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DNA Concatemers MICHAEL D Proc. Natl. Acad. Sci. USA Vol. 91, pp. 153-157, January 1994 Biochemistry Deletion of the E4 region of the genome produces adenovirus DNA concatemers MICHAEL D. WEIDEN AND HAROLD S. GINSBERG* Departments of Medicine and Microbiology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032 Contributed by Harold S. Ginsberg, September 20, 1993 ABSTRACT Two mutants containing large deletions in the by replication-induced recombination (11). The last 45 bp of E4 region of the adenovirus genome H5dl366 (91.9-98.3 map the genome contain the necessary sequence information for units) and H2dl808 (93.0-97.1 map units) were used to inves- replication, and any deletion of the terminal cytosine abol- tigate the role of E4 genes in adenovirus DNA synthesis. ishes DNA synthesis (12, 13). Infection of KB human epidermoid carcinoma cells with either Other viral gene products may be involved in viral DNA mutant resulted in production of large concatemers of viral synthesis, and other mechanisms not yet elucidated may be DNA. Only monomer viral genome forms were produced, required for completion of viral DNA replication. Some however, when mutants infected W162 cells, a monkey kidney phenomena not accounted for in the current model include cell line transformed with and expressing the E4 genes. Dif- the following: circular, supercoiled, and multimeric viral fusible E4 gene products, therefore, complement the E4 mutant DNA molecules, which were identified in electron micro- phenotype. The viral DNA concatemers produced in d1366- and scopic studies (14); the pL 5'-to-3' exonuclease required in d1808-infected KB cells did not have any specific orientation of the in vitro model (9) produces defective strands that need monomer joining: the junctions consisted of head-to-head, additional processing for maturation; a large proportion of head-to-tail, and tall-to-tail joints. The junctions were cova- viral particles produced during infection carry partially rep- lently linked molecules, but molecules were not precisely licated single-stranded DNA (15); and concatemers of input joined, and restriction enzyme maps revealed a heterogeneous DNA are formed in the in vivo plasmid model of viral DNA size distribution ofjunction fragments. A series ofmutants that synthesis (10-13). Topoisomerase, which is required in the in disrupted single E4 open reading frames (ORFs) was also vitro model, should not be needed to relieve tortional stress studied: none showed phenotypes similar to that of d1366 or in the linear molecules predicted by the current model. In di808. Mutants containing defects in both ORF3 and ORF6, addition, more than the three viral genes defined by the however, manifested the concatemer phenotype, indicating current model are needed for viral DNA replication, since redundancy in genes preventing concatemer formation. These mutations in the E4 region have a significant effect on viral data suggest that the E4 ORFs 3 and 6 express functions critical DNA synthesis (16-18). for regulation of viral DNA replication and that concatemer To gain a better understanding of the functions of the E4 intermediates may exist during adenovirus DNA synthesis. genes in adenovirus DNA synthesis, the DNA replication phenotypes of E4 mutants were investigated. These experi- Adenovirus DNA replication has been extensively studied ments revealed the formation of viral DNA concatemers, (reviewed in refs. 1 and 2). Electron microscopy demon- which suggests that multimeric genomes are a consequence strated that replication initiates at the ends of the linear of disruption of a normal viral gene function and that the genome and proceeds via strand displacement, producing a mechanism ofadenovirus DNA synthesis may differ from the semiconservatively replicated daughter genome and a single- presently accepted model. stranded (type II) molecule which may form a duplex pan- handle by virtue of inverted terminal repeats (ITRs). This type II molecule then is the substrate for another round of MATERIALS AND METHODS DNA synthesis producing a second daughter genome (3). Cell Culture and Viruses. Monolayer cultures of KB cells Insight into the mechanism of the initiation of replication (human epidermoid carcinoma cell line) and W162 cells [a has been gained from in vitro reconstitution of DNA synthe- Vero monkey kidney cell line which contains an integrated sis using isolated viral DNA and plasmid constructs. Genetic copy of the adenovirus type 5 (AdS) E4 region was supplied data and an in vitro model ofDNA replication defined the role by G. Kettner, The Johns Hopkins University] were grown of three essential viral proteins: a single-stranded-DNA- in Dulbecco's modified Eagle's medium (DMEM) supple- binding protein, a DNA polymerase, and a terminal protein mented with 10%o calf serum. E4 mutants H5dl366, in351, that is covalently linked to the 3' terminal deoxycytosine of in352, d1355, d1356, d1358, and d1359 were supplied by T. the genome and is critical for the initiation of DNA synthesis Shenk (17); H2dl808 was supplied by G. Kettner (16); and E4 (4-6). Four host proteins have also been defined as important inORF3, E4 inORF3/inORF6, E4 inORF3/dl355, and for viral DNA replication: nuclear factor I and nuclear factor d1366+ORF3 were supplied by P. Hearing (18). See Fig. 1 for III/Oct-1, which are transcription factors (ref. 7 and refer- location of mutations. ences therein); nuclear factor II, a type I topoisomerase (8); Purified virions, prepared by CsCl equilibrium density and pL, a 5'-to-3' exonuclease (9). centrifugation, were used in all infections. Material isolated An in vivo model for viral DNA synthesis used plasmid from the gradient was disrupted with 0.1% SDS and A260 was constructs to investigate the sequence requirements for rep- measured. An A2w of 1 indicates 1012 particles per ml (17). lication. The ITR is essential, and a plasmid containing a When purified Ad5 was titrated on KB cells, a 10:1 ratio of single complete copy of the repeat can be rescued by dupli- total particles to plaque-forming units was present. In all cative transfer of one end of the genome to the other (10) or Abbreviations: Adn, adenovirus type n; ITR, inverted terminal The publication costs ofthis article were defrayed in part by page charge repeat; ORF, open reading frame; PFGE, pulsed-field gel electro- payment. This article must therefore be hereby marked "advertisement" phoresis. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 153 Downloaded by guest on September 29, 2021 154 Biochemistry: Weiden and Ginsberg Proc. Natl. Acad. Sci. USA 91 (1994) 90 92 94 96 98 100 maP units --r -- -- RESULTS 3000 2000 1000 nt. Viral DNA Replication Phenotype of E4 Mutants. The studies described below used a series of twelve E4-region ORF6/7 ORF3/4 mutants (16-18). The designation of the mutant, type, and transcription ~K~ IF2 I location of the mutations are shown in Fig. 1. In summary, IF7 12IF6 IORF41ORF ORF I map d1808 is an Ad2 derivative with most ofthe E4 region deleted, and d1366 is an Ad5 derivative in which deletions affect all of *{| | | { § ~~~+Sbp in351 l~~~~~~~~~V+b -%c the E4 ORFs. Each ORF that has been identified in the E4 <g g ! i g 6^~~+bp in352 region has been individually mutated in the AdS derivatives d1355 in351, in352, d1355, d1356, d1358, d1359, and inORF3. The AdS derivatives inORF3/inORF6 and inORF3/dl355 each contain d1356 double mutations located in ORF3 and ORF6. The AdS *-20bpg d1358 derivative dl366+ORF3 has an intact ORF3 with appropriate *g -30bp 8dl3b59 splicing signals reinserted into the large E4 deletion of d1366. inUKIw.3 As previously described, d1366 and d1808 do not produce Y~+8bp luU+8bp iOF infectious virus in KB cells (16, 17), although viral DNA 44 -4s+ inORF,3/inORF6 synthesis does proceed and there is some multiplicity leak- No i-1 4bp V +8bp inORF3/dl355 iness in the replication-deficient phenotype (18). Large E4 -201 4bp d1808 deletions do multiply in W162 cells, a Vero cell line that -2269bp 4 - d1366 expresses the functional E4 region (16, 17). Fig. 2A presents a Southern blot of uncut DNAs that were isolated from KB _-_ ~~~~~~~~~d1366-+ORF3 cells 4, 24, or 48 hr after infection with AdS wild type, d1366, FIG. 1. E4 transcription map and mutant viruses. The physical or d1808. PFGE was used to separate these high molecular map ofthe E4 region is shown both in map units (above the line) and weight DNAs, and all Southern blots in Fig. 2 were hybrid- in nucleotides (below the line). The translational open reading frames ized with AdS ElA DNA, which does not cross hybridize (ORFs) are shown as boxes correctly oriented with respect to the with KB cellular DNA. At 24 and 48 hr after infection, both physical map with the ORF number either within the box or above E4 deletion mutants produced a ladder of bands with equal the box, in the case ofalternative splicing. The E4 mutants described distance between the bands (evenly spaced, individual bands in the text are labeled to the right. The vertical dotted lines locate the were apparent in the 48-hr lanes of d1366 and d1808 on mutation on the transcription map. Solid bars correspond to deletion ethidium bromide staining of the pulsed-field gel; data not mutations. Triangles above the lines correspond to insertions. The shown). Migration of viral DNA under the conditions of this size of each mutation is noted. PFGE is linearly related to the size of the DNA, and the experiments 200 particles per cell (20 plaque-forming units for splaying of the lanes is an expected consequence of the Ad5 wild type) were employed.
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