Adenovirus 6 genome types: Mapping of restriction site alterations on the genome

(~) INST1TUTPASTEUR/ELSEVIER Paris 1989

Res. ViroL 1989, 140, 545-550

ADENOVIRUS 6 GENOME TYPES: MAPPING OF RESTRICTION SITE ALTERATIONS ON T H E GENOME T. Adrian (*) and U. Wolf

Abteilung fiir Virologie, Universitiitskliniken, Haus 47, D-6650 Homburg, Saar (FRG)

Human adenoviruses (AV) have been classified into the subgenera A-F with respect to biophysical, biochemical and immunological characteristics (Wigand et al., 1982). The differences between the subgenera are well correlated with the DNA sequence heterology, as probed by hybridization or restriction site mapping (Garon et al., 1973 ; Green et al., 1979) and, more recently, by DNA restriction analysis of adenovirus prototypes 1 to 41 (Adrian et al., 1986). Besides the great number of prototypes, numerous AV variants were described (Adrian et al., 1989) ; these findings raise the question of the molecular basis of the genetic variability of adenoviruses. In a previous report (Adrian et al., 1985), we presented DNA restriction analyses of 14 genome types found in 26 isolates, which point to the high genetic variability of subgenus C adenoviruses and, in the present paper, we locate the variations on the genome. AV6 prototype (Ton99) and the 24 wild-type isolates were listed previously (Adrian et al., 1985). The 14 genome types found in these 24 strains, and the proposed designation of the representative strains, are shown in table I. The enzyme code contains the different restriction patterns ("restriction variants" or "R-variants") for individual enzymes in alphabetical order, as proposed previously (Adrian et al., 1985). R-variant 1 corresponds to the pattern of the prototype. The physical maps of the enzymes B a m H I , BglII, EcoRI and HindIII served as a reference (Naroditsky et al., 1980) for the mapping of the BstEII, KpnI and Sinai fragments with the following techniques:

Submitted August 21, 1989, accepted September 4, 1989. (*) Present address: Nationales Referenzzentrum fiir Adenoviren, Institut for Virologie und Seuchenhygiene, Medizinische Hochschule, Postfach 610180, 3000 Hannover 61 (FRG).

T. A D R I A N

546 TABLE I.

Genome type D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 Dll D12 D13 D14

Strain code Ton99 2580 3812 4757 2501 331 1508 21862 10027 3497 13304 1039 119491 127931

-

-

ET AL.

Genome types, representative strains and enzyme code of AV6 isolates.

Origin Place

Year

Washington, D.C. 53 Netherlands 61 Netherlands 62 Netherlands 63 Netherlands 64 Netherlands 65 Netherlands 67 Netherlands 71 Netherlands 72 Netherlands 74 Netherlands 79 Netherlands 82 Hannover 82 Hannover 82

Enzyme code (*) 1 1 1 1 1 1 1 1 2 1 1 1 1 1

1 2 2 2 1 3 1 2 4 1 1 5 2 6

1 2 3 3 1 1 1 4 5 2 I 1 1 1

1 1 2 1 1 3 1 1 4 1 1 1 1 1

1 1 1 1 1 2 1 3 4 1 1 5 1 1

1 2 2 2 3 4 1 5 6 2 1 1 2 1

1 1 1 1 2 2 3 (**) 4(**) 4 (**) 1 5 1 1 1

Number of isolates 6 1 1 2 3 1 2 2 1 1 1 1 1 1

(*) Enzyme order: BamHI, BgllI, BstElI, EcoRI, HindlII, KpnI, Sinai. DNA restriction patterns correspond to those shown in figs. 2 and 3 in a previous publication (Adrian et aL, 1985). (**) The numbers of Sinai variants (3) and (4) were reversed in with respect to data in Adrian et aL (1985).

1) identification o f terminal fragments was based on digestion with the nuclease Bal31 (Legerski et al., 1978); 2) the left or right position o f each terminal fragment was determined by hybridization with 32p-labelled D N A fragments o f some o f the four mentioned enzymes (Rigby et al., 1977; Southern, 1975); 3) the linear a r r a n g e m e n t o f the internal restriction fragments was based either on hybridization experiments or was deduced f r o m double-digest patterns, using a second restriction e n z y m e with k n o w n cleavage sites. A s u m m a r y o f the restriction site m a p p i n g is given in figures 1 and 2. Figure 1 shows the restriction site mapping o f the p r o t o t y p e (below the line), and the altered sites o f the R-variants (above the line) for each o f the seven endonucleases. A comparison o f the altered restriction sites for all g e n o m e types, summarized for all enzymes, is shown in figure 2. Deviations f r o m AV6 prototype D1 vary between one site (D7, D11, DI4) and multiple sites (D6, D8 and D9). Eleven g e n o m e types (fig. 2A) apparently f o r m genomic clusters (Li and Wadell, 1988), as they have between 88 and 98 °70 c o m m o n restriction sites. Within the groups, the g e n o m e types D1, 5, 7, and 14 as well as D2, 3, 4, and 10 are particularly closely related (95 to 98 070 for each group). O n the other hand, D6, D8 and D9 have only 70 to 87 070 sites in c o m m o n with the clusters and 71 to 80 070 c o m m o n sites a m o n g themselves (table II). It appears possible that g e n o m e types o f the cluster have arisen by stepwise

ADENOVIRUS 6 GENOME TYPES

547

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FIG. 1. - - Restriction site maps o f A V6 variants. Below lines: physical maps o f prototype (D1); above lines: arrows show new (1) or lost (I) sites. N u m b e r s m a r k the restriction variants according to the enzyme code in table I. For mapping, all enzymes were purchased f r o m Boehringer M a n n h e i m (FRG) and applied according to published protocols o f this company.

TABLE II. - - P e r c e n t a g e o f c o m m o n

Cluster I II

Genome type(s) 5, 7, 11 12, 14 2-4, 10, 13 8 6 9

Cluster I 92-97a

restriction sites.

Compared with: Genome type II 8 6 9

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70-71

55-57

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72-75 77 80

56-58 59 53 57

84-87

a: D5 and DI2 of cluster I shared only 92 % of common restriction sites; all other genome types shared 95-97 °70. b : main value = 90-93 070.

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FIG. 2. -- Restriction site alterations o f A V6 genorne types. A) Comparison of DI (prototype) with the genome types of the genomic cluster, as indicated by symbols. Arrow without symbol: all indicated genome types have a common altered restriction site. a=common B s t E I I site of D2-4 and DI0; b=common B g l l l site of D2-4 and D13. B) Comparison of D1 with genome types D6, 8 and 9, which have multiple altered restriction sites. 1= new sites, 1= lost sites. Numbers below the lines : 1 = BarnHI ; 2 = BglII ; 3 = BstEII ; 4 = E c o R I ; 5 = H i n d I I I ; 6 = K p n I ; 7 = Sinai.

m u t a t i o n f r o m the p r o t o t y p e or f r o m t w o ancestor strains, which m a y have evolved by recombination. The origin o f the 3 deviating genome types m a y be by r e c o m b i n a t i o n f r o m u n k n o w n ancestor strains. Our results give no hints to the existence o f " h o t s p o t s " on the genome, as suggested b y Aird e t al. (1983) for AV2 at the hexon gene (0.52-0.61). On the contrary, the variations appear to be randomly distributed over the genome, as s h o w n for AV3 g e n o m e types ( O ' D o n n e l l e t a l . , 1986) and A V I , 2 and 5 genome types (Adrian e t a l . , 1989), with the exception o f b o t h terminal regions. The variability o f AV6 (30 o f a total o f 74 sites were f o u n d variable in only 24 isolates studied) is at least as high as in the other types o f subgenus C, i . e . , AV1, 2 and 5 (Adrian e t a l . , 1989), although A V 6 occurs m u c h m o r e rarely. M o r e o v e r , several o f the g e n o m e types o f A V 6 exhibit the same extent o f genetic heterogeneity as do AV1, 2, 5 and 6 p r o t o t y p e s a m o n g

A D E N O V I R U S 6 G E N O M E TYPES

549

themselves, which show between 61 and 77 070 co-migrating fragments (Adrian

et al., 1986). KEY-WORDS: Adenovirus, D N A , G e n o t y p i n g ; Physical maps, Restriction site alterations, Classification.

RI~.SUMI~. TYPES GI~NOMIQUESD'ADI~NOVIRUS6: CARTOGRAPHIEDES ALTI~RATIONS DU SITE DE RESTRICTIONDU GI~NOME

Des &udes ont 6t6 r6alis6es afin de d&erminer les profils de restriction de 14 g6nomes d'ad6novirus, type 6. Les cartes g6nomiques ont 6t6 d6termin6es avec 7 enzymes de restriction (BamHI, BglII, BstEII, EcoRI, HindIII, KpnI et Sinai). La carte g6nomique du prototype 6tait disponible pour 4 enzymes; 11 g6nomes forment un groupe, avec un pourcentage de 88 fi 98 °70 de sites communs. Les g6nomes de types D6, 8 et 9 ont seulement 70 h 87 07o de sites communs avec le groupe pr6c6dent et 71 tt 80 °7o de sites communs entre eux. Les virus appartenant au groupe g6nomique commun pourraient avoir 6volu6 par mutations ponctuelles. Les 3 autres types .g6nomiques pourraient r6sulter d'une recombinaison entre des souches ancestrales lnconnues. MOTS-CLI~S:Ad6novirus, ADN, G6notypage; Cartes physiques, Modification des sites de restriction, Classification.

ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of Barbara Best and the photographic work of Ines Meusel. We also thank Dr R. Wigand for helpful criticism. The study was supported by grants from the "Deutsche Forschungsgemeinschaft" (Wi 3/21-3) and the "F6rdererverein der Deutschen Vereinigung zur Bektimpfung der Viruskrankheitein", Munich (FRG).

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