Characterization of adenovirus type 5 insertion and deletion mutants encoding altered DNA binding proteins

VIROLOGY 172, 634-642 (1989)

Characterization of Adenovirus Type 5 Insertion and Deletion Mutants Encoding Altered DNA Binding Proteins HANS L . VOS,' DOUGLAS E . BROUGH,* FREDERIQUE M . VAN DER LEE, ROB C . HOEBEN, 2 GIJS F . M . VERHEIJDEN,' DENNIS DOCKS, DANIEL F . KLESSIG,* AND JOHN S . SUSSENBACH 3 Laboratory for Physiological Chemistry, State University of Utrecht, Vondellaan 24a, 3521 GG Utrecht, the Netherlands ; and *Waksman Institute, Rutgers, the State University of New fersey, Piscataway, Newlersey 08854 ReceivedApril25, 1989;acceptedlune2l, 1989 We have introduced insertion and deletion mutations in the cloned DNA binding protein (DBP) gene of adenovirus type 5 . The mutated DBP genes were subsequently introduced in the viral genome by a combination of in vitro and in vivo methods. The resulting mutant viruses were tested for their viability in human 293 cells and an initial characterization of these viruses was performed . Viable mutants with insertions in the carboxyl-terminal portion of the gene could not be obtained . In contrast, a number of viable mutants were constructed that contained insertions or deletions in the aminoterminal half of DBP. Several of these, which covered the region between amino acid (gal residues 39 and 81, were phenotypically wild type, implying that this segment is completely dispensable for DBP function . However, mutations altering the region encompassed by as 2-38 were, at least, partially defective suggesting that this region is important for full activity of the protein . 01989 Academic Press, Inc .

H2hr400 and several identical mutants (Kruijeretal ., 1981 ; Anderson stall 1983 ; Brough et al., 1985) have an entirely different phenotype . They are host-range mutants that, in contrast to wild-type virus, can multiply efficiently in monkey cells as well as human cells (Kiessig, 1977 ; Klessig and Grodzicker, 1979) . Wild-type adenovirus fails to grow in these cells due to a complex block in viral late gene expression . This block includes a reduction in the rate of transcription of late genes (Johnston et al., 1985), alteration in the pattern of RNA splicing of the transcripts for the L1 and L5 late regions (Klessig and Chow, 1980 ; Anderson and Klessig, 1984 ; Anderson et al., 1988), and drastic reductions in the production of the fiber and Illa polypeptides encoded by the L5 and L1 regions, respectively (Anderson and Klessig, 1984 ; Anderson et al ., 1988) . Although the mutants listed above have been essential in identifying the functions of DBP, our understanding of the structure-function relationship of this complex protein is still rather limited . A more extensive set of mutants is needed for this purpose . Therefore we have constructed insertion and deletion mutations in the DBP gene of AdS, introduced the mutations into the virus via in vivo recombination, and have completed an initial characterization of the mutants .

INTRODUCTION The adenovirus single-stranded DNA binding protein (Ad DBP) is a multifunctional protein that is essential for viral DNA replication (van der Vliet and Sussenbach, 1975) . In addition, it appears to be involved in regulating early and late gene expression and in modulating the efficiency of cellular transformation (Lazarides et at, 1988 ; Klessig and Grodzicker, 1979 ; Rice et at, 1987) . Most of these functions have been revealed by the study of DBP mutants, such as H5ts125 and H2hr400 . H5tsl25 is temperature sensitive for growth and encodes a thermolabile DBP (van der Vliet et al., 1975) . At the nonpermissive temperature viral DNA replication is blocked (van der Mist and Sussenbach, 1975) . This mutant is also characterized by a higher frequency of transformation of rodent cells (Ginsberg et al ., 1974 ; Rice et al., 1987) and increased levels of early viral messengers (Carter and Blanton, 1 978a,b ; Babich and Nevins, 1981) . The increased levels of mRNAs and the higher frequency of transformation may arise from an abnormal activity of the mutant H5tsl25 DBP, since these effects do not occur in cells infected with a completely DBP-negative mutant (Rice and Klessig, 1985 ; Rice et al., 1987) .

MATERIAL AND METHODS ' Present address : Netherlands Cancer Institute, Plesmanlaan Enzymes and linkers

121, 1066 CXAmsterdam, the Netherlands . 2 Present address : Sylvius Laboratories, State University of Leiden, P .O . Box 9503, 2300 RA Leiden, the Netherlands . I To whom requests for reprints should be addressed . 0042-6822/89 $3 .00 Copyright 5 tgag by Academic Press, Inc . All rights of reproduction in any form reserved .

All enzymes used were from Boehringer-Mannheim or GIBCO/BRL . The Xhol linker 5'CCTCGAGG 3' was 634



MUTANTS IN THE Ads D8P GENE

from New England Biolabs ; both the C/al-linker 5'CCCATCGATGGG 3' and the sequencing primer 5'GGGCTGGGTGTGCGCGGCA 3' used for the deletion mutants (complementary to positions 2089 2107 in the Ad5 DBP gene (see Kruijer et al., 1981)) were gifts from Dr . 1 . H . van Boom (State University of Leiden) . Construction of starting plasmids Essentially the same plasmids were used for the insertion and deletion mutagenesis . The plasmid for the deletion mutagenesis, pAd51, contained the BamHlXhol (59-70 m .u .) fragment of the Ad5 genome comprising the entire coding region of the DBP gene (66 .562 m .u .) . This DNA fragment was cloned in a modified pUC12 vector that contained an Xhol site in place of the Smal site in the polylinker region . The plasmid used for the insertion mutagenesis, pAd53, only differed from pAd51 by the absence of the Sall site in the linker region . This site was removed to facilitate the introduction of a linker in the Hindll site of the DBP gene . Introduction of the mutant DBP genes into the viral genome required the use of two plasmids : pSB and pdeIXE (Fig . 3) . The plasmid pSB contained Ad5 sequences from the Sall site at 46 m .u . to the BamHl site at 59 m .u . cloned in pEMBL8 (Dente et at, 1983) . The plasmid pdeIXE was derived from the plasmid pPF446 (a generous gift of Dr . P . I . Freimuth) and contained all the Ad5 sequences between the BamHl site at 59 m .u . and a linker-derived Sall site at 100 m .u . cloned in pBR322 . To facilitate introduction of the mutation into the viral genome, the Xhol and the EcoRl sites in the E3 region at 83 and 84 m .u ., respectively, were removed by deletion of the 260-bp intervening fragment . Deletion of this region has been shown to have no effect on the viability of the resulting viruses in tissue culture (Kelly and Lewis, 1973 ; Lewis et al., 1974) . Mutagenesis Insertion mutations . A 12-bp Clal-linker 5'CCCATCGATGGG 3' was inserted in the DBP gene after linearization of pAd53 with restriction enzymes that leave blunt ends (Ball, Smal, Pvull, Hindll, EcoRV, Aosl, and Dral) (Fig . 1) . Several of these enzymes (Ball, Pvull, EcoRV, Aosl, and Dral) required a partial digestion for the insertion of the linkers at specific sites . The method we have used for the linker insertion has been described previously (Lathe et al., 1984) . In brief, unphosphorylated double-stranded Clal linkers were ligated to both ends of the linearized pAd53 . This resulted in the covalent addition of the linkers at the 5' ends only, while those at the 3' ends joined only by base pairing . After removal of the free linkers by isopropanol precipitation of the plasmid DNA, the base-paired linkers at the 3'

635

ends were removed by heating to 80° and subsequent gentle cooling . In this way the resulting 1 2-bp sticky ends could reanneal . The circularized plasmids were directly transformed into Escherichia colt C600, without prior ligation . The nucleotide sequences at the site of linker insertion were confirmed by the dideoxy chain termination sequencing method (Sanger et al., 1980) . Deletion mutations . Most deletion mutations were made by digesting pAd51 with Smal (position 2180 in Kruijer et al., 1981 (identical to the Smal site in Fig . 1 A)) and subsequently treating with the exonuclease Bal 31 . Following ligation, the DNA was redigested with Smal prior to the transformation to select against the presence of this site . The deletion clones were sequenced by the dsDNA sequencing method of Chen and Seeburg (1985) using a specific primer . Two additional deletion mutations were made by digesting pAd51 with Smal, followed by a partial digestion with Ball and religation, resulting in the deletion of either one of the two small Ball-Smal fragments . The identities of the viable deletion mutants were confirmed by recloning the BamHl--EcoRl fragment (59-75 m .u .) from the viral DNA and determining the nucleotide sequence using the dideoxy sequencing method (Sanger et al., 1980) with the primer complementary to positions 2089-2107 in Kruijer et al. (1981) . The deletion mutations are listed in Fig . 2 . Extension of mutated DNA fragments The BamHI-Xhol ( 59-70 m .u .) fragment used in the various mutagenesis procedures was enlarged by first adding theXhol-EcoRl ( 70-75 m .u .) and then the SallBamHl fragments (46-59 m .u .) (Fig . 3) . For the deletion mutants this could be performed directly in the original vector . The insertion mutants required a more elaborate, though essentially identical, pathway, because the Sall site in the polylinker of the original pAd53 plasmid was lacking . Following the addition of the two flanking fragments, a few constructs (carrying the two Ball and the Dral insertion mutants) were further extended by the addition of the EcoRI-Salt (75-100 m .u .) fragment of pdeIXE, but in most cases this part of the viral genome was added in vivo by recombination with the Xhol-Salt (70-100 m .u .) fragment of pdeIXE (Fig . 3) . In all cases the remaining part of the viral genome was provided by the left-terminal BamHl (0-59 m .u .) fragment of the Ad5 DNA-protein complex . Cell cultivation Human 293 cells, that contain the adenoviral El region (Graham et al ., 1977) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with glutamine and 10% fetal calf serum (Seralab) . HeLa cell



6 36

VOS ET AL .

B 1 3 H5in800 MetAlaHisArgTrpAlaSer Ball ATGGCCCATCGATGGGCCAGT 2300 2292

39 41 H5in801 ProProHisArgTrPGlyAla Smal CCCCCCCATCGATGGGGGGCG 2186 2178

80 82 H5in802 LeuAlaHisArgTrpAlaIle TTGGCCCATCGATGGGCCATT 2063 2055

230 232 H5in803 LeuGlnProlleAspGlyLeu Pvull TTGCAGCCCATCGATGGGCTG 1613 1605

414 416 LysPheProSerMetGlyThr AAGTTCCCATCSATGGGGACT 1061 1053

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FIG . 1 . (A) Schematic representation of the coding sequences of the Ad5 DBP gene and location of the restriction sites used for the linker

insertion mutagenesis . The 5' proximal Ball site is located in the second codon of the DBP gene, while the Dral site overlaps the stop codon . Only the relevant restriction sites are shown . The location of the mutations leading to a viable phenotype is indicated by giving the name of the corresponding mutant below the restriction site . The chymotrypsin cleavage site corresponds to the border between the N-t and C-t domain in DBP (Tsernoglou et al ., 1985) . The nucleotide and amino acid sequence around the site of insertion are shown in part (B) of the figure . The official name (if appropriate) and the site of insertion are given . Novel amino acids that result from the linker insertion are shown in bold, while the nucleotides of the linker are underlined . Since the insertion of the Clal linker in the Dral site results in the removal of the stop codon, 22 novel amino acids are added to the DBP molecule . The numbering system for the nucleotides are those used by Kruijer et a! . (1981) . The numbering of amino acid residues has not been changed to account for the linker .

monolayers were cultured as previously described (Rice and Klessig, 1984) . Transfection procedure The Ad5 DNA-protein complex was purified as previously described (Sharp etal., 1976 ; van Bergen et at, 1983), with the addition of a CsCI cushion to the sucrose/guanidinium chloride gradient to concentrate the complex . The DNA-protein complex was digested with BamHl and EcoRl to generate the required fragments for the in vivo recombination . The digest was performed at least 1 day before the transfection, so that the enzymes would have lost their activity by the time of the transfection experiment . The cotransfection procedure was performed according to Graham and van der Eb (1973) or by a slight modification of this procedure including a short glycerol shock, using 0 .5-1 .0 tg of digested complex and about 3 tg of both the Sall-EcoRl fragment carrying the mutated DBP gene and the Xhol-Sail fragment of pdelXE . Viral stock preparation and phenotypic characterization Plaques were picked after 4-7 days at 37° and virus stocks were obtained from 293 cells cultured in 96-well microtiter plates 4 to 7 days p .i . These stocks were used to infect cell monolayers for DNA analysis using a modified small-scale Hirt procedure (Hirt, 1967) and

for further multiplication of virus from the isolated plaque . Mutant viruses that proved to be of interest, based on restriction enzyme analysis of their DNAs, were plaque-purified once more according to the same procedure and high titer stocks were prepared for further characterization experiments . HeLa cell monolayers were infected with mutant viruses at a m .o .i . of 10 PFU/cell . Viral DNA accumulation at 48 hr p .i ., virus yield at 72 hr p .i ., and viral protein synthesis at 24 and 48 hr p .i . were analyzed as previously described (Hirt, 1967 ; Rice and Klessig, 1984 ; Klessig and Anderson, 1975 ; Klessig etal., 1984) . RESULTS Construction of the mutations To determine which regions of the DBP molecule are sensitive to structural changes insertion mutations were constructed by the addition of a 12-bp Clal linker at various unique restriction sites throughout the gene (see Materials and Methods for details) . The location of the insertion sites and the resulting alterations in the amino acid sequence of DBP are shown in Fig . 1 . The deletion mutations were generated around the Smalsite in the amino-terminal (N-t) domain of DBP (Fig . 1) using the exonuclease 8a131 . This site was chosen because the function of the N-t domain is relatively poorly characterized compared to that of the carboxyl-termi-



637

MUTANTS IN THE Ads DBP GENE

A.

B.

MIN ACID SEQUENCE

MIN ACIDS DELETED

Ads Mt

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dl(2-40) H5d1807 dl(32-42) 01(32-40) 01(37-40) H5d1009 1541906 8541808

M G APPKKRMRRRIESEDEEDSSQOALVPRTPSPRPSTSAADLAiAPKKKKKR 9 SREEEQRETTPERGRGAARRPPT IESEOEEDSSQOALVPRTPSPRPSTSAAOLAIAPKKKKKR MASREEEQRETTPERGRGAARRPPTMEDVSS PKKnMRRRIESEDEEDSSQDALVPRTPSPRPSTSAADLAIAPKKKKKR H SREEEQRETTPERGRGAARRPPTREOVSS APPKKRMRRRIESEDEEO5SQDALVPRTPSPRPSTSAAOLAIAPKKKKKR i SREEEQRETTPERGRGMRRPPTMEOVSSPSPSP----APPKKRMRRRIESEDEEOSSQDALOPRTPSPRPSTSAAOLAIAPKKKKKR MASREEEQRETTPERGRGAARRPPTMEOVSSPSPSPPP--PPKKRMRRRIESEDEEDSSQOALVPRTPSPRPSTSAADLAIAPKKKKKR MASREEEQRETTPERGRGMRRPPTMEDYSSPSPSPPP H SSQOALVPRTPSPRPSTSAADLAIAPKKKKKR MASREEEQRETTPERGRGAARRPPTMEOVSSPSPSPPPP P IAPKKKKKR

mutant : 1 43 Met(llyAlaProPro 01(2-40) GAMTGGGGGCGCCCCCA 2303 2172 29 43 VaiSerSerslaProP^o d1(32-40) GTGTCGTCC6C6000CCA 2216 2172 37 60 ProProProHisSerSer 115,11806 CCGCCTCCCCACTCATCA 2192 2121

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00541009

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33

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123

FiG. 2 . Nucleotide and amino acid sequences of the DBP gene of the deletion mutations . (A) The amino-terminal amino acid sequence of the wt DBP and the deletion clones used in the transfection experiments . Only the viable deletion mutants have been officially named ; the other mutations are indicated by giving the position of the first and the last deleted amino acid residues . The mutants are ordered according to the position of the first amino acid residue that is lost . H5d1808 and dl(2-40) result from the removal of one of the two Smal-Ball fragments . Novel residues that are the result of the presence of a previously non-existing triplet at the deletion junction are shown in the middle of the deleted area . The positions of the deleted amino acids of all clones is shown at the right side of the figure . Amino acids are shown in the one letter code : A, Ala ; C, Cys ; D, Asp ; E, Glu ; F, Phe ; G, Gly ; H, His ; I, lie ; K, Lys ; L, Lou ; M, Met ; N, Asn ; P, Pro ; Q, Go ; R, Arg ; 5, Set; T, Thr ; V, Val ; W, Trp ; Y, Tyr. (B) The amino acid and nucleotide sequences of the regions spanning the deletions . The order of the deletions is the same as in A and it should be read from left to right . The underlined sequence corresponds to the region 5' of the deletion breakpoint, while the 3' region is printed in bold lettering . In some cases the actual site of the deletion borders could not be accurately determined, because the nucleotide sequences around both deletion breakpoints is the same . In those cases the underlined and bold regions overlap . The coordinates given are those of the first and last nucleotides shown . Numbering is according to Kruijer or al . (1981) . The actual size of the deletion in nucleotides is given at the right of each sequence,

nal (C-t) domain . Previously, only three different mutant types had been isolated in this region, H2hr400 and identical mutants (Klessig, 1977 ; Kruijer et al., 1981 ; Anderson et al., 1983 ; Brough et al., 1985), H2ts400 (Rice and Klessig, 1984 ; Brough et al., 1985), and H5ts19 (Williams et al., 1971 ; Young et al ., 1984)) . Eight in-frame deletion mutations were isolated (Fig . 2) .

complex, digested with BamHl and EcoRl, and the Xhol-Sail fragment of pdeIXE, that provided the left and right end of the viral genome, respectively (Fig . 3) . In the single recombination experiments, the right end of the viral genome, provided by pdeIXE, was attached to the mutant fragment before the cotransfection with digested wt Ad5 complex was performed .

Introduction of the mutated DBP genes into virus

Effects of the mutations on viability

The mutations were introduced into the complete viral genome of Ad5 by either single or double in vivo recombination of the mutated DBP clone with viral DNA fragments . This type of recombination occurs with a relatively high frequency in infected cells (Volkert and Young, 1983 ; Berkner and Sharp, 1983) . Since a substantial overlap between DNA fragments is required for efficient recombination, the BamHl-Xhol fragment (59-70 m .u .) used in the mutagenesis procedures was extended by the addition of the 2 .5-kb XholEcoRl fragment (70-75 m .u .) and the 4 .8-kb SallBamHl fragment (46-59 m .u .) (Fig . 3) . In the case of the double recombination procedure, the resulting fragment was cotransfected with both the wild-type Ad5

The effects of the various mutations on virus viability were assayed by transfection efficiency and confirmed by restriction enzyme analyses of the progeny viral DNA . In the absence of the mutated Safl-EcoRl (4675 m .u .) fragment (for double recombination) orthe Sail (46-100 m .u .) fragment (for single recombination) very few viral plaques were obtained after transfections with the digested Ads DNA complex . Inclusion of these fragments in the transfection procedure resulted in increased numbers of plaques, if the mutation did not dramatically depress the viability of the resulting virus . When addition of a mutated fragment did not enhance the number of plaques and restriction enzyme analysis of the DNA of progeny viruses confirmed the absence



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type of these mutants was determined with respect to (i) the level of DNA accumulation, (ii) the rate of synthesis of DBP and late proteins, (iii) plaque morphology, and (iv) virus production . The four viable insertion mutants have been numbered H5in800 through H5in803 in the order of the insertions from the 5' to the 3' end of the DBP gene . H5in801-3 behaved very similar to wild-type virus with respect to viral DNA accumulation (Fig . 4A), production of DBP and late proteins (Fig . 5A), plaque morphology, and virus yield (Table 1) . In contrast, H5in800 exhibited a 5- to 10-fold lowered yield of infectious particles (Table 1), that correlated with a lowered production of DNA (Fig . 4A) and smaller-sized plaques . However, the synthesis of neither DBP nor late proteins appeared to be affected (Fig . 5A) . The properties of these mutants are summarized in Table 2 .

P

loChh FIG . 3 . The strategy off ragment extension and of the in vivo recombination . The DNA fragments are shown in the conventional adenovirus DNA orientation resulting in a 3' to 5' orientation of the DBP gene (shown as a bar), Only the insert sequences and the relevant restriction sites in the vector sequences are shown . Note that the top and bottom half of the figure are not drawn to the same scale (see both scale bars) . In step (1) theXhol -EcoRl (70-75 m .u .) wt fragment was added, while in step (2) the Sall-BamHl (46-59 m .u .) fragment was added . The third step was only used in the single recombination procedure and consisted of the addition of the EcoRl-Sail (75-100 ml u .) of pdelXE . For the single and double recombinations Ad5 TP-DNA complex digested extensively with BamHl and EcoRl was used to prevent fortuitous restoration of intact complex by religation of DNA fragments. Abbreviations used : B, BamHl ; C, region of the DBP gene encoding the C-terminus of the DBP protein ; F, EcoRl ; N, N-terminus of DBP ; S, Sall ; TP, terminal protein ; X, Xhol .

of a mutated DBP gene, we tentatively concluded that the mutation dramatically reduced the viability of the virus . Viable insertion mutants were identified and confirmed by the presence of a Clal site at the appropriate position in the DBP gene (Fig . 4A) . All viable deletion mutants lacked the Smal site at 66 1 and those containing larger deletions were confirmed by a reduction in the size of an Mlul/BstEll fragment covering the deletion (Fig . 4B) . The distribution of the viable and nonviable insertion mutations exhibited an interesting pattern . The three N-t domain mutants were viable, while four out of five insertion mutants in the C-t domain appear to be nonviable . Relatively small deletion mutations were also tolerated in the N-t domain . Of the eight deletions only the very N-t one, dl(2-40), was lethal (Table 1) . Characterization of the viable DBP mutants After isolation and plaque purification, 8 of the 11 viable mutants were further characterized . The pheno-

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FRG . 4. Restriction digests of the mutant viruses . (A) Restriction digests of DNA of the insertion mutants with Bglll and Clal . DNAs from an equivalent number of cells were fractionated in each lane . Repeated experiments showed that the DNA accumulation for 5in801803 was within a factor two of that observed with Ad5 and these were considered to be phenotypical wild type . The open arrow head indicates the 2151-bp wild-type fragment that has been changed by the mutagenesis . Introduction of the Clal linker results in cleavage of this fragment into 1170- and 993-bp fragments for H5in800, 1284 and 879 by for H5in801, 1407 and 756 by for H5in802, and 1859 and 304 by for H5in8o3 . These fragments have been indicated in the figure with squares . The fragment that contains the deletion in the E3 region is indicated by the solid arrow heads . The deletion results in a loss of approximately 260 by from the 2335-bp wild-type fragment . Note the reduced level of H5in800 DNA in the infected cells . (B) Restriction digests of the viable deletion mutants with Mlul and BstEll . The open arrow head indicates the position of the Ad5 wildtype fragment . The mutant fragments migrate just below that position . The sizes of the fragments are (in base pairs) : Ads wild-type, 1136 ; H5dI809, 1127 ; H50806, 1082 ; H5d1807, 1061 ; H5d1808, 1013 . Note the reduced level of the H5d1807 DNA .



639

MUTANTS IN THE Ad5 DBP GENE TABLE 1 VIABII. I

rI AN

D VIRUS YIE LDS U'- I HL MU IAN I S

Virus'

Description

Viability'

Virus yield'

Ad5

wt

-f

1710

+ + + +

350 1640 1930 2210

+ + + + + + 1

270 N .D, N. D. N .D . 4080 3330 2140

H5in800 H5in801 H5in802 H5in803

Insertions Ball-I Smal

Ball-2 Pvull Hindll EooRV Aosl

Dral

H5d1807

H5d1809 H5d1806 H5d1808

Deletions dl(2-40) dl(26-50) dl(32-42) dl(32-40) dl(37-40) dl(39-41) dl(40-58) dl(40-81)

loss of a number of amino acids, but also to the disappearance of potential phosphorylation sites . Furthermore, the regions that are lost in some of the mutants are very rich in proline residues, which dramatically affect protein secondary structure . This could exert a relatively large influence on the migratory behavior of the DBP molecule during SDS-PAGE, since the N-t domain is probably responsible for the aberrant migration of the 59-kDa wild-type DBP as a 72-kDa protein in gel electrophoresis . While the deletions had a marked effect on the migration of the DBP molecules in SDSPAGE, neither deletion or insertion mutations altered the stability of the resulting mutant proteins (data not shown) . DISCUSSION

a Only the viable mutants that have been characterized have been given systematic names . "' -" indicates that the mutants are viable, since they multiply in 293 or HeLa cells . Mutations from which mutant viruses could not be obtained were termed nonviable (- }, HeLa cells were infected at m .o .i . 10, and the virus yield (given as PFUlcell) was determined at 72 hr p .i . The results are an average of two to tour experiments . N .D ., not determined .

Most of the deletion mutants in the N-t domain are viable (Table 1) . The four mutants, that have been characterized in more detail, have been numbered H5d1806 through H5d1809 . Three of the mutants, H5d1806, H5d1808, and H5d1809, were not deficient in any of the properties we have tested (Table 2), such as the level of viral DNA (Fig . 4B), synthesis of DBP and late proteins (Fig . 5B), virus yield (Table 1), and plaque morphology . The fourth mutant, H5d1807, contains a deletion of amino acid residues 26 to 50 and is affected in several of the parameters tested . H5d1807 exhibited a reduced virus yield (Table 1) and altered plaque morphology (Table 2) . In some experiments a reduced level of viral DNA (Fig . 4B) and late protein synthesis (Fig . 5B, C) was observed . The level of DBP synthesis in the H5d1807-infected cells does not appear to be reduced and was higher than wild-type in some experiments (Figs . 513 and 5C) . The smaller size DBP in mutant-infected cells con firmed the presence of the deletion (Fig . 5B) . The decrease in the apparent size of the mutant DBP molecules in some cases was more pronounced than expected . However, the deletions do not only lead to the

A set of insertion and deletion mutations in the Ad5 DBP gene have been constructed in order to define regions of the protein necessary for its activities . The mutated gene was introduced into the viral genome via in vivo recombination, taking advantage of the high recombination frequency of adenoviruses . Viable virus could not be generated with a number of these mutations suggesting that some of the alterations were lethal . The eight insertion mutations were constructed using a Clal linker that encoded helix-breaking proline or glycine residues in two of the three reading frames . These insertions therefore were expected to cause moderate to severe local structural changes . Virus viability varied with the site of linker insertion . Insertions in the N-t domain were tolerated, while those in the C-t domain did not allow production of viable virus, with a single exception (H5in803) . In fact, of the five C-t domain insertions, H5in803 is nearest to the N-t domain (at as 231) . This distribution of viable and nonviable mutations is consistent with nucleotide sequence comparisons of DBP genes of several adenovirus serotypes (Quinn and Kitchingman, 1984 ; Kitchingman, 1985 ; Vos et al., 1988) . The C-t domain is highly conserved and, in general, appears unable to tolerate relatively large structural changes . In contrast, the poorly conserved N-t domain does not exhibit such constraints . Eight N-t deletion mutants were generated around the Smal site (aa 40) . Seven of these were viable and in total encompassed as 26-81 . This suggests that the 26 81 as region of the protein is not essential for viral growth . In fact, removal of the 3' two-thirds of this region (aa 39-81 ; H5d1806 ; H5d1808 ; H5d1809) or insertion within it (H5in801 and H5in802) had no effect ; all of these mutants had phenotypes similar to wtAdS (Table 2) . Thus, as 39-81 appear to be completely dispensable for DBP function .



640

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x '

x x <

FIG . 5 . DBP and total protein synthesis in HeLa cells infected with the insertion (A) and deletion (B, c) mutants . Cells were infected in separate experiments with a m .o .i . of 10 at either 24 hr p .i . (A and B) or 48 hr p .i . (C) proteins were labeled with 50 ACi of [ 35S]methionine in a total volume of 1 ml for 1 hr prior to analysis . DBP levels were determined after immunoprecipitation with a polyclonal antiserum (Klessig et at., 1984) . The two bands indicated by the arrow heads represent DRIP and a 48K proteolytic fragment (Ariga et al., 1980 ; Tsernoglou et at, 1985). The main bands in the panels showing total protein synthesis represent the adenovirus late structural proteins . Two major capsid proteins, hexon (II) and fiber (IV), are indicated .

Unfortunately, no other mutants were available with a deletion extending further in the direction of the C-t domain . It is likely that such larger deletions would affect DBP function because a very basic region is located just downstream of the deletion endpoint of H5d1808 (aa 81) . This region (aa 84-90) has the highly conserved sequence Pro(Lys) 6Arg, that resembles the SV40 large T antigen nuclear localization signal (Kalderon et at, 1984) . Its loss would probably have severe

consequences for the functioning of DBP in DNA replication and other nuclear processes . A region that shows a similar preponderance of basic residues is located between residues 42 and 49 in the Ad5 DBP gene . It has the sequence ProLysLysArgMetArgArgArg . This region is deleted in H5d1806, H5d1807, and H5d1808, yet they yield functional DBPs . Apparently, this signal is redundant or does not function as a nuclear localization signal . Interestingly, Cleghon et al .

TABLE 2 SUMMARY OF THE RESULTS OBTAINED WITH THE VIABLE MUTANTS

Mutant

Location'

Virus yield

DBP synthesis

DNA accumulation

Late protein synthesis

Plaque morphology

Insertions H5in8OO 1-15i1-801 H5in802 1-15in803

2 40 81 231

red . ° wt wt wt

wt` wt wt wt

red . wt wt wt

wt wt wt wt

small wt wt wt

Deletions H5d1806 H5d1807 H5d1808 H5d1809

dl(40-58) dl(26-50) dl(40-81) dl(39-41)

wt red . wt wt

wt wt° wt wt

wt red a wt wt

wt red . wt wt

wt small Wt Wt

' The numbers refer to the position of the amino acid residue(s) affected by the mutation .

The reductions (red .) in virus yield, DNA accumulation, and late protein synthesis were approximately 5 to 10-fold . `Values were considered to be wild type (wt) when they were within a factor of two of the Ads control . ° Variable levels have been observed ; in some experiments these were higher than wild type . e Variable levels have been observed ; in some experiments these were nearly equal to wild type .



MUTANTS IN THE Ad5 DBP GENE

(1989) have recently described a mutant that contains a deletion of amino acid residues 23 to 105, thus lacking both possible nuclear localization signals . This mutant is severely affected in nuclear transport of its DBP . In contrast to the 39-81 as region, insertions and deletions in the very N-t portion of DBP (aa 2-38) depressed DBP activities . Deletion of this entire region in dl(2-40) resulted in the failure to isolate infectious virus suggesting that this alteration is lethal . Removal of part of this segment in H5d1807 (aa 26-50) resulted in a partially defective virus that exhibited decreased yields of infectious virus and altered plaque morphology (Table 2) . Since the synthesis and accumulation of the H5dl807 DBP is similar to Ad5, the above phenotype must be due to partial loss of DBP activity, rather than simple reduction in the amount of this protein . The deleted region unique to H5d1807 (aa 26-38) contains the sequence (Ser) 2 ProSerProSer(Pro), . A region with a similar serine- and proline-rich character can be found in the N-t domains of most adenovirus DBP genes (Vos et al., 1988), although not always in the same relative position . Loss of this region might influence DBP activity, perhaps due to the removal of potential phosphorylation sites of this highly phosphorylated protein . An insertion at as 2 in H5in80O also led to a partially defective phenotype (Table 2) . This implies that the N-t part of the 2-38 as segment is also important for DBP function . Perhaps the very N-t residues, that exhibit sequence conservation between serotypes (Quinn and Kitchingman, 1984 ; Kitchingman, 1985 ; Vos et al., 1988), are important in the properfolding of the protein . However, if DBP folding is altered in H5in800, it has not affected the stability of the protein . In summary, structural alterations in the highly conserved C-t half of DBP dramatically depressed the protein's activity . On the other hand, mutations in the poorly conserved N-t domain are more readily tolerated . For instance, the region between as 39 and 81 appears to be completely dispensable, while more N-t alterations result in partially defective DBPs . ACKNOWLEDGMENTS This research was supported in part by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (NWO) to J .S .S . and by Public Health Service Grant A123691 from the National Institutes of Health to D .F .K . D .F .K . is a recipient of Faculty Research Award 270 for the American Cancer Society .

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