Characterization of a highly transcribed DNA region of herpesvirus of turkeys

361

Gene, 79 (1989) 361-367 Elsevier GEN 03038

Characterization (Protein

of a highly transcribed DNA region of herpesvirus of turkeys

A; multiple

nucleotide

RNA

species;

Marek’s

disease;

recombinant

DNA;

gene library;

phage

I vectors;

sequence)

Pradip K. Bandyopadhyay Synergen, Inc., Boulder, CO 80301 (U.S.A.)

Received

by J.A. Engler:

Revised:

5 February

Accepted:

15 November

Tel. (303)938-6200:

Fax (303)938-6270

1988

1989

7 February

1989

SUMMARY

A highly transcribed DNA region of turkey herpesvirus (HVT) codes for multiple species of RNA. Although the RNA is transcribed from both strands of DNA, no overlapping complementary transcripts have been detected. The complete nucleotide sequence corresponding to the cDNA of one of the HVT transcripts was determined. This sequence is highly homologous to the nucleotide sequence of Marek’s disease virus antigen gp57-65-coding gene and appears to represent the corresponding HVT-coding gene.

INTRODUCTION

Herpesvirus of turkeys (HVT), an apathogenic field isolate from turkeys, is antigenically related to MDV, the herpesvirus that causes MD in poultry. Vaccination with HVT strain FC126 (Witter et al., 1970) protects chicken against MD (Okazaki et al., 1970; Purchase et al., 1971; 1972). HVT has a ds DNA genome of about 155 kb (Ross, 1985) and

Correspondence to: Dr. P.K. address:

1645

14th

Street,

Bandyopadhyay, Boulder,

CO

at his present 80302

(U.S.A.)

Tel. (303)443-8223. Abbreviations:

aa, amino acid(s); bp, base pair(s); CEF, chicken

embryo tibroblast;

ds, double strand(ed);

gen; HVT, herpesvirus MD, Marek’s otide(s);

oligo,

frame; protein

disease;

of turkeys;

MDV, Marek’s

oligodeoxyribonucleotide; A, MDV-A

0378-l 119/89/$03.50

antigen;

0 1989 Elsevier

gp57-65, MDV-A anti-

kb, kilobase disease

or 1000 bp; virus; nt, nucle-

ORF,

open

reading

ss, single strand(ed).

Science Publishers

B.V. (Biomedical

shares considerable homology to the nucleotide sequences of MDV (Buckmaster et al., 1988). Antigenie determinants common to both MDV and HVT have also been described (Ikuta et al., 1983a; Silva and Lee, 1984). Little is known about the pattern and control of gene expression in MDV- or HVT-infected CEF. The MDV gp57-65-coding gene has been characterized and sequenced by Coussens and Velicer (1988). It is now possible to study the various aspects of the control of expression of this gene. This protein, also identified as MDV-A antigen, is antigenitally related to an HVT-coded protein, HVT-A. To study the details of the control of expression of an HVT-coded gene, a highly transcribed region of HVT DNA was identified. In this report, the isolation and characterization of this highly transcribed region of the HVT genome has been described. The nucleotide sequence of this region was found to be highly homologous to the nucleotide sequence of the MDV-A antigen-coding gene. Division)

362

EMBL3-Q8 T7

b

PROMOTER

SP6

a:: i

\

pGem 10

\

\

Fig. 1. Maps of cloned HVT DNA. (a) Map of i EMBL3-QS. I DNA. The HVT DNA fragments

obtained

QB, QC, and QD in the text) in order of decreasing with respect

to the T7 and SP6 promoters

EXPERIMENTAL

t

1 EMBL3-QS

size. (b) Map of plasmids

to indicate

orientation

AND DISCUSSION

PROMOTER

1. SalI SP6

The solid line represents

by digesting

is shown. A, B, C, D correspond

and A. D next to the dots have been included

1

57

Sal I

0:

the vector

PROMOTER

PROMOTER

HVT nucleotide

pGEM2

and pGEMl0.

to the Sal1 fragments

and the dotted lines

as A, B, C, and D (QA,

The orientation

of QC and QD

shown in a, above. Fragments

of D and C in these plasmids

m

sequences

with Sal1 are designated

in the context

12345

B, C

of the HVT DNA.

67

8

4 origin

(a) Analysis of infected cell RNA A genomic library of HVT DNA, from strain FC126, was constructed in vector I EMBL3. The library consisted of overlapping 15-20-kb HVT DNA fragments inserted into BarnHI-cleaved 1 EMBL3 DNA (Frischauf et al., 1983). DNA from members of the HVT-EMBL3 library were digested with SaZI. Southern (1975) blot analysis of this DNA with a cDNA probe prepared from total RNA isolated from CEF infected with HVT (Maniatis et al., 1982) indicated that nucleotide sequences contained in 1EMBL3Q8 were highly transcribed, in particular fragment C, designated Q8.C (Fig. la). DNA fragments C and D (Q8.C and Q8.D) were isolated from a Sal1 digest of 1EMBL3-Q8 DNA and used to probe Northern blots of poly(A)+ RNA isolated from HVT-infected CEF. Fig. 2, lanes 1 and 2, show

-( 28s -18s

Fig. 2. Northern-blot from HVT-infected

analysis

pathy.

The 32P-labeled

lanes:

1, Q8.D;

pGEM10 merase; pGEM2

probes

2, Q8.C;

transcribed

from HindHI-digested 5, transcript

with T7 RNA

used for the various

3, transcript

pGEMl0

transcribed

from HindHI-digested

from

6, transcript

with SphI.

60% cytolanes are:

SacI-digested 4, transcript

with T7 RNA polypGEM2 from

with SP6 RNA polymerase;

ment from Q8.C digested Q8.C digested

poly(A) + RNA isolated

with SP6 RNA polymerase;

polymerase;

transcribed

of total

CEF when the cells exhibited

with SphI; 8, minor

transcribed

SacI-digested 7, major fragfragment

from

363

Hind111 was transcribed with T7 RNA polymerase and DNA linearized with Sac1 was transcribed using SP6 RNA polymerase. (T7 and SP6 RNA polymerases were purchased from Promega Biotec and radioactive transcripts were synthesized using [ IX-~~P]CTPaccording to procedures recommended by the vendor.) 32P-labeled transcripts were used to probe Northern blots of poly(A)+ RNA isolated from CEF infected with HVT (Zinn et al., 1984). Fig. 2, lanes 3, 4, 5, and 6 show the results of such an analysis. T7 RNA polymerase transcripts of both pGEM 10 and gGEM2 hybridize to multiple species

that both Q8.C and Q8.D

a ~~~8~~~

\p. (1)

Xho I

(374) ATG

Q8C

(737)(1019) Sal1 Sal I

(1493) (1835)

(2450)

------I{

(2827)

1

lkb

.

.

.

l

. Fig. 3. Partial

maps of HVT DNA. (a) Detailed

are shown (see Fig. la). The two transcripts whose nucleotide and stop codons cDNAs.

have been reported

of the largest ORF. The position

The major and minor fragments

for transcript primer.

sequences

I and its 5’ sequences.

Sequences

oligos as primers.

for the complete

map of region of HVT DNA used in the sequencing

in opposing

orientations

here. The numbers

are marked represent

of the poly(A)-addition

nt positions,

cDNA

of Suu3AI

subfragments

in both orientations

(b) Schematic

of the cDNA

were determined

Q8.C and part of Q8.A

box shows the region of DNA

the ATG (374) and TAG (1493), the start

sites has been determined

within SphI digest of Q8.C has been indicated. Sequences

studies.

I and II. The blackened

from the sequence of overlapping

were obtained

from a full-length

of the respective

sequences

obtained

using the M13mp18

universal

clone using appropriate

synthetic

364

of RNA of similar molecular size. The SP6 RNA polymerase transcript pGEM10 hybridizes primarily to a RNA species about of these experiments HVT. Nucleotide

indicate

sequences

major transcript To localize

that both strands

of

in CEF infected with complementary

to one

in Q8.C is part of a family of tran-

scripts, four or more ranging 6 kb to approx.

in size from approx.

1.8 kb, the other strand codes for a of approx. regions

analysis

1.8 kb in size. The results

DNA in Q8.C are transcribed of the strands

(b) Nucleotide sequence determination and sequence

nucleotide

primers

for the

synthesis

of the various

RNA species, Q8.C DNA

fragment

was digested with SphI and the two sub-

fragments, approx. 1.4 kb and 2.0 kb, were isolated (Fig. la). Radioactive nick-translated probes prepared from both fragments were used for Northernblot analysis of poly(A)+ RNA isolated from CEF infected with HVT. Fig. 2, lanes 7 and 8 show the results of the experiments. The approx. 1.4-kb fragment hybridizes predominantly (approx. 75%) to an approx. 1.8-kb transcript while the 2.0-kb fragment participates in the transcription of the family of four transcripts mentioned earlier. Together with the results described in Fig. 2, lanes 3,4, 5, and 6, these observations indicate that the ,!$hI subfragments of Q8.C are predominantly transcribed in opposite

sequences

and Sequenase

subcloned

in

using synthetic oligos as

(Biggin et al., 1983; Tabor

and Richardson, 1987) purchased from United States Biochemical Corporation, Cleveland, OH. The nucleotide

sequences

tiguous with M13mp18

1.8 kb.

in Q8.C responsible

HVT

M 13mp 18 were determined

determined new primers

of the HVT DNA

using the Ml3 synthesized

con-

DNA in these clones were universal

primer

while

on the basis of previously

determined sequences were used to obtain overlapping nucleotide sequences of the HVT DNA. A schematic representation of overlapping nucleotide sequences in both orientations obtained by means of the complete cDNA of transcript I is shown in Fig. 3b. The isolated cDNA of transcript I was also digested with Sau3A1, subcloned into M13mp18 and the nucleotide sequence determined. Nucleotide

60% in a

sequences from the longest cDNA corresponding to transcript I together with sequences 5’ to it are shown in Fig. 4. Sequences from nt l-465 were derived from a genomic clone XhoI-Sal1 in Q8.A (Fig. 3a) and sequences from nt 280-1835 were obtained from the longest cDNA clone. Sequences between nt 280 and 1835 were determined from both strands of DNA and sequences from nt l-279 were determined from one strand. Each nt from each strand was determined at least twice from separate sequencing reactions. The sequences between nt 280 and 465 obtained from both the genomic and the cDNA clones were identical. Based on this observa-

vector, AZap (Stratagene, La Jolla, CA). The library was screened with probes prepared from the 2.0-kb and 1.4-kb ,SphI fragments of Q8.C. The cDNAs thus identified were subcloned into M 13mp18 (Yanisch-Perron et al., 1985) for sequencing. Both of the SphI fragments of Q8.C were also cloned into M 13mp 18 for sequencing. An XhoI-Sal1 DNA fragment from ;1 EMBL3-Q8 (subfragment of Q8.A) was subcloned into M 13mp18 for sequencing. Fig. 3a shows the map of this region of DNA. Position and orientation of two transcripts are indicated as I and II, respectively. I corresponds to the abundant transcript identified by the approx. 1.4-kb SphI subfragment of QS.C, while II corresponds to one of the transcripts detected by the approx. 2.0-kb SphI subfragment of Q8.C.

with the genomic nucleotide sequences at this position. Sequences from nt 1781-3030 were determined from genomic clones of Q8.C digested with SphI (not shown). The location of the poly(A) signal for transcript II at nt position 2827 was determined by comparing sequences of the corresponding cDNA with the sequences of the genomic DNA in this region. The nucleotide sequence was analyzed using a computer program, PC Gene, from IntelliGenetics, Mountain View, CA. The longest ORF codes for a protein of 373 aa. The hydrophobicity of the predicted protein was analyzed using the methods described by Klein et al. (1985). Fig. 5 shows the plot of the hydropathic index of the predicted protein. A eukaryotic secretory signal sequence is predicted

directions. To further characterize the RNA species transcribed from QS.C, a cDNA library was constructed from total poly(A)+ RNA isolated from HVTinfected CEF. The RNA used was isolated from infected cells when the cells showed approx. cytopathy. The cDNA library was constructed

tion the 5’ end of the cDNA

appears

to be colinear

T

TGC

CGC GAG

GGG TGT

CAA

TTC

GGC

TGC

CM

ATT

GGC AGC AGC

CGG CAT GCT TAC --

TM

TTT

GAT GTC ATG

CAG TGT

b4 * TTG (1 CGT GCT ATT ACC ---

CM

ATG TAT

GTT TTA

&FG

CCC. GGC GGG

A%

CGC MG

TAT

CGC TAC TM

TTG AGC 1 Tnh -- AGA

181

* CAA TGA GGA GCT GCA ATT TM AGC TM +A CCG CAT .-_1_2e--. 77 A& - XTA CGC ATC TAT -CGA -MC TTG TTC GAG - TTA -

ATG GTT TCC AAC ATG MVSNMRSTRTALTGWV

CTA GTT L V

CTG TCT TTA L S L

GAT ACC D T

CAT H

CAT ATC H I

CTA ACT TTC AAC L T _F N

TCA GAG S E

GTG V

CCC MT 1' N

TCG CCT ACG S -P T

CAG Q

GTG

421

CM

GTC V

401

CCT TTG P L

541

ACA GCT T A

GO1

Q

ACC

TCT

TGT

GCC

T

S

C

A

CCT TCT P

ACC GM T E

TTA TCT ACA ACT T L u

CCT GAT ATA ATC TGC GAC D I P 1 c

CGA GAA GAA R E E

GTA TTC GTA TTC V F V F

GTC GAC CCC CCT TCA V D P I'S

CCA ATC I' 1

GCC GGT A G

GGA GTA CCG GGG TCG GnT G V P G S D

CTA TAC L Y

GCG

A

AAG

GAC D

GTA V

CAT ATT Ii I

TAC ATG Y M

CCT CCA GTT I’ P V

cTc AGC L 5

GGA CAA AAC G E N

CCC GGA 'XT P G S

GTC TAC V Y

GTA TCT TGG AGA C& V 5 W 11 R

CGT GAC R D

GGG AGT G S

GGC TAC TTT G Y F

II

701

MT

CGA GTG V

841

TTA

ATA

901

L

I

GTA

N

V

ACA)GGA T G

GAG GTT GCC E V n TCG GM

CCT AGG MA P R K

S

I3

CAA GTG ATA Q V I

CGT GTG T& R V C

CM Q

CTG CGT GAT AGA TTT AAT L F N II D R

CGC CCA T'i'G R P L

TAC ADA GCA TCT TGC ATc Gm Y S C I V K A

12Gl

TTT TGG TGG TTC GAA T;T GGC CGC GGG GCC ACA CTA GTA TCC ACA ATA F W W F E S G R G A T L V S T I

1321

TCT GGA CTC GM S G L E

1381

C&l’

ATA AGC ACA TCC AAT]GCT ACA GCTlGTA CCG ACG GTA TAT TAT I S T S N A T A V P T V Y Y 3.104 CTG GCA TTT &A GAT GGG iv_2'CTG CAG GA; CAT CGA TCG rend L A F K T$C

-A

J-

'3

ATA TI;(LGTT CGA

S

CCA AAG GTT TCC TGC TTG GTA GCG TGG ALG P K V 5 C L V A W K

P

f&A

A$T

CGT GGA TAG

ATT

A:&

FT;r

T;

ATC -

A~~G&$

-&A &AT

sequences

CGA

from the HVT sequences. at comparable

at comparable

positions

in a different

reading

The numbered

sites have been boxed.

TGA

1501

GTG

CAG ACT MT

15Gl

TGC

AAC

lG21

L

GCC

r

AAA

code. The underlined

positions.

starting

TTA

1441

GTC

lGO1 1741

CGA -TCG CAT TCT TCT GTTTCG

1001

MA

1850

AAA MA

ACG GM

TTC

I (Fig. 3a) and region 5’ to it. The largest ORF starts at nt 374. The corresponding arrows Identical

at and extending

aa indicate

identical

above the nucleotide

The numbered

in the MDV sequences. frame

ATA

P

E Y Y D .A T ?;--.% TAC GGC CG; TCT AGG ACT GGC CTT GTT TTT

GCT -- ATT GTA ATC AGC AAA AM

of transcript

ORF are given in the one-letter

in MDV sequence

TTC

CCC

H

TCA

ti CAT TAT CAC $$T_A@&&C--.&G~R~~T&!~ TAT Il--r-.-? ‘GTA TGT GAT ATA i$A ATT ATT MG TGT-- TAT MC--

AT'J SAC MT-AA

Fig. 4. Nucleotide

TGT MC

CAC

yx

-R-N5

""A" MG

1201

GAC D

TCT

AAC GGA AAC ATT GCC ACA CCC CGC MG N G N I A T P R K

GAT ATG D M

G fU

1141

AGA CAT cri?r TAT CCC R II F lz

GGC G

‘KG K

1081

CGA CCC GCA TCC GTG GAT GTA TTG GCC I< PA S \r D V L A

CM Q

CAG

1021

GTT AGA RAT GTC CAC ACC ATG GGC GTG GM V I? N V 11 T M G V 1:

AAC N

CGC

961

CCT ACA CCC CGT GGA MT K I' T 1' G

CTC GGA L G

TCi S

I

TGG TCC .UC TTC GCT CTT GAC F A W S N DL

MT N

ACC T

CGT ATC I -R

K

721

K

TAT GCA TAh Y A Y

ACA ATG T M

GAC TAC MA D Y K GAA MA E K

MC N

MA K

661 S

S

AGA ATT H I

CTTfMC L N

TCC TCC ATT CGA CGG GAT CCC CAG GGT TCT TTC TGG ACT AGT .I S S S I II R D l' 0 G S AAA TAT TTC ATA TGG ATTjAAT K Y F I W I N

CCC CAT MC P H N

GTC GCC ACC MG V A T K

CAC CGCrM II 1~ -N

GAC GAT GM D D E

TAC CAC GCC AAC GM E Y 11 A N

GTC ACG TTC MT V T F N

GGA TTG G L

CCC ATT TCG GCC GAT GGC GTT r A D V P -G r

TCC GM S E

GM

3Gl

CGT ACT GCG CTG ACG GGA TGG

ACT AGT TCC S S 2

TTG TGT GAC CTT ATA L D I c L

301

CGT TCT ACG

GM AGC 1; s

glycosylation

121

CGT GAA _. ._ 1. ATC'GTG ATA GAT CGT CGG TCT GCG CAT-- CGC _~._ -

. ACT -

Gyr CGA MA .CCA TTT TAT

TTT F

ACC

Gl

TM

241

GGC ATA G I

GTA CCG ACG ACT V 1' T T

predicted

TCG

---

CTT TGT

largest

TAT

residues

sequences

lines below the sequences

between

indicate

indicate

MDV gp57-65

the additional

the position

number

of nt present

and number

of nt missing

nt 5’ and 3’ to the ORF have also been underlined. beyond

the C terminus

of this ORF

have

aa in the

and the protein

Identical

aa predicted

also been underlined.

Potential

366

(c) Conclusions 40 30 1

o:& E :T :

s

ZO-

i / Aq /+jb

io-

8

-lO-

I

v’

-20:

-3o-

-4o-

The cDNA transcript

corresponding

to a highly expressed

from HVT has been characterized.

protein

that is highly homologous

to that predicted

for MDV gp57-65. The high homology

-5O-,,,,,,,,,,",,,,'",',,"'"',,'"',,","',,"",,,c 1

70 '40

WI

'lo

280

350

Fig. 5. Plot of the hydropathic index of the HVT protein predicted by the largest ORF in nucleotide sequence in Fig. 4, computed using an interval of 17 aa (Klein et al., 1985). The aa placed above the midpoint line are hydrophobic and those below are hydrophilic.

with a potential

cleavage site between aa residues 25 and 26. There are four potential sites for N-linked glycosylation (Asn-X-Ser/Thr) shown as boxed residues in Fig. 4. The amino acid sequence bears extensive homology to the predicted amino acid sequence

of the MDV protein gp57-65 (Coussens and Velicer, 1988), also known as A antigen. Both MDV and HVT share a number of virally encoded antigenic determinants of which the A antigen is one (Ikuta et al., 1983a; Silva and Lee, 1984). The sequences of the MDV gp56-65 gene were aligned with the corresponding HVT sequences. The predicted site of initiation of the HVT protein is at nt 374 (Fig. 4), the corresponding position for the MDV protein is 24 nt 5’ to this position. The identical aa have been underlined. The MDVgp57-65 gene has an insertion of 104 nt between nt 1469 and 1470 in Fig. 4; this deletion shifts the corresponding ORF further in the MDV than in the HVT. The ORF in HVT terminates at nt position 1493, however, the nucleotide sequence homology continues further downstream (underlined). When a change in the reading frame is allowed to accommodate insertion of the 104 nt in the HVT sequences, the aa homology in the predicted ORF is extended. There is also considerable sequence homology 5’ to the predicted start codons of the MDV and HVT proteins. These sequences have been underlined. However, the positions ofthese sequences are not identical with respect to the predicted start codons.

The

location of the transcript in the HVT genome has also been determined. The sequences code for a to the MDV

gp57-65 suggests that the cDNA reported encode MDV-A

the HVT protein

that corresponds

here may to the

antigen.

It is not possible observations

to conclude

from the above

if the ‘deletion’ observed

in the HVT

cDNA sequences compared to the MDV genomic sequences is characteristic of the strain of HVT used in the present study or a feature of wild-type HVT. In the case of MDV, the gp57-65 gene product is gradually lost, as the virus is passed in cell culture, and it also loses its pathogenicity for chickens (Nazerian, 1984). The strain of HVT used in the present study may be in the process of losing this antigen. However, the presence of this antigen at high passage levels of HVT in cell culture has also been reported (Ikuta et al., 1983b). The characterization of the HVT ‘protein A’ gene makes possible the identification of the transcriptional and translational control elements for the expression of this gene in CEF infected with HVT. A comparative study of MDV- and HVT-coded proteins and the control elements involved in their synthesis,

can now be attempted.

ACKNOWLEDGEMENTS

I thank S.L. Martin for the use of the cDNA library, D.I. Aparisio for technical assistance, J. Vannice and M. King for critical reading of the manuscript, J. Cox and D. Hirsh for support, T. Klein and C. Worland for help in preparing the manuscript.

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regions

T.: Identification

adjacent

gene. Cell 34 (1984) 856-879.

to the human

of two beta-