Proc. Nati. Acad. Sci. USA Vol. 83, pp. 4690-4694, July 1986 Biochemistry Defective RNA splicing in the absence of adenovirus- associated RNA, (regulation of differential RNA splicing/mRNA abundance control/transation control) CATHARINA SVENSSON AND G6RAN AKUSJARVI Department of Medical Genetics, Box 589, Biomedical Center, S-751 23 Uppsala, Sweden Communicated by Peter Reichard, March 12, 1986

ABSTRACT We have analyzed late gene expression in 293 proteins may modify the efficiency by which RNA splice sites cells infected with an adenovirus type 5 mutant d1331, which is are used. It has also been proposed (3, 5) that the adenovirus- defective in production of the low molecular weight virus- associated RNAs play a similar role in splicing of late viral associated (VA) RNA,. The results show that several steps in mRNA, as has been suggested and proven for the small late gene expression are affected. In addition to the previously nuclear U RNAs (see ref. 6 and refs. therein). characterized defect in late mRNA translation, mutant infected The virus-associated (VA) RNAs are small RNA polymer- cells also show an aberrant selection of RNA splice sites and a ase III transcripts, designated VA RNA, and VA RNA], (7, substantially reduced L2, L3, and L5 mRNA accumulation. 8), which are produced in large amounts late during an Normal or even slightly elevated amounts of mRNA from adenovirus infection. Both RNAs are =160 nucleotides long region Li are produced. However, the Li pre-mRNA is spliced and are transcribed from two closely spaced transcription only to generate the mRNA encoding the Mr 52,000-55,000 units located within the Li cotermination family (see Fig. 1A) polypeptide and no detectable mRNA for polypeptide Ma. (9). Studies of viral mutants have shown that VA RNA, is Cotransfection of a plasmid encoding VA RNA, complemented required for efficient translation of viral mRNAs late after the splicing defect in trans, suggesting that the abnormalities infection (10). VA RNA, rescues the translational capacity of are due to the absence of VA RNA,, rather than to a cis-acting infected cells by suppressing the activity ofa double-stranded change in the nuclear precursor RNA. In a HeLa cell variant, RNA-dependent protein kinase, which phosphorylates the which allows late protein synthesis also in the absence of VA a-subunit ofthe eukaryotic initiation factor 2 (eIF-2) (11-13). RNAI, because of a lack of eukaryotic initiation factor 2a Previous studies have indicated that the absence of VA kinase expression, a normal repertoire of late mRNA was RNA, does not significantly perturb late viral transcription produced. We conclude that a soluble factor, most likely a late (10). We have reexamined this in more detail and show that viral protein, controls differential RNA splicing and late the lack of VA RNA, expression results in severe defects in mRNA accumulation during an adenovirus infection. late mRNA expression. We present evidence for the exist- ence of a late viral protein that controls the specificity of A lytic adenovirus infection is by convention divided into two RNA splicing and the efficiency oflate mRNA accumulation. phases, an early and a late phase, which are separated by viral DNA replication and characterized by the expression of MATERIALS AND METHODS specific subsets ofthe viral genome. Most ofthe late proteins are translated from mRNAs originating from the major late Virus Infection and Radioactive Labeling of Cells. transcription unit (see Fig. 1A), which extends in the right- Subconfluent monolayers of HeLa and 293 cells were infect- ward direction from a promoter at coordinate 16.8 to a ed at a multiplicity of 10 fluorescence-forming units per cell position near the right-terminal end of the genome. The late (14) with mutants d1327 and d1331 (Fig. 1C). At different time mRNAs are grouped into five families, each consisting of points, cells were labeled with [35S]methionine (20 juCi/ml; species with coterminal 3' ends (L1-L5; Fig. 1A) and having 1000 Ci/mmol; 1 Ci = 37 GBq; New England Nuclear), a common tripartite leader attached to the 5' end (reviewed fractionated into cytoplasm and nuclei by IsoB-Nonidet P-40 in ref. 1). extraction and used to prepare cytoplasmic RNA and total Expression of the late mRNA is regulated at both the level protein extracts as described (15). of transcription termination and RNA processing. At early RNA Blot Analysis. Total cytoplasmic RNA (1 lg) was times, transcription from the major late promoter terminates electrophoresed in 1% agarose containing 0.7% formalde- near the middle of the genome, whereas at late times this hyde and transferred to a nitrocellulose filter (16). Hybrid- block is alleviated, allowing expression of the distal parts of ization was at 430C in a buffer containing 40% formamide, the transcription unit (L2-L5; Fig. 1A) (reviewed in ref. 1). 10% dextran sulfate, 4x SSC (1x SSC = 0.15 M NaCl/0.015 More interestingly, the virus infection also induces a M Na citrate), lx Denhardt's solution (lx Denhardt's change in the splicing pattern oflate mRNA. Whereas a single solution = 0.02% bovine serum albumin/0.02% Ficoll/0.02% Li mRNA body (species Llb; Fig. LA) is produced early after polyvinylpyrrolidone), 6.5 mM Tris'HCl (pH 7.5), with re- infection, three alternatively spliced mRNAs are generated gion-specific adenovirus type 2 DNA fragments 32P-labeled from the same primary transcript at late times (2-4). This by nick-translation (16) as hybridization probes. change in RNA processing has previously been shown to be Complementation Analysis. Subconfluent -monolayers of a regulated process, probably at the level of RNA splice-site 293 cells (6-cm Petri dishes) were transfected by the calcium selection (3, 4). The identity and origin of the factor(s) phosphate coprecipitation technique (17) with either 10 jig of controlling differential RNA splicing is not known. Events pBR322 or plasmid pHindB. Twenty-five hours post-trans- linked to viral DNA replication, a cellular factor, or late viral fection, cells were infected with mutants d1327 or d1331. After an additional 23 hr of incubation, cells were labeled with The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: VA RNA, virus-associated RNA; eIF-2, eukaryotic in accordance with 18 U.S.C. §1734 solely to indicate this fact. initiation factor 2; hpi, hours post-infection. 4690 Downloaded by guest on October 4, 2021 Biochemistry: Svensson and Akusjdrvi Proc. Natl. Acad. Sci. USA 83 (1986) 4691 [35S]methionine and used to prepare total RNA and protein d1331 infection, RNA from all late regions was expressed extracts (15). (Fig. 2), showing that the late phase had commenced. However, the spliced structure of late mRNA differed sig- RESULTS nificantly from a wild-type infection. For example, in region L2 (Fig. 2), the percentage of RNA species L2b was dramat- Aberrant Splicing in dl331-Infected 293 Cells. To study the ically reduced and in region L3, the polypeptide pVI mRNA effect of VA RNA, on late mRNA expression, we used two (species L3a; Fig. 2) was almost completely absent. deletion mutants of adenovirus type 5-dI327 and d1331. The steady-state concentration of mRNA varied also Mutant d1331 lacks 29 base pairs within the transcriptional considerably between d1327- and d1331-infected cells. Where- control region of the VA RNA, gene and fails, therefore, to as mRNA from regions Li and L4 accumulated with a similar produce VA RNA, (10). Both viruses have a deletion =2 if not slightly elevated efficiency, mRNAs specific to late kilobases long, which removes almost the entire E3 region regions L2, L3, and L5 were substantially reduced (by a (Fig. 1C). Since E3 is dispensable for viral growth in tissue factor of 5-10; Table 1). culture cells (10), d1327 display a wild-type phenotype. In summary, our data show that the phenotype of mutant The transition from early to late phase of an adenovirus d1331 is much more complex than previously believed, and infection is characterized by the onset of viral DNA replica- the results suggest that VA RNA&, by some mechanism, tion, which occurs around 8-9 hr post-infection (hpi) in both participates in the control of mRNA production from the wild-type and d1331-infected cells (ref. 10; data not shown). major late transcription unit. To quantitate late mRNA accumulation, total cytoplasmic Complementation of the RNA Processing Defects by VA RNA was isolated from 293 cells infected with either of RNA, in trans. Since mutant d1331 has a 29-base-pair deletion mutants d1331 or d1327 and analyzed by RNA blot analysis. within the intragenic promoter element for VA RNA, tran- As expected (2-4), accumulation ofLi mRNA during a d1327 scription (10), it was necessary to show that the observed infection resulted in a successive change in the RNA splice RNA processing defects were not caused by an altered pattern. At 12 hpi, the major Li mRNA was the Mr structure of the nuclear pre-mRNA. 52,000-55,000 mRNA with the "i"-leader sequence (2) 293 cells transfected with plasmid pHindB (encoding spliced in between the second and the third leader segments adenovirus type 2 VA RNA, and VA RNA,,; see ref. 15) were (species Llb+i, Figs. 1A and 2). As the late phase developed, superinfected with either ofmutants d1331 or d1327. RNA blot the relative amount of this mRNA diminished in favor of an analysis showed that pHindB cotransfection resulted in the increased production of the Mr 52,000-55,000 mRNA (spe- recovery of the Li MIIa mRNA (species L1C) production in cies Llb) and the synthesis ofanother mRNA, the IIIa mRNA d1331-infected cells, as well as a reestablishment of the splice (species L1J). During a d1331 infection, in contrast, approx- pattern and steady-state concentration of L2 mRNA (Fig. imately the same steady-state concentration of Li mRNA 3A). Thus, we conclude that the defects in RNA accumula- accumulated (Table 1). However, mutant infected cells tion result from the absence of VA RNA, rather than from a showed a defect in Li pre-mRNA splicing. Thus, still at 24 cis-acting change of the nuclear pre-mRNA. hpi, half of the Mr 52,000-55,000 mRNA population retained Wild-Type Phenotype of dl331 in a HeLa Cell Line Lacking the i-leader segment (Fig. 2). More interestingly, even at this the eIF-2a Kinase Activity. Since pHindB cotransfection also late stage undetectable levels ofthe IIIa mRNA were present. rescued the translational capacity of the d1331-infected cells Hence, we conclude that the lack of VA RNA, expression to wild-type levels (Fig. 3B), we could not distinguish results in an atypical Li mRNA pattern during a productive between the possibilities that (i) VA RNA, directly partici- adenovirus infection. pates in RNA processing or (it) VA RNA, indirectly affects Decreased Accumulation of mRNA from Regions L2, L3, maturation oflate mRNA by allowing synthesis ofa late viral and L5 in d1331-Infected Cells. The transition from the early protein(s) that is required for mRNA maturation. to the late phase of infection is associated with an activation To address this question, we analyzed d1331 growth in a of L2-L5 mRNA production (reviewed in ref. 1). During a HeLa cell line that lacks the eukaryotic initiation factor 2a 5'-eaders A 1 2 i 3 LI L2 L3 L4 LS _N_ d LATE _mo _ __m c -(*{_ -I b a

loI 3 I I 0 10 20 30 40 50 60 70 80 90 100

EARLY - - I-) -

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FIG. 1. Organization of the adenovirus genome. (A) Structure of mRNAs originating from the major late transcription unit early and late after infection. The mRNAs are divided into five families (L1-L5), each having coterminal 3' ends and a common set of 5' leaders spliced onto the RNA body. The i-leader, which is found on a fraction of the mRNAs, is shown in parentheses. The positions of the VA RNA genes are shown by open arrows. (B) Position ofadenovirus type 2 DNA fragments used as region-specific hybridization probes. L1, HindIII-I [32.15-37.94 map units (m.u.)]; L2, Kpn I-Sph I (42.76-49.52 m.u.); L3, Sma I-Kpn I (56.81-61.86 m.u.); L4, Sma I-L (76.07-76.71 m.u.); L5, Sph I (86.82-88.49 m.u.). (C) Position ofdeletions in adenovirus type S mutants d1327 and d1331. Both viruses share a common deletion removing almost the entire E3 region (78.5-84.3 m.u.). In addition, d1331 lacks 29 base pairs within the transcriptional control region for VA RNA,. Downloaded by guest on October 4, 2021 4692 Biochemistry: Svensson and Akusjarvi Proc. Natl. Acad Sci. USA 83 (1986) LI d1327 d1331 14 d1327 d 1331 A - r L1 L2 (N C0 Ca (N (N 0 w C4 00 V _- _- C _ _I N,- '- N- N Clf) N cV) C() C) 'a m _ w r m ab-- - Vl b+i mm M _+ - + m+- b -- a i *

C _ c - 0 a d- a a- "" - b + Lsl b- 3 5 of C- pos9 L2 d1327 d1331 L5 d1327 d1331 c - dej* 4A I , I --I I -I 'I ei co IV e4 o IV (N Go 14 O (CN _ _ N _- _- C B N,- N Co

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FIG. 3. Complementation of the growth defect of d1331 by cotransfection of a plasmid encoding VA RNA,. Monolayers of 293 cells transfected with pHindB (+), encoding the adenovirus type 2 VA RNA&, or pBR322 (-) were infected with either ofmutants d1327 or d1331. Cells were pulse-labeled 23 hpi with [35S]methionine and used to prepare cell extracts and total cytoplasmic RNA. (A) RNA blot analysis of the mRNA specific to late regions Li and L2. Symbols are as described in legend to Fig. 2. (B) Analysis of FIG. 2. RNA blot analysis of late mRNA accumulation in d1327- [35S]methionine-labeled proteins by NaDodSO4/polyacrylamide gel and d1331-infected 293 cells. Total cytoplasmic RNA isolated 12, 18, electrophoresis. The positions of some late viral proteins are and 24 hpi was separated on a formaldehyde gel and transferred to indicated. nitrocellulose paper. Hybridization was with 32P-labeled DNA probes specific for the five late mRNA families (Fig. 1B). Arrows showed that in the presence of a functional translational indicate the positions of the 18S and 28S ribosomal RNAs. The machinery, wild-type levels of both Li and L2 mRNAs were lettering on the left denotes the position of the late mRNAs, which produced in the absence ofVA RNA, (Fig. 4B). We therefore are depicted in Fig. IA. conclude that the defects in differential RNA splicing and late mRNA accumulation observed during a d1331 infection of293 (eIF-2a) kinase activity (data not shown). Schneider et al. cells (Fig. 2) are caused by the absence ofa regulatory factor, have shown that in the absence of eIF-2a kinase expression, probably a protein, rather than by direct involvement of VA d1331 growth is phenotypically normal (12). As shown in Fig. RNA, in RNA processing. 4A, d1331 infection resulted in the synthesis of structural Synthesis of Correctly Processed Li mRNAs During a polypeptides, which coincided in time and reached levels Prolonged d1331 Infection of293 Cells. Since protein synthesis comparable to wild type. Furthermore, an RNA blot analysis is not completely blocked in dl331-infected 293 cells (reduced by a factor of =20; Fig. 3B), it may be predicted that a Table 1. Accumulation of late viral mRNAs in d1327- and prolonged infection will allow accumulation of a regulatory d1331-infected 293 cells factor in sufficient amounts to trigger the switches in RNA dl327* dl331* dl331/dl327t processing. Cell extracts were prepared at different time points from 18 hpi 24 hpi 18 hpi 24 hpi 18 hpi 24 hpi d1331- and d1327-infected 293 cells continuously labeled with Li 57 100 88 115 1.5 1.15 [35S]methionine. As expected, a slow accumulation of late L2 76 100 11 31 0.14 0.31 viral proteins could be detected also in the d1331-infected L3 75 100 9 27 0.12 0.27 cells (Fig. 5A). In addition, an RNA blot analysis of RNA L4 57 100 41 129 0.72 1.29 prepared at 50 hpi (Fig. 5B) demonstrated that the defect in L5 70 100 10 26 0.14 0.26 pre-mRNA splicing was partially corrected. Thus, we could an Quantitation of the late mRNA expression was by densitometric detect increased efficiency of removal of the i-leader scanning of the RNA blots shown in Fig. 2. sequence (compare Fig. 2 and Fig. SB) as well as a partial shift *For each 3'-coterminal family, the steady-state concentration of to maturation ofthe MIIa mRNA (species L1). Another RNA, RNA is expressed in percentage of the amount present in d1327- which from its size can be predicted to correspond to species infected cells at 24 hpi, which arbitrarily has been set to 100%. Lla+i (Fig. 1A), also accumulated to a substantial amount in tThe ratio oflate mRNA expressed in d1331- and d1327-infected cells. the d1331-infected cells. Downloaded by guest on October 4, 2021 Biochemistry: Svensson and Akusjdrvi Proc. Natl. Acad. Sci. USA 83 (1986) 4693 A A d1331 I -ICId1327 d1327 d1331 ° am CD m 2 _--Ncv _'sC4 a a N N 0 CO X rN N 0 _- C C' (t) CN C) it L() 11-- ___ 100K- ',. v - .

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C- * b-9 _ FIG. 4. Kinetics ofprotein synthesis and mRNA accumulation in c- *S d1327- and d1331-infected HeLa cells. HeLa monolayer cells were infected with mutants d1327 or d1331. At 19, 29, and 43 hpi, cells were pulse-labeled with [35S]methionine and used to prepare cell extracts FIG. 5. Effect of prolonged d1327 and d1331 infections. 293 cells and total cytoplasmic RNA. (A) Analysis of [35S]methionine-labeled infected with d1327 or d1331 were labeled continuously with proteins by NaDodSO4/polyacrylamide gel electrophoresis. Mock, [35S]methionine beginning at 12 hpi. At 18, 28, 37, 42, and 50 hpi, cell uninfected cells. (B) RNA blot analysis of the Li and L2 mRNAs. extracts and total cytoplasmic RNA were prepared. (A) Analysis of Symbols are as described in legend to Fig. 2. [3YS]methionine-labeled proteins by NaDodSO4/polyacrylamide gel electrophoresis. (B) RNA blot analysis of the Li and L2 mRNAs accumulated at 50 hpi. The symbols are as described in the legend to The effect of a prolonged d1331 infection was even more Fig. 2. dramatic for the L2 mRNA accumulation, suggesting that multiple regulatory factors may be involved. Thus, both the splice pattern and the steady-state concentrations of L2 quantities of Li mRNA accumulated in d1331-infected cells mRNA were corrected to wild-type levels under these con- (Table 1), no shift in Li pre-mRNA processing was observed ditions. (Fig. 2). Two experiments strongly suggest that this defect is caused DISCUSSION by the lack of a late adenovirus protein rather than a cellular factor(s) or VA RNA, itself. Firstly, in a HeLa cell line that Differential RNA splicing is likely to be a very important does not express the eIF-2a kinase and thus allow late viral mechanism for generation ofprotein diversity during eukary- protein synthesis also in the absence of VA RNA, (12) (Fig. otic cell development. An increasing number ofcellular genes 4A), mutant d1331 grows like wild type and produces the full are being shown to maturate alternatively spliced mRNAs. repertoire of late mRNAs (Fig. 4B). Secondly, during a However, trans-acting factors that regulate RNA splice-site prolonged d1331 infection of293 cells, the low level ofprotein selection have not yet been described. In this report, we synthesis remaining in the mutant-infected cell (Fig. 5A) present evidence for the existence of a late adenovirus allows, with time, for an accumulation of sufficient amounts protein that regulates the maturation of differentially spliced oflate polypeptides to partially trigger the shift in differential adenovirus mRNAs. We show that a mutant defective in VA pre-mRNA splicing (Fig. 5B). RNA, expression has at least three phenotypic characteris- Since early protein synthesis is unaffected in d1331-infected tics. In addition to the previously described translational cells (10, 13), the accumulation of early gene products is not defect (10), a VA RNA, negative mutant also shows an sufficient to trigger differential Li pre-mRNA splicing, im- aberrant splicing of late adenovirus mRNA and a defect in plying that the regulatory factor is synthesized late after accumulation of L2, L3, and L5 mRNA (Fig. 2; Table 1). infection. Furthermore, since adenovirus turns off host-cell The transition from the early to the late phase ofa wild-type gene expression at late times (18), it seems unlikely that the infection is accompanied by a regulated shift in the specificity regulatory factor is of cellular origin. Collectively, these of Li pre-mRNA splicing (2-4). Thus, the IlIa mRNA results suggest that adenoviruses encode a late gene product (species L1, Fig. 1A), which is absent early after infection, that regulates the specificity of RNA splicing. This factor is becomes an abundant Li mRNA at late times (Fig. 2). most likely a late viral protein, since polypeptide synthesis is Surprisingly, our analysis showed that although normal required to induce the shift in differential splicing. Downloaded by guest on October 4, 2021 4694 Biochemistry: Svensson and Akusjarvi Proc. Natl. Acad. Sci. USA 83 (1986) Interestingly, the maturation ofmRNA from all late regions exclusive accumulation of the Mr 52,000-55,000 mRNA (ref. (L1-L5) is subjected to a control at the level ofRNA splicing 22 and refs. therein). (Fig. 2). Furthermore, since all adenovirus transcription units accumulate differently spliced mRNAs early and late after We thank Dr. U. Pettersson for valuable comments on the infection (reviewed in ref. 1), the regulatory protein described manuscript. We also thank Dr. T. Shenk and his colleagues for here may have a more general function in controlling the providing us with mutants d1331 and d1327. This work was supported specificity of RNA splicing. by grants from the Swedish National Science Research Council and Very little is known about how the accumulation of the Swedish Cancer Society. alternatively spliced mRNAs is regulated. Pulse-labeling 1. Akusjarvi, G., Pettersson, U. & Roberts, R. J. (1986) in experiments have suggested that changes in RNA splice-site Developments in Molecular Virology, ed. Doerfler, W. selection rather than differential mRNA stability or delayed (Nijhoff, Boston), Vol. 5, pp. 53-95. transport times across the nuclear membrane are the cause of 2. Chow, L. T., Broker, T. R. & Lewis, J. B. (1979) J. Mol. Biol. the observed Li mRNA expression (3). However, the situ- 134, 265-303. ation may be much more complex. For example, the accu- 3. Nevins, J. R. & Wilson, M. C. (1981) Nature (London) 290, mulation of the E1B 13S and 22S mRNAs have been 113-118. suggested to be controlled both at the level ofRNA splice-site 4. Akusjarvi, G. & Persson, H. (1981) Nature (London) 292, 420-426. selection (19) and differential cytoplasmic stability (20). 5. Mathews, M. B. (1980) Nature (London) 285, 575-577. Viral DNA replication has been shown to be a prerequisite 6. Chabot, B., Black, D. L., LeMaster, D. M. & Steitz, J. (1985) for expression of mRNAs from regions L2 and L5. We show Science 230, 1344-1349. here that a replicating template and the early gene products 7. Reich, P. R., Forget, B. G., Weissman, S. H. & Rose, J. A. allow only for a limited expression of late mRNA (Table 1), (1966) J. Mol. Biol. 17, 428-439. thus suggesting that late viral proteins are also required for a 8. Mathews, M. B. (1975) Cell 6, 223-229. correct L2 to L5 mRNA accumulation. 9. Akusjarvi, G., Mathews, M. B., Andersson, P., Vennstrom, For unknown reasons, the L4 mRNAs do not conform to B. & Pettersson, U. (1980) Proc. Natl. Acad. Sci. USA 77, this general decline in mRNA accumulation. Almost wild- 2424-2428. 10. Thimmappaya, B., Weinberger, C., Schneider, R. J. & Shenk, type quantities of L4 mRNA are recovered also from d1331- T. (1982) Cell 31, 543-551. infected cells (Table 1). This indicates that the synthesis ofL4 11. Siekierka, J., Marino, T. M., Reichel, P. A. & Mathews, mRNA is regulated differently than the other late mRNAs. In M. B. (1985) Proc. Natl. Acad. Sci. USA 82, 1959-1963. theory, this can be achieved by several independent mech- 12. Schneider, R. J., Safer, B., Munemitsu, S. M., Samuel, C. E. anisms. For example, in the absence oflate viral proteins, the & Shenk, T. (1985) Proc. Natl. Acad. Sci. USA 82, 4321-4325. cellular splicing machinery might show a preference for the 13. O'Malley, R. P., Mariano, T. M., Siekierka, J. & Mathews, splice signals unique to the L4 mRNAs and the Li Mr M. B. (1986) Cell 44, 391-400. 52,000-55,000 mRNA, which are predominantly accumulat- 14. Philipson, L. (1961) Virology 15, 263-268. under these conditions (Fig. 2). Alternatively, synthesis 15. Svensson, C. & Akusjarvi, G. (1984) Mol. Cell. Biol. 4, ing 736-742. ofL4 mRNA may be under the control ofa different promoter 16. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular (21). However, differences in cytoplasmic half-lives or in- Cloning: A Laboratory Manual (Cold Spring Harbor Labora- creased transport efficiencies may also explain the unexpect- tory, Cold Spring Harbor, NY). ed behavior of the L4 mRNAs. 17. Wigler, M., Pellicer, A., Silverstein, S. & Axel, R. (1978) Cell Finally, we would like to point out the intriguing similarity 14, 729-731. between monkey cells abortively infected with adenovirus 18. Beltz, G. A. & Flint, S. J. (1979) J. Mol. Biol. 131, 353-373. type 2 and 293 cells infected with the VA RNAI-negative 19. Montell, C., Fisher, E. F., Caruthers, M. H. & Berk, A. J. mutant d1331. infected cells show (i) a (1984) Mol. Cell. Biol. 4, 966-972. Abortively monkey 20. Wilson, M. C. & Darnell, J. E. (1981) J. Mol. Biol. 148, translational defect in which synthesis of the fiber polypep- 231-251. tide is drastically reduced; (ii) a reduced accumulation ofL2, 21. Girvitz, S. C. & Rainbow, A. J. (1978) Virology 84, 75-86. L3, and L5, but not L4, mRNAs; and (iii) a defect in the 22. Johnston, J. M., Anderson, K. P. & Klessig, D. F. (1985) J. differential splicing of the Li pre-mRNA resulting in an Virol. 56, 378-385. Downloaded by guest on October 4, 2021