplasmic RNA from monkey cells infected with 5 to 10 p.f.u.! cell was performed as previously described (Klessig & Anderson, 1975). Viral RNA for electron microscopic analysis, cell-free protein synthesis, or structural analysis of the 5’ termini was purified by solution hybridization to intact Ad2 DNA or its restri&ion enzyme fragments, using the methods of Lewis et al. (1975). (c) Cell-free

protein

synthesis

ddcnovirus proteins were synthesized in a fractionated mammalian cell-free programmod with purified fiber mRNA or with total cytoplasmic RNA from cells as previously described by Lewis et al. (1975). (d) Analysis

of viral

syst.em infected

protein

‘I’11o synthesis of viral proteins in viva was assayed by labeling the cells with [35S]. methionino for 1 h at various times post infection and analyzing the products by sodium dodeoyl sulfate/polyacrylamide gel electrophoresis (Anderson et al., 1973). (e) Immunoprecip&tion

of$ber

Tho lato viral proteins were labeled between 36 and 38 h after infection with 5 to 10 p.f.u./cell by addition of 100 to 200 &i of [35S]methionine in 1 ml of DMEM minus by scraping methioninc: per 100 mm x 20 mm dish of CVl cells. The cells were harvested in phosphate-buffered saline (PBS), resuspended in 100 ~1 of PBS and 300 pg phenylmethylsulfonyl fluoride (PMSF)/ml, and disrupted by freeze-thawing and sonication. After treatment with 30 pg of RNAase A/ml and 20 pg of DNAase I/ml in 5 m&I-MgCl, at room temperature for 10 min, the lysate was diluted with 900 ~1 of 10 mM-sodium phosphate (pH 6.8), 150 mM-NaCl, 1 M-urea, 1% Triton Xl00 and 300 pg PMSF/ml (buffer A) and centrifuged 10 min at 12,000 g or 2 h at 80,000 g. Fiber from varying amounts of the supernatant was precipitated in buffer A using anti-serum prepared against purified native fiber (a generous gift from C. Anderson) and unlabeled late viral proteins as carrier to maintain the proper fiber concentration for efficient precipitation. After incubation at 37°C for 30 min, the reaction mixture was centrifuged for 10 min at 12,000 g and the pellet was washod twice with buffer A. The precipitate was analyzed by sodium dodecyl sulfate/ polyacrylamidc gel electrophoresis. (f) Electron

microscopic

unalysis

of late viral

RNAs

Late viral RNA was hybridized to denatured Ad2 DNA and prepared for electron microscopic analysis as described previously (Chow & Broker, 1978). +X174 DNA and open circular ColEl were included as single and double-strand length standards, respectively. Electron micrographs were taken with a Zeiss EM 1OA electron microscope. Molecules were traced and data analysis was performed as described by Chow & Broker (1978). (g) Biochemical

analysis

of late viral

RNAs

The 5’ termini of the fiber and 100 K mRNAs were analyzed as described by Klessig (19773). The different size classes of fiber RNAs were determined as follows: polyadenylated, cytoplasmic RNA prepared from CVl or HeLa cells 36 or 24 h post infection, respectively, was fractionated by electrophoresis for 16 h at 25 mA on a 20 cm x 20 cm x 0.3 cm, 1.6% agarose gel containing 5 to 10 mM-methylmercuric hydroxide (Bailey & Davidson, 1976). The RNA was transferred to diazobenzyloxymethyl (DBM) paper according to the method of Alwine et al. (1977). The length of treatment of the gel with 50 m&r-NaOH, 5 m&I-2-mercaptoethanol was increased to 90 min to enhance RNA hydrolysis and thus the transfer of large RNA, The resulting “RNA blot” was hybridized to a cloned segment of DNA consisting of the plasmid pBR322 into which a BamHI

I>. F. KLXSSIG

224 fragment personal between

AND

I,.

T. (‘HOW

of the Ad%SV40 hybrid virus, Ad2+NDldpl (T. C:rodzickc-r CA J. Sumbrook, communication) was inserted. This fragment cont,ains Ad2 scqu~~ncrs c~~cod~tl co-ordinates 86 and 89 and is flanked on both sides by 200 to 400 base-pairs of’

SV40 DNA.

3. Results (a) Production

qf$ber protein and its messesqrr KNA

in abortive infectio?ls

The synthesis of many of t’he late viral proteins is significantly reduced in abortirc infections or when RNA from these infections is used to program a cell-free t)rannlation system. The synthesis of fiber, however. could not be det,ected in abortively infcct,ecl cells, nor in cell-free systems directed by RNA isolated from these cells (Klessig KS however, was hampered due to a Anderson, 1975). The sensitivity of detection, moderate background caused by host polypeptides which migrat,ed similarly to fiber in sodium dodecyl sulfate/polyacrylamide gels. To overcome t,his problem, fiber synthesized in viva was immunoprecipitated using rabbit’ antibodies prepared against the native fiber. Figure 1 shows an autoradiogram of in vi,vo labeled polypeptides that were fractionated by electrophoresis before and aft,er immunoprecipitation with antifiber serum. Although there was some non-specific precipitation, fiber was grcaat)ly purified from other polypeptides of similar molecular weight. Quantitation by densitometry of the autoradiogram indicat,ed t,hat the synthesis of fiber was between 100 and lOOO-fold lower in infections of monkey cells wit)h Ad2 alone than in those with Ad2 +NDl, Ad2hr400 or Ad2 plus SV40. Immunoprecipitation of fiber synthesized in a cell-free system was unsuccessful. A second approach bo overcome the background problem was at#tempted. Fiberspecific RNAs were selected from cytoplasmic RNA prepared from abort,ively (Ad2) or productively (hr400) infected cells by hybridization t’o EcoRI E (co-ordinat’c+ 83.4 to 89.7), a rest,riction fragment containing bhe main body of t,he fiber gene (coordinates 86.2 to 91.3) (Chow & Broker. 1978; Lewis c:t al.. 1975). The purified mRR’LZ was then translat,ed in a mixed mammalian cell-fret> system (Lewis et al.. 1975). Figure 2 shows that there was translatable fiber mRNA in Ad2-infected monk(‘,v cells, although it was in much lower amount, (at least, 5 to IO-fold) than found i11 hr400-infect,ed cells. Precise quantitation of the rclativt, levels of translatable mRNA in the two t,ypes of infection was not, possible because t,hc efficiency of selection of t IW mRNA by hybridization depends on the relative concent’ration of RN,4 c~nlplementary to the fiber gene, which is five- to 20-fold lower in abortJive infections as compared with productive infections (Klessig & Anderson. 1975). Thus; this method could overestimate the relative amount, in abortive infections, particularly if tih:~ mRNA from productive infections was in excess during h.ybridization. That this wan probably the case was suggested by experiments in which total cptoplasmic mRh’A from a productive infection was diluted lo-fold with RNA from uninfected cells: synthesis of fiber was still readily observed (data not, shown). This indicates that the relative abundance of translatable message in productive ‘L’W~SUS abortive infections differs more than lo-fold. However, it, cannot be ruled out t,hat the fiber mRNA from abortive. but not) productive infect’ions may competca poorly \zith other mltNAs fol the ribosomes of the host or the cell-free system. Only whclt thcsc* compotinp 1-1X14x are removed might, the fiber mRNA be efl’icient,ly translated.

INCOMPLETE

SPLICING

OF

FIBER

225

mRNA

Total lysate Immunopreclpltate hr400

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Fro. 1. Quantitation of the fiber production in monkey cells. The late viral and cellular proteins were labeled between 36 and 38 h post infection with [35S]methionine. A portion of the labeled cell lysate was directly analyzed by electrophoresis on a sodium dodecyl sulfate/polyacrylamide (17.5%) gel, while another portion was first immunoprecipitated with serum made against native fiber. The lysate from productive infections (hr400, Ad2+NDl(NDl), or Ad2 plus SV40) were diluted 0, 10, 100 or 1000.fold (1, 10-l, 10-a, 1O-3). These diluted lysates and the undiluted lysates from abortive infections were immunoprecipitated in the presence of unlabeled late viral proteins from productive infections as carrier to maintain the proper fiber concentration for efficient precipitation. The amount of labeled fiber was determined by densitometry of the resulting autoradiogram. Exposures of varying duration were used to ensure a linear response of the X-ray films. In addition, the density of the fiber band from Ad2 infections was also directly compared on the same exposure with one of the dilutions from each productive infection where the band intensity was similar. This eliminated the problem of variation in film response and development from exposure to exposure. Analysis of 3 experiments with 2 different sets of lysates indicated that fiber synthesis was reduced 200 to 1000.fold in Ad2 compared to Ad2 plus SV40 co-infected monkey cells. The synthesis of fiber in hi-400 or Ad2 +NDl-infected ceils was 1.5 to 3.fold lower than in Ad2 plus SV40 co-infections.

226

1).

Fiber

F.

KLESYIG

AND

L.

T.

CHOW

-

FIG. 2. Cell-free synthesis of fiber. The fractionated mammalian cell-free transla.tion synttmI was programmed with total cytoplasmic RNA from Ada-infected human cells, ikhwichiu coli ribosomal RNA (rRNA), or hybridization-purified fiber mRNA isolat.ed from Ad2 nt‘ hr4UOinfected monkey cells. The proteins synthesized in vitro were fractionated on a sodium tlodrcyl sulfate/polyacrylamide (17.5%) gel and visualized by autoradiography.

INCOMPLETE

SPLICING

OF FIBER

(b) Presence of a capped 5’ oligonucleotide

mRNA

227

in fiber RNA

One possible explanation for the poor translatability of fiber mRNA in aboitive infections might be that this mRNA lacks the 5’-terminal 7mG5’ppp5’N1mNz cap which is found on most eukaryotic mRNAs and is required for their efficient translation (for a review, see Shatkin, 1976). To test this possibility, fiber mRNA was isolated from abortively (Ad2 or 140) or productively (Ad2+NDl) infected cells by hybridization to the EcoRI E fragment. To ensure that we were looking only at the fiber mRNA rather than other RNAs complementary to EcoRI E, RNAs from Ad2+NDl or 140-infected as well as Ad2-infected monkey cells were examined. Ad2+NDl is a non-defective Ad2-SV40 hybrid virus which grows well in both human and monkey cells. In this virus, a small segment of the Ad2 genome between co-ordinates 80 and 86 (which includes the non-fiber part of the region covered by the EcoRI E fragment) is replaced by a SV40 sequence encoded between co-ordinates 28 and 11 on the SV40 map (Lewis et aE., 1969; Kelly & Lewis, 1973). Virus 140 is a point mutant of Ad2 +NDl which grows normally in human cells but no longer multiplies efficiently in monkey cells (Grodzicker et al., 1974,1976; Gesteland et al., 1977). The mRNA for the 1OOK protein was chosen as a positive control to show that at least some of the late viral mRNAs synthesized in abortive infections are capped. The 1OOK mRNA which is in the same transcriptional unit as fiber RNA (cf. Fig. 9), was analyzed after selection by hybridization with EcoRI F (co-ordinates 70.7 to 75*9), a restriction fragment partially encoding its main body which lies between co-ordinates 66 and 78 (Chow & Broker, 1978). The similar level of synthesis of both the 1OOK mRNA and its corresponding protein in the two types of infections (Klessig & Anderson, 1975) suggests that it is likely to be capped. These purified RNAs were digested with RNAase T, and chromatographed on dihydroxyboryl cellulose to select for the capped 5’ oligonucleotide (Gelinas & Roberts, 1977; Klessig, 1977b). The 1OOK and the fiber mRNAs isolated from AdS-infected human cells contain the same capped T, oligonucleotides 7mG5’ppp5’AmC(mU (C,U,)G (differing only in the degree of methylation) that are part of a common tripartite leader sequence encoded upstream from the main bodies of these genes (Chow et aZ., 1977a; Klessig, 19773). The other late mRNAs t,hat are encoded between co-ordinates 29 and 92 and transcribed in a rightward direction also contain this tripartite leader encoded at 16.6, 19.6 and 26.6 (Chow et al., 1977a; Berget et al., 1977; Chow & Broker, 1978). Figure 3 shows that, the fiber and 1OOK mRNAs contained the same capped T, oligonucleotides (spots denoted by arrows in Fig. 9) in both abortively and productively infected monkey cells as were found in human cells. These T, oligonucleotides were identified by their partial resistance to RNAase T, (an RNAase that degrades RNA to 3’ nucleoside monophosphates in the absence of methylated 2’-hydroxyl groups on the riboses) and the presence of lmG. Although accurate quantitation of the relative amounts of capped oligonucleotides was not possible by this method because of the low amount of fiber RNA in abortive infections and the lengthy procedures involved, the intensities of the spots could be used for approximation. For the 1OOK mRNA they were about equal in the abortive and productive infections, just as were the levels of 1OOK mRNA. In contrast, the amount of these oligonucleotides from fiber RNA isolated from abortive infections

8

El40

I

F140

z pH 3.5

FIG. 3. Dihydroxyboryl (DBAE)-cellulose-selected T, oligonucleotides from fiber and 100 K mRNAs. 3ZP-labeled fiber and 100 K mRNAs were selected from total cytoplasmic RNA prepared from 140 or Ad2 +NDl-infected CVl cells by solution hybridization to the EcoRI E and EcoRI F restriction fragments, respectively. The hybridized RNA was chromatographed on DBAEcellulose after RNAase T, digestion, and the bound material was fractionated by electrophoresis on cellulose acetate at pH 3.5 in the 1st dimension and by homochromatography (Homo) in the

INCOMPLETE

SPLICING

OF

FIBER

mRNA

229

was greatly depressed when compared to productive infections. This again parallels the reduction in the level of RNA complementary to the fiber gene and suggests that the amount of cap associated with the RNA from the different infections is proportional to the concentration of that RNA. Thus, the absence of a cap or a marked reduction in the proportion of capped to uncapped fiber RNAs is unlikely to be the explanation for the discrepancy between the levels of depression of RNA concentrations and protein production. Because of the severe reduction of the fiber RNA in abortive infections, we cannot rigorously exclude the possibility that the capped oligonucleotides observed in the fiber RNA preparation from abortively infected cells are contaminants from other late RNAs that contain the same oligonucleotides. (c) Normul

splicing

of most early and lute transcriptts

in monkey CVI ceUs

To determine if the late viral RNAs made in monkey cells were spliced in patterns similar to those observed in human cells and to determine the differences in splicing, if any, during productive and abortive infections, cytoplasmic RNA from hr400 or Ad2-infected CVl cells was hybridized to intact Ad2 DNA under conditions which favored the formation of RNA:DNA heteroduplexes over DNA:DNA homoduplexes (Thomas et al., 1976; White & Hogness, 1977; Casey & Davidson, 1977). The resulting heteroduplexes were then subjected to electron microscopic analysis as described previously (Chow & Broker, 1978). For the majority of the late r-strand RNAs, the splicing patterns in abortively (Ad2) and productively (hr400) infected monkey cells are indistinguishable from those seen in human cells. Most molecules had the common tripartite leader at co-ordinates 16.6, 19.6 and 26.6 (Chow et al., 1977a; Berget et al., 1977; Chow & Broker, 1978) (Fig. 4). A variable proportion of the mRNAs had an extra leader segment from co-ordinate 22,O to 23,2 interposed between the second and the third leader as was seen in human cells infected with Ad2, Ad3, Ad7 or Ad2+NDl (cf. Fig. 5(c)) (Chow et al., 1979a,b; Kilpatrick et al., 1979; Chow, Lewis, Sambrook, Grodzicker & Broker, unpublished results). RNAs from early regions lA, lB, 3 and 4 (cf. Fig. 9) that were occasionally observed in our late RNA preparations also had splicing patterns identical to those seen in human cells (Chow et al., 1979a). Also present were the early and the late forms of Z-strand mRNAs for the DNA binding protein (encoded by early region 2) with a leader sequence at co-ordinates 75.1 (early), 72.0 (major late) or 86.7 (minor late) (Chow et al., 1979a). (d) Incomplete

q&king

of the fiber transcript

Unlike the other r-strand mRNAs, fiber RNA, which is the promoter-distal member of the major late transcriptional unit, was not only present in lower quantities in abortive infection but had dramatically different patterns of splicing in productively and abortively infected monkey cells compared to human cells, when cytoplasmic RNA preparations enriched for fiber sequence were examined by electron microscopy. 2nd dimension. The capped 5’-terminal oligonucleotides (indicated by arrows) were the only 7mGMP and were partially resistant to RNA&se T,. All other oligonucleotides which contained oligonucleotides having 4 or more phosphates were digested to 3’ nucleoside monophosphates with RNAase T, (for details, see Klessig, 19776). Capped oligonucleotides from fiber RNA prepared from AdB-infected CVl cells have been analyzed similarly; the results were identical to those from 140-infected CVl cells.

230

D. Ir’. KLEGYIG

AND

L. 1’. CHOW

FIG. 4. Heteroduplexes of Ad2 DNA with late r-strand RNAs from CVl cells. Late Ad2 T-&ml mRNAs in CVI cells have the tripartite leaders as seen in HeLa cells. The leader segments src labeled 1, 2 and 3. The 3’ ends are so indicated. L and R denote the left and right termini of the Ad2 DNA, respectively. (a) Fiber RNA (co-ordinates 86.3 to 91.3) with additional ancillary leader segments y (co-ordinates 78.6 to 79.1) and z (co-ordinates 84.7 to 851). (b) 100 K mRNA (co-ordinates 66.4 to 73.7/74.1 t,o 78). The small splice near co-ordinate 74 (0) did not seem t(J exist in some of the 100 K RNAs.

INCOMPLETE

SPLICING

OF

FIBER

231

mRNA

In human cells the fiber mRNA with the tripartite leader has several forms which differ from one another by the presence or absence of different combinations of three additional ancillary leader sequences encoded at co-ordinates 77.2 to 77.6 (x), 78.6 to 79.1 (y), and 84.7 to 85.1 (z) (Chow & Broker, 1978). The y leader at 79 is very common and can be found on up to 25% of the fiber mRNAs while the other two leaders are very rare (<2%). In monkey cells, the fiber RNA had a normal tripartite leader; but the x, y and z leaders were much more common, and 48% to 70% or 77% to 85% of the fiber RNA molecules in hr400 and Ad2 infections, respectively, contained at least one of these ancillary leaders (Fig. 4(a)) (Table 1). Most striking, particularly in

TABLE I Analysis

Time$

Experiment

1. Ad2 hr400 2. Ad2 hr400 3. Ad2 hr400 4. Ad2 hr400

-

of additional leader sequencest on Jiber transcripts in infected monkey cells

- 1%)§

Y (%)

(h) 30 30 36 36 36 36 48 48

XY (%)

2 (%)I1

+ (%)a

1.8 6.1 -

17.9 28.8 18.4 13.4 18.0 17.6 23.1 21.4

48.2 12.0 50.0 7.7 54.0 5.3 50.0 7.1

23.2 30.3 21.1 44.2 13.8 52.6 19.2 38.1

8.9 22.7 10.5 28.9 10.2 21.0 7.7 26.2

5.8 4.2 3.5 7.1

Total scored 112 66 38 52 167 57 18 42

t All transcripts have the common tripartite leader. The presence or absence of additional leader segments are listed in the Table. The map co-ordinates of short leaders z, y and z have been taken from Chow & Broker (1978) and are described in the text. Leader y is open in one reading frame but has no initiation codon and is not translated (Zain et al., 1979; Dunn et al., 1978). It is not known whether leaders 2 and z have initiation codons, open reading frames, or termination codons. $ Time of RNA harvest after virus infection of monkey CVl cells. 0 Mature fiber RNA with only the tripartite leader. I/ All short ancillary leader combinations including the z component have been pooled. These include z, zz, yz and zyz. 11 Includes all transcripts with longer upstream sequences as described in the text.

abortive (Ad2) infections, is the preservation of long upstream sequences which failed to be spliced out of the initial transcript during processing of the fiber mRNA. RNA molecules containing these sequences fell into three heterogeneous classes that differed in the length of the preserved upstream sequences. Class I fiber RNAs were most abundant and contained sequences from co-ordinates approximately 66 to 7’7.6 with or without the ancillary leaders at 79 and/or 85 (39 molecules measured). Many of these RNAs contained a small deletion at co-ordinate 73.5 to 74. Class II RNAs were similar to class I except the preserved sequence was shorter and mapped eit,her from 72.5 to 77.6 or from 74 to 77.6 (18 molecules measured). Class III RNAs were infrequently observed. They retained sequences from co-ordinates 66 or 68 and 77.6, but, with some sequences between these co-ordinates removed (12 molecules

FIG. 5. Heteroduplexes of Ad2 DNA with incompletely spliced Ad2 fiber RNA in CVI cells. Molecules (a) and (b) are class I RNAs. Molecule (c) is a class II RNA with a 4-part loader; the extra leader segment between the 2nd and 3rd leader maps at co-ordinates 22 to 23.2. The DNA loops are indicated by arrowheads in the electron micrographs; (a’), (b’) and (c’) are interpretiw~

(b’)

tracings of (a), (b) and (c), respectively; (--) DNA; (- - - -) RNA. The largest DNA loop in (c) had a duplex region but was not traced in (c’). Other notations are the same as in Fig. 4. The co-ordinates for the extra sequences are (a) 66.8 to 77,6/z; (b) 66.4 to 73.7/74.1 to 77,6/y/z; (c) 72.6 to 73.6/73.9 to 77.6/y. They are approximated in the tracings for simplicity. i, denotes the extra leader of co-ordinates 22.0 to 23.2 between the 2nd and the 3rd leader segments.

FIG. 6. Heteroduplexes of Ad2 Molecuies (a) and (b) are class III larked due to pairing of the terminal initiated from the late promoter at (which would have been the main

DNA with incompletely spliced fiber and region 3 RNAs. incompletely spliced fiber RNAs. Both molecules have oircuinverted duplication (TID). (c) and (d) Early region 3 RNAs co-ordinates 16.45. In (c) and (d) the last short RNA segment body if properly spliced) maps at co-ordinates Y4.7 to 86.0. III

(b')

(c’)

(d')

(c) the first leader at 16.6 failed to hybridize. In (d) the first leader was pulled away from its DNA complement during preparation of the microscopic grid. (a’), (b’), (c’) and (d’) Interpretative tracings of (a), (b), (c) and (d), respectively. Other notations are the same as in Figs 4 and 5. The extra sequences preserved have co-ordinates: (a) 65-5 to 66.2/72-7 to 77.6/y; (b) 66.5 to 67.3/77.4 to 78; (c) 72.8 to 77.6; (d) 66.3 to 77.6.

D.

236

F.

KLESSIG

AND

L.

T.

CHOW

measured). Of a total of 192 incompletely spliced fiber RNAs scored in abortircl infections, t,he relative abundances of classes I. II and III were approximateI! 12:4:1. All the class I and II structures seen in abortive infections were also present in productive infections, alt,hough at a much lower frequency (see below). Selected electron micrographs of these incompletely spliced molecules are presented in Figures 5 and 6(a) and (b). Schematic representations of t,hese and additional molecules are shown in Figure ‘7. In t,he first two classes the additional sequences are equivalent, to an entire gene (100K for class I, 33K. or pVII1 for class II) (Chow & Broker. 1978: Chow et al., 1979a,b) upstream to the fiber sequence, t,hus forming a polycistronic mRNA. Our current knowledge of translation by eukaryotic ribosomes indicates that only the 5’ message of a polycistronic RNA is t.ranslated (Kozak> 1978). Therefore, we suspect that these molecules cannot be used for the synthesis of the fiber protein. The various species of fiber transcripts, with or without, long upstream sequences, were scored visually with electron microscopy in several blind experiments using fiber RNA isolated from monkey cells infect,ed with Ad2 or hr400 at various times between 30 and 48 hours post infection. Transcripts cont’aining the long upstream sequences represent,ed a significantly greater proportion of fiber-cont’aining RNA molecules from Ad2 (50%) compared to hr400 (5 to 12°,&)-infectcd monkey cells: irrespective of the time of RNA preparation (Table 1). 100 K, 33K

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FIG. 7. Graphic representation of Ad2 fiber RNAs in CVl cells. All RNAs have oither the tripartite leader or the 4-part leader as shown in Figs 4(a) and 6(c). The extra RNA segments conserved in classes I, II and III incompletely spliced molecules are upstream from the fiber main body. They cover the genes for lOOK, 33K, pVII1 and the early region 3 as indicated. Some molecules observed are not included; they usually differ by the presence or absence of leader y CIT z or the small splice near 74.

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mRNA

237

Early region 3 occupies a unique position on the Ad2 genome. It is embedded in the late transcriptional unit between the 1OOK and fiber mRNAs and is transcribed in the same direction. At early times after infection, region 3 has its own promoter near co-ordinate 76.6 and generates a family of RNAs with different splicing patterns (Kitchingman et al., 1977; Berk & Sharp, 1978; Chow et al., 1979a,b) At intermediate to late times, some region 3 RNAs are produced from the major late promoter at 16.45 (Ziff & Evans, 1978) and have the common tripartite leader linked to coordinate 77, 79 or 85, coincident with the sites where the tripartite leader is linked to the fiber ancillary leaders x, y and z (Chow et al., 1979a,b; Broker & Chow, 1979). This sequence identity has been interpreted as a response by the fiber mRNA maturat,ion processes to the early splicing signals that are similar at early and late times: (Chow et al., 1979aJ; Broker & Chow, 1979). Similar to the observation in human cells, some region 3 RNAs were found to have tripartite leaders 30 to 48 hours after infection of monkey cells with Ad2 or hr400. As was seen with fiber RNA, some of these retained long sequences between co-ordinates 66 (major), 72.5 or 74 (minor) and 77.6 (Figs 6(c) and (d), and 9); such long region 3 RNAs were present at much higher proportion in Ad2-infected than in hr400-infected cells (data not shown). We do not know whether region 3 proteins are also much depressed at late times in abortive infections, because region 3 mRNAs are also synthesized from the early promoter at late times. The incompletely spliced fiber molecules could also be detected biochemically. Polyadenylated, cytoplasmic RNA isolated at late times (24 to 36 hours post infection) from AdB-infected human cells or monkey cells infected with Ad2 or hr400 were fractionated by electrophoresis in an agarose gel containing methylmercuric hydroxide as a denaturing agent. After transferring the RNA to diazobenzyloxymethyl (DBM)paper by the method of Alwine et al. (1977), the filter was probed with a recombinant plasmid containing the main body of the fiber gene (co-ordinates 86 to 89) (Klessig, unpublished results; also see Materials and Methods). In addition to the lower molecular weight mature fiber mRNAs, two much longer and slightly heterogeneous size classes of RNA were detected (Fig. 8). These presumably corresponded to the first two classes of incompletely spliced fiber RNA defined by the electron microscopic analysis. These larger RNAs were also detected biochemically and by electron microscopy in the cytoplasm of AdB-infected human cells, but accounted for less than 1% of the total fiber RNAs. In hr400 and Ad2-infected monkey cells, they represented 5 to 10% and 15 to 300/,, respectively, as determined by densitometry of the autoradiogram. The concentration of these incompletely spliced molecules (per pg of total cytoplasmic RNA) was similar in Ad2 and hr400-infected monkey cells (Fig. 8). The completely spliced molecules represented a higher percentage of the total fiber RNA in abortive infections when analyzed by RNA blots rather than by electron microscopy. This discrepancy probably results from an overestimation of the amount of mature fiber mRNA in Ad2 preparations beeause of the presence of an RNA of similar size encoding the DNA binding protein. This DNA-binding protein mRNA has a leader sequence between co-ordinates 86.7 and 86.2 (Chow et al., 1979a,b) which is homologous to the probe used for hybridization. Because mature size fiber is present in much reduced levels in abortive infections, this mRNA contamination could significantly alter the quantitation in abortive but not productive infections.

238

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FIG. 8. Different size classes of RNA that contain the fiber squences. Equal amounts of polyadenylated, cytoplasmic RNA isolated from CVl cells 36 h after infection with 5 to 10 p.f.u. of Ad2 or hr400, or from HeLa cells 24 h after infection with 20 to 30 p.f.u. of Ad2 were fractionated on a methylmercuric hydroxidelagaroso gel. After transfer to DBM paper, the RNA was hybridized to a Y’-labeled cloned fragment of DNA which contain Ad2 sequences between co-ordinates 86 and 89 and is homologous to only the main body of the fiber. The bands of higher molecular weights seen on the autoradiogram presumably corresponded to the class I and IIfiber RNAs that were shown by electron microscopy to contain long sequences located between the tripartite leader and fiber gene. The lengths of class I and II RNAs were 5.3 and 3.2 to 3.0 x 1 O3 base-pairs, respectively, as determined by comparison to single-stranded Hind111 fragments of the Add genome which were used as size markers on the same gel. They are shorter than the 6 and 3.3 to 3.7 x lo3 base-pairs determined by electron microscopy. The reason for this discrepancy is, perhaps, that high molecular weight RNAs and single-strand DNAs migrate at different rates under thcsc conditions.

INCOMPLETE

SPLICING

OF

FIBER

mRNA

239

4. Discussion The nature of the block to adenovirus multiplication in monkey cells has been further characterized. A striking feature was the greater than loo-fold reduction in the fiber protein synthesis in abortive infections; other late proteins were reduced modestl.y, or not at all. Similar patterns of reduction in viral protein synthesis were found when RNA from AdB-infected monkey cells was used to program a cell-free translation system. Most notable was the inability to produce detectable levels of fiber. Analyses of both the complexity and the amounts of adenoviral-specified RNA in abortively infected monkey cells show that all species of late viral RNA are present, but the concentrations of several species are reduced up to fivefold. This correlates well with the reduced synthesis of the corresponding proteins (Klessig & Anderson, 1975; Klessig, unpublished results). An exception is the fiber protein. The five- to 20-fold decrease in the level of the cytoplasmic fiber RNA cannot account for the 100 to lOOO-fold reduction in the fiber protein. We have attempted to detect defects in the cytoplasmic fiber RNA which would explain its poor translatability. Our results showed that the fiber RNA contained a normal cap and a normal tripartite leader at its 5’ terminus. However, a large proportion of these molecules was blocked in processing in abortive infections and retained long sequences between the leader and the main coding region that normally are spliced out of the primary transcript (Fig. 6; Table 1). The long extra sequences retained in the fiber transcripts generally constitute a second gene (lOOK, 33K or pVII1; cf Figs 7 and 9) which is located 5’ to the fiber sequences, thus forming a polycistronic mRNA. Since eukaryotic ribosomes appear to translate only the 5’ message of a polycistronic RNA (Kozak, 1978), these molecules probably cannot be utilized for the synthesis of the fiber protein, The same incompletely spliced RNAs were also found in similar concentration (per pg of cytoplasmic RNA), but represented a lower percentage of the fiber transcripts in productively infected monkey cells (Fig. 8; Table 1). We feel that these long fiber molecules might represent intermediates in the processing pathway of the fiber mRNA. In abortive infections, most primary transcripts destined to become fiber RNA were trapped as these processing intermediates due to delays or blocks in further processing. The more severe reduction of the fiber transcripts as compared to other late RNAs in the same transcriptional unit in the abortive infection might reflect a greater instability of the incompletely spliced molecules. Alternatively, it could be due to premature termination of transcription or an imbalance in polyadenylation of t)he primary transcript which normally results in the equitable distribution of the r-strand transcription into five major families of late RNAs (Chow et al., 19776; Chow & Rroker 1978; McGrogan & Raskas, 1978; Nevins & Darnell, 1978; Fraser $ Ziff, 1978). At late times many of the RNAs encoded just upstream from the fiber gene in early region 3 (co-ordinates 76 to 86) also contained the same long sequences as fiber RNA, especially in abortive infections. This suggests that there is a general defect in splicing in the region between 66 and 77.6 which affects all mRNAs encoded downstream. The splicing patterns of the other late viral mRNA.s made in monkey cells were normal. A comparison of splicing patterns seen in region 3 and the fiber region in infected human and monkey cells is presented in Figure 9.

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FIG. 9. Comparison of splicing patterns of Ad2 RNA in human (HeLa) and monkey (CVl) cells. A simplified transcriptional and translational map is presented at the top of the Figure. Thin arrows depict early regions; thick arrows depict late regions. Arrows also indicate the direction of transcription with arrowheads placed at the 3’ termini of the families of RNAs. There arc 5 early regions, ElA, ElB, E2, E3 and E4, each with its own promoter. All late r-strand mltNAs downstream from 16 initiate from the same promoter at co-ordinate 16.45 and have the tripartite. leaders (1, 2, 3). In HeLa cells, the late r-strand unit and each of the early regions produce families of RNA (Chow & Broker, 1978; Chow et al., 1979a,b). Only one RNA from each family is shown. Early RNAs that were occasionally seen in the late RNA preparations isolated from infected monkey cells have identical splicing patterns as seen in human cells. Most r-strand late RNAs have the tripartite leader and are identical in the 2 systems. The only exceptions are the fiber RNA and early region 3 RNAs that have.initiated from the late promoter as shown in the bottom portion of the Figure. For simplicity not all the observed fiber molecules have been listed (cf. Fig. 7). Some region 3 RNAs with 3’ co-ordinates near 83 were not included either. For comparison of splicing patterns, the 1OOK RNA family has been included. A, The tripartite leader; DBP, DNA-binding protein.

INCOMPLETE

SPLICING

OF FIBER

mRNA

241

Whether the live- to 20-fold depression of the fiber RNA concentration together with the failure to remove long upstream sequences in 50% of the fiber RNA can completely account for the drastic reduction in the synthesis of fiber protein is uncertain. First, these incompletely spliced RNAs were also present in productive infections of monkey cells. However, in this case they represented less than 12% of the fiber RNAs. In addition, the remaining fiber RNAs in abortive infections appear to be normal by the criteria thus far applied (presence of capped, three-part leader and normal splicing). These “normal” RNAs could amount to 5% of the mature fiber mRNA found in productive infections. That some translatable fiber mRNA was made was shown by the cell-free synthesis of a small amount of fiber from purified fiber mRNA prepared from abortive infections. However, if a linear or near linear relationship exists between fiber mRNA concentration and the amount of fiber synthesis, then only a 20-fold rather than 100 to lOOO-fold reduction should be observed. This suggests additional, more subtle abnormalities (e.g. methylation and/or splicing differences) of this RNA that interfere with its translation. Another possibility is that there might be aberrations in the translational machinery of the monkey cells that, prohibit efficient utilization of the remaining fiber mRNA, in addition to the defects seen at the RNA level. In any case, the expression of the fiber gene is at least partially controlled at the level of RNA processing, namely RNA splicing. This is one of the first systems that suggest a role for RNA splicing in modulation of gene expression. Recently, Segal et al. (1979) reported that the failure of SV40 to grow in teratocarcinoma cells was due to a block in viral mRNA splicing. The host, range mutant of adenovirus, hr400, expresses its late genes and grows efficiently on monkey cells, presumably as a result of proper processing of the fiber RNA. The mutation resides in a small region between co-ordinates 62.9 and 65.6 (Klessig & Grodzicker, 1979) which is part of the coding region of the DNA binding protein. DNA replication which requires the DNA binding protein occurs normally in abortive infections. Thus, the DNA binding protein may have a second function which is impaired in monkey cells infected with wild-type adenoviruses. The data presented in this paper suggest that this second function involves RNA processing. We are grateful to Dr Thomas Broker of the manuscript, Dr James Lewis for Dr Carl Anderson for providing anti-fiber assistance, and to MS Marie Moschitta for This work was supported by Cancer National Cancer Institute.

for the graphic illustrations and critical review help with the in. vitro translation system, to serum, to MS Margaret Quinlan for technical secretarial assistance. Research Center grant no. CA13106 from the

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