VIROLOGY 186,496-506

(1992)

Analysis

of Immediate-Early

Transcripts

of Equine Cytomegalovirus

BOONYOS RAENGSAKULRACH AND JOHN STACZEK’ Department

of Microbiology

and immunology,

Louisiana State University

Received June 7, 199 1; accepted

Medical Center, Shrevepon

October

Louisiana

71130-3932

16, 199 1

Equine cytomegalovirus (ECMV) contains a linear, double-stranded DNA genome composed of a 146-kbp unique region flanked by a pair of l&kbp direct repeat (DR) sequences at the termini. Cycloheximide, actinomycin D, and phosphonoacetic acid were applied to infected cell cultures to divide viral transcription into immediate-early (IE), early, and late phases. Eight IE transcripts were identified and mapped to two regions (I and II) of the viral genome. Two of these IE RNAs (13.0 and 5.5 kb in size) were transcribed from region I, which is located within the DR regions; these IE genes are diploid. The other IE transcripts (17.0, 9.0, 7.2, 6.8, 4.5, and 4.2 kb) originated from region II. IE region II is adjacent to region I and spans both unique and DR sequences at the left terminus of the genome. Region II IE transcripts are spliced and transcribed in the opposite direction from region I IE transcripts. IE transcripts from region I were present throughout the replication cycle, whereas those from region II were more abundant during the IE stage than at the early and late stages of infection. These studies demonstrate that ECMV differs from other herpesviruses in o 1992 Academic PUS, I~C. the organization and unusually large transcription units of its IE genes.

moter (Cherrington et a/., 199 1; Thomsen et al., 1984), enhancer (Cherrington eta/., 1991; Ghazal eta/., 1987; Hunninghake et al., 1989; Sambucetti et al., 1989; Thomsen et a/., 1984), and modulator (Lubon et al., 1989; Mach et al., 1989) elements. Furthermore, viral transacting factors that upregulate the HCMV IE gene have been identified through the use of regulatory-reporter gene constructs (Spaete and Mocarski, 1985). While much progress has been made in describing and understanding HCMV IE gene regulation, much less progress has been made in understanding viral gene regulation of nonhuman cytomegaloviruses (Staczek, 1990). Murine cytomegalovirus (MCMV) transcription is coordinately regulated (Misra et al., 1978). Several viral mRNAs, ranging in size from 1.05 to 5.1 kb, are detected at IE times and originate from specific areas of the genome (Dorsch-HBsler et a/., 1985; Keil et a/., 1984, 1985; Koszinowski et a/., 1986; Marks et al., 1983; Spector, 1985). MCMV early transcripts originate from all areas of the genome except map position 0.278-0.305. Although the major MCMV IE RNA is detected throughout the early phase, it decreases in abundance (Marks et al., 1983). The most abundant late transcripts mapped to a region spanning 0.440-0.770 map units. Transcription of the guinea pig cytomegalovirus genome (GPCMV) has recently been characterized (Yin et a/., 1990). Seventeen GPCMV IE transcripts have been identified. Yin et a/. (1990) have suggested that the major GPCMV IE genes may be placed collinearly with other major CMV IE genes including the 1.95-kb IEl and 2.25-kb IE2 RNAs of human CMV (0.739 to 0.755 map units; (Stenberg et al., 1984, 1985; Stinski, 1983), the 2.5-kb IE94

INTRODUCTION Transcription in herpesvirus-infected cells is operationallydivided into three phases: (1) during the immediate-early (IE) phase, the first de ~OL/Oviral transcripts are made in the absence of concurrent protein synthesis; (2) the early (E) phase is initiated by IE gene products before viral DNA replication; and (3) late (L) RNAs are transcribed abundantly after the commencement of viral DNA synthesis. For parochial reasons, the gene regulation of human herpesviruses has been the focal point of many laboratories. One transcriptional theme that emerges from these studies is the reliance of viral gene expression on the initial transcription and translation of a few select genes during the IE phase. Consequently, the identification and characterization of IE gene products have become a priority. The cascade regulation of genes in several strains of human cytomegalovirus (HCMV) is well documented (DeMarchi, 1981; Wathen et al., 1981; McDonough and Spector, 1983) and several IE gene regions have been identified (Wathen and Stinski, 1982). The IE genes that encode the most abundant IE transcripts are located in the unique long region of the HCMV genome (Stenberg eta/., 1989; Stinski, 1983; Stinski et al., 1983). These IE transcripts are post-transcriptionally modified (Stenberg et al., 1984, 1985) and their translation products are readily detected in infected cells (Gibson, 1981; Michelson-Fiske et a/., 1977; Stenberg et al., 1989; Stinski, 1983). Analyses of the upstream regions of the IE genes have identified pro’ TO whom reprint requests should be addressed. 0042-6822/92

$3.00

Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.

496

497

ECMV IE TRANSCRIPTS I I 0.2

I I 0.1

I 0

I I 0.5

1 I 0.4

I I 0.3

I I 0.6

I I 0.6

I I 0.7

I I 0.9

I 1 .o

DR

U

DR

Map Units

Structure 18.0

kb9

145.0

&@,K,

F

kbl,

18.0

, , L

G

QH

A

N

I, 20.5

1

D I

4.2

8.0

15.0

,p, 1 I

B

3.4

24.0

4.8

19.0

D

E

I

M I

kbp Fl2-

0

c

’

I

BamHl

3.6 2.1

I I 3.0!

I I li.7

5.9

II I, 4.5!

III III 2.6

21.5

I I

A 60.0

I I

I 15.0

I 13.5

K2@ II

6.5

Hindlll

12.0

FIG. 1. Structure of the ECMV genome and restriction endonuclease maps. The second line illustrates the ECMV genome, which consists of a 146-kbp central unique sequence (U) flanked by a pair of 18-kbp direct repeats (DR) at both termini. BamHl and HindIll restriction maps of the genome are shown on lines 3 and 4. Uncloned fragments are circled. El (bottom left) and C4 (bottom right) are homologous BarnHI-HindIll subfragments of BamHl E and C, respectively. The BamHl F region (middle) is enlarged to show detailed maps. Fl , F2, and F3 are subclones of the BamHI F. Jl, J2, and J3 are subclones of the F2 (HindIll J). H, B, and S on the bottom map represent restriction sites for HindIll, BarnHI, and Smal, respectively. Sizes in kilobase pairs are shown under each fragment.

RNA of simian CMV (0.71 to 0.73 map units; Jeang et a/., 1984) and the 2.75-kb iel and 1.75-kb ie2 RNAs of murine CMV (0.769 to 0.815 map units; Keil et a/., 1984, 1987a,b). Equine cytomegalovirus (ECMV or equine herpesvirus type 2) is a ubiquitous infectious agent that has been associated with conjunctivitis and respiratory diseases in horses (Browning and Studdert, 1988; Staczek, 1990). Like other cytomegaloviruses, ECMV is associated with latent and persistent infections in its natural host. In tissue culture, ECMV demonstrates a restricted host-cell tropism; only cells of horse, rabbit, or cat are permissive for viral replication. Infection of permissive cells leads to delayed cytopathology, formation of cytomegaly, and a low titer of virus (Wharton er a/., 1981). In contrast, infection of hamster embryo cells with uv-irradiated ECMV at a high multiplicity of infection results in the coestablishment of oncogenic transformation and/or persistent infection (Staczek er a/., 1984). ECMV has been used in our laboratory as a model for studying the molecular biology of herpesvirus infection. The genome of ECMV is a double-stranded DNA with a buoyant density of 1.716 g/ml that corresponds to a guanosine-cytosine content of 57.7% (Wharton et a/., 1981). It contains 182 kbp organized into a 146-kbp central unique region bracketed by 18-kbp direct repeated (DR) sequences at both termini (Colacino et al., 1989; Raengsakulrach, 1991; Raengsakulrach et al.,

1992). Therefore, the ECMV genome resembles those of channel catfish virus (Chousterman era/., 1979) and human herpesvirus 6 (Martin et al., 1991). In this article, we describe the transcription of ECMV IE genes. The ECMV IE transcripts are characterized by their map locations, strand specificity, precursorproduct relationship, and kinetics of expression. These studies suggest that the ECMV IE transcription is complex and involves two large transcription units, one of which is diploid. MATERIALS AND METHODS Virus and cell culture The LK strain of ECMV (Plummer and Waterson, 1963) was propagated and assayed for infectivity in RK-13 (rabbit kidney) cells as described previously (Colacino et a/., 1989). RK-13 cells were cultured in Eagle’s minimal essential medium supplemented with penicillin (100 U/ml), streptomycin (100 pg/ml), nonessential amino acids, and 5% fetal calf serum. Virus infection and use of metabolic inhibitors RK-13 cells at 95-l 00% confluence were infected with ECMV (multiplicity of infection = 5-l 0 PFU/cell), and poly(A) RNA was isolated from the infected cells. To prepare IE RNA, cells were treated with cycloheximide (CH, 100 pg/ml) for 1 hr prior to infection with

498

RAENGSAKULRACH

AND STACZEK

B

A

M IE E L

M IE E L

M IE E L

9.0

9.0

7.1 6.0

7.2 6.8 5.6

M IE E L

M IE E L

M IE E L

BamHl G M IE E L

BamHl Q M IE E L

BamHl H M IE E L

5.b 4.5

BamHl E M IE E L

BamHl Fl M IE E L

BamHl J M IE E L

8,6 7.0 5.7 50

6.6 62 5.6 4.9 41

3.7

23

BamHl K

BamHl F

BamHl L

BamHl N

BamHl A

BamHl I

FIG. 2. Northern blot analysis of ECMV mRNA by BarnHI fragment probes. Poly(A) RNA was isolated from mock-infected cells (M) or infected cells under immediate-early (IE), early (E), and late (L) conditions. Following electrophoretic separation, the RNA was transferred onto GeneScreen Plus filters. The DNA probes indicated at the bottom of each autoradiograph were either cloned or gel-purified uncloned BarnHi fragments. Sizes of transcripts are shown to the right of each panel in kilobases.

ECMV; RNA was isolated at 4 hr postinfection (hr p.i.). Under this condition, greater than 96% of protein synthesis was inhibited (data not shown). To accumulate early transcripts, RNA was harvested at 30 hr p.i. from infected cells that were maintained in the presence of phosphonoacetic acid (PAA, 100 Kg/ml). Under this condition, PAA inhibits ECMV DNA synthesis by greater than 99% (Flowers, 1989). Late RNA was isolated at 30 hr p.i. from ECMV-infected cells maintained in medium without metabolic inhibitors. Preparation

of poly(A) RNA

Poly(A) RNA was prepared by a modified procedure of Badley et al, (I 988). Cells were incubated in lysis buffer (0.2 M NaCI, 0.2 M Tris-HCI, pH 7.5, 1.5 mM MgCI,, 2% SDS, and 200 pg/ml proteinase K) for 1-2 hr at 45”. Subsequently, the NaCl concentration of the

lysate was adjusted to 0.5 M. Oligo (dT) cellulose equilibrated in binding buffer (0.5 M NaCl and 10 mM TrisHCI, pH 7.5) was added to the lysate. The mixture was rocked gently at room temperature for 20 to 60 min. After centrifugation, the pellet was washed twice with binding buffer and loaded into a spun-column (1 ml disposable syringe with glass filter at the bottom, Sambrook et a/., 1989). The column was centrifuged at 1000 g for 5 min and washed with binding buffer. The poly(A) RNA was eluted by adding warm elution buffer (10 mM Tris 7.5, 65”) to the column. The eluate was collected, and the poly(A) RNA was precipitated in ethanol and stored at -70”. Northern

blotting

Poly(A) RNA was fractionated by electrophoresis into 1.2% agarose gels containing gel-running buffer (0.2 M

499

ECMV IE TRANSCRIPTS

C M IE E L

M IE E L

M IE E L

Hybridization with riboprobes

.4.7

-2.4

BamHl D

BamHl P

BamHl B

M IE E L

M IE E L

M IE E L

6.4

BamHl M

65” and the blots were washed according to the protocol of the manufacturer (NEN). The hybridization solution contained 10% dextran sulfate, 1.O% SDS, 1 M NaCI, and 1 X 1O6cpm/ml denatured probe. The blots were washed, air-dried, and exposed to Kodak X-Omat film with intensifying screens at -70”.

5.2

6.5 5.5

40

4.5

BamHI C

BamHlO

FIG. P-Confinued

morpholinopropanesulfonic acid, 50 mM sodium acetate, and 5 mlLl EDTA, pH 8.0) and 2.2 Mformaldehyde (Sambrook et a/., 1989). In all cases, 5 pg of RNA samples was dissolved in gel-running buffer containing 50% deionized formamide-15% formaldehyde and heated for 15 min at 55” before loading onto the gel. The size markers included a 0.24- to 9.5-kb RNA ladder (BRL, Gaithersburg, MD). After electrophoresis, the RNA in the gel was transferred to GeneScreen Plus membranes (New England Nuclear [NEN], Boston, MA) by a capillary blot procedure. Hybridization with DNA probes Cloned DNA fragments or uncloned restriction fragments of ECMV DNA purified from the agarose gels with a GENECLEAN kit (Biol 01, La Jolla, CA) were labeled with r’P]dNTP by random priming (USB, Cleveland, OH). The labeled DNA probes were chromatographed through Sephadex G-50 and recovered by ethanol precipitation. Hybridization was performed at

SP6 or T7 RNA polymerases were used to generate strand-specific riboprobesfrom DNAfragments cloned into the pGEM-3Z vector (Promega, Madison, WI). In vitro transcription included 0.5 pg of template DNA, [a-32P]CTP(800 Ci/mmol, NEN), and reagents supplied in the riboprobe Gemini II kit (Promega). Following the transcription reaction, the DNA templates were removed by digestion with DNase (0.12 pglpl). The solution was extracted once with phenol-chloroform, once with chloroform, and chromatographed through a Sephadex G-50 spun-column. The riboprobes were recovered by ethanol precipitation and resuspended in TES buffer (10 mM Tris-HCI, pH 7.4, 5 mM EDTA, and 1% SDS). Hybridization was performed at 65” in a solution of 50% formamide, 10% dextran sulfate, 1% SDS, 100 pg/ml yeast tRNA, and 2-5 X lo5 cpm/ml riboprobe. Following hybridization, blots were washed once each time with 2X SSC (0.3 M sodium chloride and 0.03 M sodium citrate) at room temperature (RT) for 5 min, 1X SSC-0.19/o SDS at RT for 30 min, 1X SSC-0.1% SDS at 70” for 30 min, 1X SSC-0.1% SDS at RT for 30 min, 1X SSC-0.19/o SDS at 70” for 30 min, 2X SSC at RTfor 15 min, 2X SSC plus 100 pg/ml RNase A at RT for 30 min, and 2X SSC at RT for 15 min. The blots were air-dried and autoradiographed. RESULTS Cascade regulation of ECMV transcription The eclipse period of ECMV in RK cells in approximately 24 hr (Wharton et al., 1981). Viral DNA synthesis commences at approximately 14 hr p.i. (Colacino et a/., 1989). As with other herpesviruses, ECMV transcription can be divided into immediate-early, early, and late phases (Flowers, 1989). Poly(A) RNA isolated from ECMV-infected cells maintained under conditions designed to accumulate each class of viral transcripts was analyzed by Northern blot hybridization (Fig. 2). IE transcripts were defined as viral RNAs present at 4 hr p.i. during a CH block. Early transcripts were defined as RNAs detected under early, but not under IE conditions. Late RNAs were detected only under late, but not under IE or early conditions. BarnHI fragments representing 100% of the ECMV genome [Fig. 1; (Colacino et a/., 1989; Raengsakulrach, 1991; Raengsa-

500

RAENGSAKULRACH

AND STACZEK

TABLE1 ECMV MRNA MAPPING TO INDIVIDUAL ECMV RE FRAGMENTS ECMVBamHl RE fragments (m.u.)

EarlymRNA (kb)

L(O.22-0.25) G(0.25-0.31) Q (0.31-0.33) H (0.33-0.38) A (0.38-0.54)

13.0, 5.5, 13.0-5.5 smear 17.0.8 9.0, 7.2, 6.8, 4.5 17.o,a9.0, 7.2, 6.8, 4.5 17.0,' 9.0, 7.2, 6.8, 4.5, 4.2 -

N (0.54-0.56) 1(0.56-0.61)

-

D (0.61-0.69) P(O.69-0.71) B (0.71-0.84)

-

M (0.84-0.87) C (0.87-0.97)

13.0, 5.5, 13.0-5.5 smear 17.0.* 9.0,

E (0.00-0.08) Rl (0.08-0.09) J (0.09-0.12) K(O.12-0.16) F(O.16-0.22)

R2 (0.97-0.98) 0 (0.98-1.00)

'This

IEmRNA (W

transcript

is detected

3.0, 2.4 -

7.1, 5.6 6.5, 5.6, 0.9

2.7

1.9, 1.2

6.6, 5.1, 2.9, 1.7 11.5, 5.1, 3.5, 2.5, 0.8

2.0 12.5, 4.0, 2.8, 1.3 12.5 6.2 8.8, 6.6, 4.4, 2.5, 1.2 3.7, 2.3 4.7 2.1

5.2, 4.0, 1.1 4.5, 1.3

3.4, 2.7, 2.0, 1.5 10.5, 5.8, 1.0 10.5 11.5, 7.6, 3.3 8.0, 3.1, 1.6, 1.0 8.6, 6.6, 3.0, 6.6, 5.7, 9.3, 2.9, 9.2, 6.5,

7.0, 6.2, 2.5, 2.4 4.8, 8.5, 2.4, 6.4 3.1,

13.0, 5.6

5.7, 5.0, 1.5, 0.9 5.6, 4.9, 4.1, 1.8 2.1, 1.4 6.6, 5.1. 4.2, 1.7, 1.0 2.4, 1.8 -

7.1, 5.6

7.2, 6.8, 4.5 17.0,# 9.0, 7.2, 6.8, 4.5 upon extended exposure

4.6

Late mRNA kb)

1.5

of the autoradiograph.

kulrach et a/., 1992)] were used as probes. These hybridization results (Fig. 2) are summarized in Table 1. During a CH block, two groups of IE transcripts were identified (Fig. 2). Group I consisted of easily identifiable 13.0- and 5.5-kb transcripts and a smear ranging from 13.0 to 5.5 kb (referred to as the 13.0-5.5 smear). These group I transcripts originated from the DRs (BarnHI C and E). Group II IE RNAs included the readily detectable 9.0-, 7.2-, 6.8-, and 4.5-kb transcripts. The group II IE RNAs originated from the DR (BarnHI Rl and J) and nearby unique sequences (BarnHI F) associated with the left terminus. Because the BarnHI K probe (BarnHI K is located between BarnHI J and F) did not hybridize to any of the group II transcripts, these group II transcripts must be spliced. Most regions of the viral genome were transcribed during early (PAA block) and late times. At least 33 early and 63 late ECMV transcripts were identified (Table 1). However, this count may be an underestimation for several reasons. Some of the probes (e.g., the 29.5kbp BarnHI A) were large and probably hybridized to several discrete transcripts of similar size. Hybridiza-

tion of the radioactive probe to these closely migrating bands might produce a smear that would not be included in the enumeration of transcripts from the Northern blots or a diffuse band that would be scored as a single band rather than several discrete bands. Furthermore, the distinction between early and late RNAs was defined by the presence or absence of drug inhibitor. This procedure does not permit quantitative discrimination of early and late gene products. The specific aim of this study was to demonstrate temporal regulation rather than to quantify RNAs. Some of the IE gene regions were also used for transcnbrng early and late RNAs. For example, a 5.6-kb early and a 2.7-kb late transcript originated from the BarnHI J region (Fig. 2). Transcription of IE, early, and late RNA species from the same region throughout the infection indicates a complex pattern of regulation. IE RNA mapping

by ECMV HindIll

probes

To more precisely map the genes encoding the IE transcripts, we used the ECMV HindIll fragments as

ECMV IE TRANSCRIPTS IE

Hindlll

L

IE

L

Hindlll IE

IE

L

Hindlll

K

L

IE

Hindlll

C IE

L

L

501 IE

H

Hindlll IE

L

L

IE

J

Hindlll

L

G

L

9.3

6.4 5.3

2.9

tlindlll

E

Hindlll

I

Hindlll

F

FIG. 3. Northern blot analysis of ECMV mRNA by Hindlll fragment probes. IE and L mRNAs were analyzed as detailed in the legend to Fig. 2. Probes from the left terminus are HindIll L, K, C, H, J, and G (top row). Probes from the right terminus are HindIll E, I, and F (bottom row).

probes for Northern analysis of IE poly(A) RNAs (Fig. 3). Late RNAs were also included on the blots. Overall, these results agree with the transcription maps deduced when the BamHl probes were used. However, the Hindlll J probe also detected an additional 4.2-kb group II IE transcript. This band has been detected in several blots probed with the HindIll J or subclones of the Hindlll J (data not shown). Furthermore, the 4.2-kb transcript is detected if the immediate-early conditions are extended beyond the 4 hr of CH block routinely used in this laboratory (Fig. 6). Data obtained from these IE RNA mapping studies are summarized diagrammatically in Fig. 4 and detailed below. Localization

of group I and group II IE transcripts

To map the group I IE transcripts more precisely, the BamHl C of the right DR was subcloned as Cl (BarnHIHindlll fragment adjacent to BamHl M), C2 (HindIll I),

C3 (Hindill K2), and C4 @amHI-HindIll fragment adjacent to BamHl R2, Fig. 1). The C4 is approximately 6 kbp and was subcloned by the restriction endonuclease Smal into six subfragments (2.05, 1.8, 0.7, 0.6, 0.4, and 0.3 kbp). All of these C4 subclones hybridized to the 13-and 5.5kb IE transcripts and to the 13.0-5.5 smear (data not shown). We predict that the 5.5-kb IE transcript is derived primarily from the C4 region. However, this 5.5-kb RNA band may represent multiple species of same-sized transcripts generated by alternative splicing. For example, a faint 5.5-kb RNA band was also detected by the HindIll L probe (Fig. 3). This transcript might be the same 5.5-kb transcript identified by the C4 subregion probes. To address this possibility, more detailed subclones and analysis for RNA splicing are required (in progress). To determine whether the group I IE transcripts map only to the DR region, we used H13.5 and H13.0

502

RAENGSAKULRACH

AND STACZEK /,

DR

Kbp

16.0

E 13.0

u

RlJ I I 2.1

K I 7.0!

I

LKl I I 3.0

3.1

F

6.1

1 I

16.7

H I 6.9

w------f+ i-y-----+-y

IE Region I

-----j-l ______ *

J

14.51

I

DR

Kbp

M

B I

//

i

C

!

'

11.5

146.0

BamHl /I

L

I

//

G I

Hindlll // ,,

Kbp

R20 I I

C I

4.0

16.0

19.0

2.1

3.6 I

I I

I

E I

K2 I II

Fi

I

13.5

lo.0

7.2 6.8 4.5 4.2 IE Region I : .,:.;;;.,c I

IE Region II

FIG. 4. Transcription map of ECMV IE genes. Structure at the termini of the ECMV genome is shown on the top, followed by BarnHI and Hindlll restriction maps on lines 2 and 3. Uncloned fragments are circled. El is homologous to C4. Arrow lines indicate the map position and direction of IE transcripts. Arrowheads show the 3’ end. Horizontal dashed lines represent putative intron sequences of the transcripts. Sizes in kilobase pairs are shown next to the transcripts. The 5.5-kb transcript is the most abundant IE species. The presence of the 4.2.kb IE transcript is questionable since it is not always detected. Two IE transcription units are designated IE regions I and II.

(HindIll-SalI subfragments of HindIll I) as probes in northern hybridization analyses (Fig. 5). The Sal1 restriction site in HindIll I was used previously to set the junction between the unique and the DR region at the right genomic terminus (Raengsakulrach et al., 1991). A large IE transcript (13.0 kb or slightly larger) could be detected with the H13.5 probe upon prolonged autoradiographic exposure. This large transcript might represent a very minor IE RNA species that extends through the unique and DR junction. When typical autoradiographic exposures were analyzed (Fig. 5) the H13.5 probe did not hybridize to the 13.0-5.5 smear, whereas the H13.0 probe did. The H13.0 (3.0 kbp) probe contains DNA sequences homologous to HindIll L (also 3.0 kbp) in the left DR. We concluded that the genes encoded by the group I IE transcripts (13.0, 13.0-5.5 smear, and 5.5 kb) were located entirely within the DR regions of the viral genome. The group II IE transcripts were detected by the HindIll J probe but not by its flanking fragments WindIll H and G (Fig. 3). Similar results were obtained when subclones of BarnHI F (Fl, F2, and F3; Fig. 1) were used. The F2 (HindIll J) hybridized to the group II IE transcripts, while the Fl and F3 did not (data not shown). These data positioned the group II IE tran-

scripts within the /-/indIll J of the BarnHI F region and suggested that these transcripts were spliced (Fig. 4). The HindIll C and F fragments encompass parts of both IE regions I and Il. When the HindIll C and F fragments were used to probe the IE RNAs (Fig. 3), IE transcripts from IE region I (13.0 and 5.5 kb) were more readily detected and, therefore, presumably more abundant than those from region Il.

Precursor-product transcripts

relationship between IE

Multiple species of IE RNAs are transcribed from each of the ECMV IE regions. Experiments were performed to determine whether smaller transcripts were derived from larger transcripts. CH and actinomycin D (Act D) were employed to control the level of transcription (Fig. 6). CH was present from 1 hr before infection to the time at which poly(A) RNAs were isolated. Act D was added to infected cells at 3 hr p.i., and poly(A) RNA was isolated at 3, 4, 5, and 6 hr p.i. (Fig. 6B). For comparison, another set of IE RNAs was isolated from infected cells maintained in medium supplemented with CH (Fig. 6A). The C4 and BarnHI F probes, representing IE region I and II, respectively, were used to detect the IE transcripts.

ECMV IE TRANSCRIPTS

503

DR (Right)

A

B 4

3 --

E

K2

I I

I

II

F

5

6

4

6

9.0

IE

h&i.

s.0 7.2 8.6

7.2 6.6

L

4.5 4.2

L 4.5 4.2

13.0 13.0

BamHl 6.8 5.5

6.5 4.5

3

4

F 5

BamHl 3

6

4

F 5

6 h&i.

4.5

c4 H13.0

H13.5 FIG. 5. Localization of group the direct repeat. Radioactive H13.0) of the HindIll I restriction hybridize to IE and L transcripts. detailed in the legend to Fig. 2.

5

Hindlll

H13.5 H13.0 I I SalI IE

3

I immediate-early transcripts within HindIll-SalI subclones (H13.5 and enzyme fragment were permitted to IE and L mRNAs were analyzed as

During the CH block, the band intensity of each IE RNA species increased with time (Fig. 6A). During 3 hr of Act D block from 3 to 6 hr p.i., all IE transcripts appeared to be stable (Fig. 6B). Between 4 and 6 hr p.i. of Act D block, the ratio among the IE transcripts originating from either IE region I or II did not change. Therefore, we concluded that the smaller transcripts were not processed products from the larger transcripts. However, the transcripts must contain overlapping exon sequences because the summation of the lengths of the transcripts exceeds the coding capacity of each IE region. Levels of the 4.5-kb transcript from region II appeared to increase from 3 to 4 hr p.i. (Fig. 6B). What caused this increase was unknown since there was no reduction of the levels of other IE RNA species observed. We predict that this increase took place before Act D exerted its full transcription inhibitory effect because Act D was added to the infected cells at 3 hr p.i., which is the same time that the first set of RNAs was isolated. Time course of transcription

from the IE regions

The C4 and HindIll J probes were used in Northern hybridization analyses to examine the transcription from IE regions I and II throughout the viral replication cycle (Fig. 7). Poly(A) RNAs were isolated from infected cells at 4, 10, 15, 30, and 48 hr p.i. The 13.0- and

c4

FIG. 6. Precursor-product relationship among the IE mRNAs. The C4 and BamHl F DNA, representing the IE regions I and II, respectively, were used as probes in Northern hybridization. The amount of RNA loaded per lane was standardized by ethidium bromide staining (data not shown). (A) IE Poly(A) RNAs were isolated at 3, 4. 5, and 6 hr p.i. from ECMV-infected cells maintained in the presence of cycloheximide from 1 hr before infection to the times of RNA isolation. (6) Another set of poly(A) RNAs was isolated as described for A except that actinomycin D was added to infected cells at 3 hr p.i. and kept until the times of RNA isolation.

5.5-kb transcripts from IE region I were detected by the C4 probe at each time point examined, with slight increases in the band intensity for each successive inter-

4

10

15

c4

30

48

4

10

15

Hindlll

30

48

h.p.i.

J

FIG. 7. Time course of transcription from IE gene regions. Poly(A) RNA isolated at various times after infection was analyzed by Northern hybridization. Equal amounts of RNA were loaded into each lane. The C4 and Hindlll J probes represent the IE regions I and II, respectively. Sizes of the transcripts are shown to the right of the autoradiographs.

RAENGSAKULRACH

504 IE L

from the same strand at both IE and late times p.i. Interestingly, while one strand of IE region II was transcribed at IE times, both strands were utilized at late times.

IE L

IE L 13.0

2.4

CCds

C4-SP6

c4-T7 IE L

9.0 Z2 6.8

Jl-ds

Jl-Ti’

FIG. 8. Strand specificity of the IE transcripts. Single-stranded riboprobes were used in Northern hybridization to determine the directions of the IE transcription. Riboprobes were obtained from in vitro transcription of the C4 or Jl DNA that had been cloned into pGEM32 vectors. SP6 and T7 designate which RNA polymerase was used to synthesize riboprobes. Double-stranded (ds) probes were prepared by randomly primed labeling of the DNA clones. The C4 and Jl DNA represent the IE regions I and II, respectively.

val in the replication cycle. A 2.4-kb RNA was transcribed abundantly from the C4 region at 30 and 48 hr p.i., which is consistent with the previous classification of this transcript as a late transcript (Table 1, BarnHI C and E regions). In contrast, the IE transcripts from IE region II were more abundant at earlier stages of infection (4, 10, and 15 hr p.i.) than at later stages. The 9.0and 4.5-kb transcripts were detected at high levels at 4 hr p.i. and then declined thereafter. The highest level of the 7.2- and 6.8-kb IE transcripts was observed at 10 hr p.i. At later times (30 and 48 hr p.i.), other species of RNA were predominantly transcribed from IE region II. Strand-specificity

AND STACZEK

of the IE transcripts

Riboprobes were obtained by the in vitro transcription of the C4 and J1 fragments cloned into the pGEM32 vector. C4 (BarnHI-HindIll fragment) and Jl (HindIll-Smal fragment, Fig. 1) represent IE regions I and II, respectively. Autoradiographs of blots containing IE and late RNAs probed with double-stranded (ds) probes or riboprobes are shown in Fig. 8. These data indicate the directions of the IE transcription as shown in Fig. 4. Transcripts from IE region I were generated

DISCUSSION In these experiments, we have confirmed that ECMV transcription, like that of other herpesviruses, is sequentially regulated. At least 8 IE, 33 early, and 63 late RNA species were identified and mapped to specific regions of the viral genome. Two of the IE transcripts (13.0 and 5.5 kb) that originate from the DR regions are located at the termini of the genome. Therefore, these IE genes are diploid and are designated group I IE genes. In contrast, the genes for the IE RNA 17.0-, 9.0-, 7.2-, 6.8-, 4.5-, and 4.2-kb transcripts are haploid and are designated group II IE genes. The group II IE genes are located next to IE region I at the left DR and encompass adjacent unique regions. Transcription from IE regions I and II occurs in opposite directions. Because of the proximity of IE regions I and II at the left genomic terminus, it is likely that these IE genes use the same regulatory-enhancer region for their transcription. The arrangement of ECMV IE genes is similar to the major IE gene cluster of murine CMV (0.7690.81 8 map units) in which iel (2.75 kb) and ie3 (1.O to 5.1 kb) RNAs are transcribed in an opposite direction from the ie2 (1.75 kb) RNA (Keil et al., 1987a,b). Among the ECMV IE RNAs, the 5.5-kb IE region I transcript is the most abundant species as indicated by the band intensity on the Northern blots. Using subclones of IE region I as probes, we mapped this major IE transcript to the C4 and El regions of the viral genome (Fig. 4; the BarnHI-HindIll digestion product of BarnHI C and BarnHI E, respectively; the El fragment is identical to C4 by hybridization and restriction enzyme analyses; data not shown). We predict that the smear observed between the 13.0- and the 5.5-kb IE RNA bands on the Northern blots (Figs. 2, 3, and 5) is derived from the extension of the 5.5-kb transcript into the HindIll Kl and L regions of the left genomic terminus or into the corresponding regions of the right terminus (Fig. 4). This extension may terminate at various positions within these regions. Another possible cause of the 13.0-5.5 smear is the existence of complicated patterns of splicing for the group I IE transcripts. Note that the HindIll L also hybridizes, albeit faintly, to a 5.5-kb RNA band (Fig. 3). This suggests that a small exon sequence(s) of the 5.5-kb IE transcript may be present in the HindIll L region. From IE region II, five species of IE transcripts are detected consistently and unambiguously. The detection of the 4.2- and the 17.0-kb species is more variable. The 4.2-kb species is detected more consistently when the immediate-early conditions are extended beyond the 4 hr used for CH block (compare Figs. 6

505

ECMV IE TRANSCRIPTS

and 2) and when subclones of the BamHl F and HindIll 1 are used (data not shown). On the other hand, the 17-O-kb species can be seen more often after longer autoradiographic exposure times (data not shown). Mapping data for the IE region II transcripts suggest that a large region corresponding to the 8.9-kb HindIll H is spliced out to generate the smaller IE RNA species. This intron is relatively large; however, it is not unusual if compared to the intron sequences present in some Epstein-Barr virus genes [for example, EBNA (nuclear antigen) genes; see (Kieff and Liebowitz, 1990)]. During the ECMV replication cycle, the expression of each IE region is differentially regulated. The region I IE RNAs persist throughout the infection, while the region II IE RNAs are more abundant at the early stages of infection. Furthermore, the levels of each species of region II IE RNAs (9.0 and 4.5 kb; 7.2 and 6.8 kb) vary individually during the infection (Fig. 7). The variation in abundance of region II IE RNAs may be due to differential regulation at the transcriptional level. For example, the 9.0- and 4.5-kb transcripts may use the same RNA start sites and, therefore, may be subjected to the same transcriptional controls. Alternatively, modulation in transcript abundance may occur at the posttranscriptional level (e.g., RNA degradation). During the late phase of virus replication, the abundance of all region II IE RNAs declines. Concomitant with this decline is the detection of RNAs originating from the opposite strand of the IE template. It is tempting to speculate that these late transcripts, which are antisense to the region II IE RNAs, may play a role in modulating the expression of the region II IE genes. It should be noted that late RNAs from this IE region II [HindIll J (Fig. 3) and its collinear BamHl F (Fig. 2)] appear as distinct bands against a highly smeared background. This high background may reflect complicated patterns of splicing and/or the existence of both sense and antisense transcripts. We have demonstrated previously that ECMV has a genome structure (Raengsakulrach er al., 1991) that is distinct among equine herpesviruses (EHVs). The other EHVs (including EHV-1, EHV-3, and EHV-4) have genomes that are topographically similar and collinear. Their genomes are characterized by a long unique component linked to a short component consisting of a unique sequence bordered by inverted repeat sequences (Atherton et a/., 1982; Baumann et a/., 1986a,b; Cullinane et al., 1988; Henry et a/., 1981; Sullivan et a/., 1984). With respect to transcription, EHV-1 encodes one 6-kb IE RNA species within the inverted repeat regions of its genome (Gray et al., 1987a,b); likewise, a single 5.7-kb IE RNA species has been reported for EHV-3 (Sullivan et a/., 1990). In contrast, ECMV encodes multiple IE RNA species.

In conclusion, ECMV appears to be distinct from other EHVs or CMVs in genome structure and IE gene organization and expression. Our other recent studies have suggested that ECMV has a genome structure that resembles the genomes of CCV and HHV-6 (Raengsakulrach et al., 1991); thus, it will be of interest to see whether the IE genes of these three viruses are similar or different. ACKNOWLEDGMENTS We thank Susanna Ponder for technical assistance and Joseph Colacino and Catherine Breeden for critical reading of the manuscript. This study was supported by Public Health Service Grant Al21 996 from the National Institutes of Health, Biomedical Research Support Grant 2S07RR05882-05, an American Cancer Society Institution Grant, and a grant from the American Heart Association.

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