G~ATl`[xtATTGTTT~G~GGGAGGGGGCTACAGGGCG~CGGGGTCC~CGC~TC~AG~CGCT~GTAGTGCCGG~GG~G. AAATAA. GAT. H pa I I poly(A). gII- terml nat ...
J. gen, Virol. (1989), 70, 1239 1246.
Printed in Great Britain
1239
Key words: P R V/glycoprotein gll/repeat units
Pseudorabies Virus Displays Variable Numbers of a Repeat Unit Adjacent to the 3' End of the Glycoprotein glI Gene By A R T U R S I M O N , T H O M A S C. M E T T E N L E I T E R AND HANNS-JOACHIM RZtHA* Federal Research Centre for Virus Diseases of Animals, P.O. Box 1149, D-7400 Tiibingen, F.R.G. (Accepted 4 January 1989)
SUMMARY
The coding region of the glycoprotein complex glI of pseudorabies virus (PRV) is located in the unique long part of the genome on Sail subfragments 1A and 1G of BamHI fragment 1 (map units 0.105 to 0.130). Fragment 1G which includes the 3' end of the gII gene displays a size heterogeneity among different PRV strains and also within plaque isolates of a given strain. To reveal the cause of this heterogeneity and whether it might affect the gII-coding region we sequenced different l G fragments of the PRV strains Ka, Phylaxia and Dessau, and determined the 3' end of the glI mRNA by SI analysis. These data show that the size heterogeneity is caused by the presence of a variable number of tandemly repeated DNA sequence downstream but adjacent to the coding region of the glycoprotein gII gene. The 3' end of the gII mRNA was mapped about 24 bp upstream of the first repeat unit. A 15 bp sequence 5' G G G A C G G A G G G G A G A 3' is repeated from three to over 50 times in different 1G fragments. It is the only repeat unit present in strain Ka, whereas the Phylaxia and Dessau strains show additional modifications. Pseudorabies virus (PRV) or suid herpesvirus 1 (Roizman et aL, 1981) contains a linear, double-stranded DNA genome of approximately 150 kbp. It is composed of two sets of unique sequences, UL and Us. Us is bounded by inverted repeats and is found in two orientations relative to UL. After PRV infection several glycoproteins are synthesized and incorporated into the viral envelope and the plasma membrane of the infected cell. One glycoprotein, gX, is secreted into the culture medium (Rea et al., 1985). Up to now, six glycoprotein genes have been mapped on the viral genome and sequenced (Mettenleiter et al., 1985a, 1986; Petrovskis et al., 1986a, b; Rea et aL, 1985; Robbins et al., 1986, 1987; Wathen & Wathen, 1984). The genes for gI, gp63, gp50 and gX reside in Us whereas the genes for gII and gIII are localized in UL (Mettenleiter et al., 1986; Robbins et al., 1986, 1987). The glycoprotein gII, a homologue of the herpes simplex virus type 1 (HSV-1) glycoprotein gB, is a major constituent of the viral envelope (Hampl et al., 1984; Luk~tcs et al., 1985) and is represented by a complex of three related glycoproteins that are derived from a common precursor protein (Mettenleiter et al., 1986). Two of these glycoproteins are considered to arise by proteolytic cleavage of the larger precursor polypeptide and are linked by disulphide bonds (Hampl et al., 1984; LukS.cs et al., 1985). The structural gene for the glI complex has been mapped to Sail subfragments 1A and 1G of BamHI fragment 1 (map units 0.105 to 0-130) (Mettenleiter et al., 1986), and the 3' end of the coding region has been located in Sail fragment 1G (Robbins et aL, 1987). BamHl fragment 1 of PRV DNA, which encompasses the gene for the glycoprotein glI, is about 30 kbp in size. In order to map the glI gene more precisely Sail subfragments ofBarnHI 1 of PRV strain Ka (Kaplan & Vatter, 1959) have been cloned into pBR325 and designated 1A to IH according to their size (Mettenleiter et al., 1986) (Fig. 1). After separation in agarose gels the 0000-8710 © 1989 SGM
Short communication
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Fig. 1. (a) Physical map of the genome of PRV. Locations of the BamHl restriction fragments are indicated. (b) Sail subfragments of BamHI fragment 1 are shown (Mettenleiter et al., 1986). Arrow denotes the open reading frame of glycoprotein glI. (c) HpalI (H) restriction map of Sail (S) fragment IG. Boxes represent the reiteration in HpalI fragment A. (a) 1
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Fig. 2. Size heterogeneity of Sail fragments 1G of different PRV isolates. DNA of PRV strain Phylaxia (lane 1), three single plaque isolates (lanes 2 to 4) of strain Ka, strain A57 (lane 5), Phylaxia reisolated after passage in cattle (lane 6) and swine (lane 7), two single plaque isolates of strain Dessau (lanes 8, 10) and parental Dessau virus stock (lane 9) was isolated and digested with either BamHI and Sail (a) or Sail alone (b). After separation in an 1-0~ agarose gel fragments were transferred onto nitrocellulose and hybridized with 32p-labeUed cloned fragment 1G (a) or fragment 1H (b). SalI subfragment 1G did not a p p e a r as one distinct band but seemed to consist of two submolar restriction fragments. Both fragments were isolated and cloned. In Southern blot analyses these two fragments cross-hybridized with each other in contrast to the other SalI fragments, which suggested that two size variants of the same SalI fragment 1G had been cloned. To examine this heterogeneity more closely P R V D N A of different origins was digested with SalI or SaII/BamHI and blot hybridized with the 32p-labelled, cloned fragment 1G. In the different virus isolates tested, including the different P R V strains Phylaxia, A57 and Dessau (Mettenleiter et aL, 1985 b), and several single plaque isolates of strains K a and Dessau, the labelled fragment 1G recognized fragments varying in size between 0.9 and 1-6 k b p (Fig. 2). This size heterogeneity
Short communication
1241
Table 1. Repeat units in Sall fragment 1G Type
Sequence
Type I
5' GGGACGGAGGGGAGA 3'
Type II
5' GGGACGGGGGGGGGAGA 3" b
Size (bp)
PRV strain
15
Ka, Phylaxia, Dessau
17
Phylaxia
a
Table 2. Different 1G fragments cloned in M13mpl9 Clone
Size (bp)
Repeat numbers
Type
MKGI MKG2 MKG3 MPG3 MPGt MDGI MDG2
1166 1136 596 1378 674 863 788
41 39 3 54 8
Type I Type I Type I Type I, type II Type I Modified type I Modified type I
was found in fragment 1G only and not in the other Sail subfragments of BamHI fragment 1. For example, fragment 1H with a size similar to 1G and located immediately adjacent to it (Mettenleiter et al., 1986) exhibited one distinct size in all viral DNAs tested (Fig. 2). To analyse the size heterogeneity of fragment 1G in more detail we cloned BamHI fragment 1 of virus strains Ka, Phylaxia and Dessau into pBR325. After Sali digestion the resulting 1G fragments were isolated and cloned into phage M13mp19 (Norrander et al., 1983). The recombinant phages MKG1 and M K G 2 contained K a 1G fragments of 1166 bp and 1136 bp, respectively. Fragment 1G of Phylaxia DNA, 1378 bp in size, was obtained in MPG3. Dessau 1G fragments of 863 bp and 748 bp were cloned giving rise to M D G 1 and MDG2. After plaque purification spontaneous deletions led to the isolation of a smaller Ka 1G fragment of 596 bp (MKG3) and a smaller Phylaxia 1G fragment of 674 bp (MPG 1). Thus, we were able to isolate seven 1G fragments of different size from three PRV strains. After HpalI digestion of MKG1 and M K G 2 the size heterogeneity could be localized in the large HpalI fragment A (Fig. 1) of both 1G fragments indicating that the variability is contained in a distinct part of fragment 1G. To reveal the cause of the size heterogeneity we sequenced the HpalI fragment A of all seven cloned Sail 1G fragments and in addition one complete 1G fragment of each strain to confirm that no other site might contribute to the size difference. Sequencing was done by the dideoxy chain termination method of Sanger et al. (1980) using [35S]dATP (Biggin et al., 1983), and deoxy-7-deazaguanosine triphosphate (Boehringer) instead of d G T P to avoid sequencing artefacts (Mizusawa et al., 1986) due to the high G + C content of PRV DNA. Sequencing of both strands of the HpalI fragment A of the K a 1G fragment of M K G 3 (596 bp) revealed the existence of three tandem repeat units of the 15 bp sequence 5' G G G A C G G A G G G G A G A 3' in its middle part. The same repeat units were found in the larger Ka fragments in MKG1 (1166 bp) and M K G 2 (1136 bp) and were designated type I repeat units (Table 1). In a 6 ~ wedgeshaped sequencing gel the reiteration was resolved as far as possible. To determine the overall length of the repetition the easily recognizable repeated fragment pattern in the A track was counted and resulted in the determination of 39 and 41 repeat units for M K G 2 and MKG1, respectively. In 1G fragments of Ka D N A the first repeat unit starts 261 bp downstream from the Sail site separating fragment 1G from fragment 1A (Fig. 1). The 6 bp incomplete type I repeat sequence 5' G G G A C G 3' precedes the first repeat unit. The last repeat unit is followed by a duplication of the 6 bp sequence 5' G G T C G C 3'. These border sequences separate the repeated from the non-repeated area in fragment 1G (Fig. 3). Sequence analysis of both strands of the smaller Phylaxia fragment 1G cloned in M P G 1 (674 bp) revealed eight units of the same basic type I repeat as in Ka. By sequencing the largest fragment MPG3 (1174 bp), we detected a different fragment pattern interspersed seven times
1242
Short communication 10 20 30 40 50 60 70 •TCGA•TC••CC•C•GCCCCC•CCACCCCCGCGGTCCCCGCCCAG••C•AGCCC•ACCACGT•TACCA•CACCCCCGCCC
80
Sail 90 100 110 120 130 140 150 GCGGA•CG•GCGGACG•CGG•CTGTACCAGCA•C•cCGACCCGTCATCGACCT•AcCGG•CACCGcG•GTCGCGC•GCA HpaII 170
180
190
200
210
220
160
230
240
AGAGCTGGCGCGTGTGACGGCCCCGCGGACCCCCCGTTGTATGGTTTCCCCCCGTTGCCCCCGTGTGTGGAM~.T~GTT 250
260
270
280
290
300
310
TTTTTCT/~TTCTGTACACACGGCGTGCGTGTCATCGTGGTCGC GGGACGGAC,GGGAGA GGGACGGAGC,C~AGA GGG border a a 320 330 340 350 360 370 380 390 ACGGAGGGGAGA GGGACGGAGGGGAGA GGGACGGAGGGGAGA CC,GACGGAC,C,C~AGA C a ; G A C G G A ~ G A GGG a
a
a
400 410 ACC,C,ACC,GC,ACA ~ A ~ A G G C G A C , a
a
470
480
a
a
420 430 44Q A ~ACCGA~AGA----~C(;A~AGA
a490
[
500
450
i
b
510
460 I~ A C G G A C G C G A G A G
520
a
530
540
cc^cc,ca_c,o~c^ Ic c ~ _ eoc.~_.c~ca x, I occ^c~Aoccc^o^F c o ~ ^ ~ ^ G ^ l a
[
b
J
a
t
,,
b
c,c,c^c0cA_c,c,cc^ l
a
550 560 570 580 590 600 610. 6 GA C,C~ACGGAC~7~AGA CI?X;ACGGA~GA GGGACC,GAGGGGAGA C~ACCAgAGGGC~GA GGGACC,C~GC,GGA a
a
20 630 CA ICCC,A ~ A G A I [
a
a
640 650 660 C,GC,ACC,GAC,C,C GAGA [ C C C ^ ~ A G A
I~
I
a
[
b
a
670
680
ICG , G^CGG^GGGG^G^
I
690 GC,GAC.GGAG
a
a7
700 710 720 730 740 750 760 GGGAGA GGGACGGAGGGGAGAGGGACC~AGGGGAGA GGGA~AGGGCAGA GGGACGGAGGGGAGAGC,GACC,GAG a
70
a
780
790
a800
a
810
1~2Q
a830
840
L~--GAGA]CGGA _CGGC, C~GC~_ACA]GC~ACCC , A_GGGC^G^G I C,CA_CC, C ~ ^ C A l GGCACGGACG , C~AG^GGGAC l t~ J a [ b l ~ -
-
860 870 880 890 900 910 9 GGAGGGGAGA GGGAC,C,GAGGGGAGA C,GGACGGAGGGGAGA GGGA~AGGGGAGA GGGACGGAGGGGAGAGC,GAC 850
20 a 930 950 a 960 990 a 940 a 970 980 a GGAGGGGAGA GC,GACGGAGGGGAGA GC~ACGGAC,C,GGA(;A GGGACGGAGC,C~AGA GGGACGGAGGGGAGAGGGAC a
a
a
a
a
1000 1010 1020 1030 1040 1050 1060 10 GGAGGGGAC~. GGGACGGAGGGGAGAGGGACGGAGGGGAGAGGGACGGAGGGGAGA C,GGAC_.GGAGGGGAGAGGGAC a
a
a
a
a
70 1080 1090 1100 1110 1120 1130 1140 GGAGGGGAGA CGGACGGAGGGGAGAGC~ACC,GAC,GC,GAGA GGGACGAGACGGACGCGAGACGTGTTGCCA~CAAGC a a a border AC AC 3'-end rnRNA 1150 1160 1170 1180 1190 1200 1210 1220
G~ATl`[xtATTGTTT~G~GGGAGGGGGCTACAGGGCG~CGGGGTCC~CGC~TC~AG~CGCT~GTAGTGCCGG~GG~G AAATAA
GAT Hpa II g I I - terml nat i o n - c o d o n < ............................
poly(A)
1230 1240 1250 1260 1270 1280 1290 1300 CGTGGCCATCGCCCCGACGCGGCTGGCCAGCA•CGCGG•CCCGCTGTTCTTCTTGC•CGCCTTGTGCTCCTGCTGCTCGA
