Identification and characterization of an equine herpesvirus 1 late gene encoding a potential zinc finger

VIROLOGY

188, 704-713

(1992)

Identification and Characterization of an Equine Herpesvirus 1 Late Gene Encoding a Potential Zinc Finger V. ROGER HOLDEN, RAMANA R. YALAMANCHILI,’ Department

RONALD N. HARTY, AND DENNIS J. O’CALLAGHAN2

of Microbiology and Immunology, Louisiana State University Medical 150 1 Kings Highway, Shreveport, Louisiana 7 1130-3932 Received December

9, 199 1; accepted

Center,

February 24, 1992

In this report, we present the DNA sequence and transcriptional characterization of a gene (lR5) that maps within each of the inverted repeat (IR) segments of the equine herpesvirus type 1 (EHV-1) genome. The IR5 open reading frame (ORF) is located within both IR sequences (nucleotides 9932-10,642 of the IR). DNA sequence analyses of the IR5 gene region revealed an ORF of 236 amino acids (24,793 Da) that showed significant homology to ORF64 of varicella-zoster virus and ORF3 of EHV-4 both of which map within the inverted repeats and to the US1 0 ORF of herpes simplex virus type 1 (HSV-1) which maps within the unique short segment. Additional analyses of the nucleotide sequence failed to reveal any overlapping ORFs that would correspond to US1 1 or US1 2 of HSV-1. Interestingly, the IR5 ORF of EHV-1 possesses a sequence of 13 amino acids (CAYWCCLGI-JAFAC) that is a perfect match to the consensus zinc finger motif (C-X2-4-C-X2..,5-C\H-X2-4-C\H). Putative &-acting elements flanking the IR5 ORF include a TATA box (nucleotides 9864-9870), a CAAT box (nucleotides 9709-9714), and a polyadenylation signal (nucleotides 10,64510,650). Northern blot and Sl nuclease analyses identified a single 0.9-kb mRNA species that first appears at 2 hr postinfection, and whose synthesis is reduced in the presence of phosphonoacetic acid, an inhibitor of EHV-1 DNA synthesis. Thus, the IR5 gene of EHV-1 exhibits characteristics representative of a late gene of the y-l class. The characterization of the IR5 gene at the DNA and RNA levels will facilitate ongoing studies to identify and characterize 0 1992 Academic Press, Inc. the IR5 polypeptide.

1987a,b; Grundy et a/., 1989; Harty eta/., 1989, 1991); however, multiple IE proteins have been detected both in vivo and in vitro (Caughman et a/., 1988; Robertson eta/., 1988b). Recently, the EHV-1 IE gene product has been shown to possess transactivating and negative autoregulatory functions similar to those of ICP4 of HSV-1 (Smith et a/., 1992). The gene arrangement within the IR sequence of EHV-1 is unique in comparison to those of other alphaherpesviruses. The IR sequence of EHV-1 contains at least six genes (Gray et a/., 1987a,b; Grundy et a/., 1989; Harty et a/., 1989, 1991; Harty and O’Callaghan, 1991; Holden et a/., 1992; C. A. Breeden, R. R. Yalamanchili, and D. J. O’Callaghan, in preparation; Holden et a/., in preparation), while HSV-1 and varicella-zoster virus (VZV) contain one and three IR genes, respectively (Davison and Scott, 1986; Davison and McGeoch, 1986; MeGeoch et al,, 1985, 1986). Three of the EHV-1 IR genes (IRl, IR4, and IR5) have homologs within the S regions of HSV-1 and VZV. IRl, the only immediate-early gene of EHV-1, (Gray et al., 1987a,b; Grundy et al., 1989; Harty et al., 1989, 199 1) is homologous to lCP4 of HSV-1 and open reading frame 62 (ORF62) of VZV. The IR4 gene of EHV-1 is homologous to the HSV-1 immediate-early gene ICP22; however, IR4 is differentially expressed as a 1.4 kb early transcript and 1.7 kb late transcript (Holden et

INTRODUCTION Equine herpesvirus type 1 (EHV-1) has a doublestranded, DNA genome consisting of two regions: a long (L) region (1 12 kbp) and a covalently linked short (S) region (32 kbp). The S region consists of two identical inverted repeat sequences (IRS = 12.8 kbp) that bracket unique sequences (Us). The IR segments allow the S region to invert relative to the fixed L region, resulting in the formation of two isomeric DNA molecules (Baumann et al., 1986; Henry eta/., 1981; O’Callaghan and Harty, 1992; Ruyechan et a/., 1982; Whalley et al., 1981). EHV-1 gene expression has been shown to be temporally regulated at the level of transcription (Cohen et a/., 1975, 1977; Huang et al., 1971), and the gene products have been classified into three kinetic classes designated immediate-early (IE), early (E), and late (L) (Caughman et al., 1985, 1988; Gray et al., 1987a,b). EHV-1 expresses a single immediate-early gene (IRl) that encodes a 6.0-kb transcript (Gray eta/.,

Sequence data from this article have been deposited with the EMBUGenBank Data Libraries under Accession No. M84521 ’ Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115. ’ To whom correspondence and reprint requests should be addressed. 0042-6822192

$5.00

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

704

705

THE IR5 GENE OF EHV-1

al., 1992). IR2 is an early gene mapping within IRl and may encode a truncated form of the IE polypeptide (Har-ty and O’Callaghan, 1991). Finally, IR3 and IR6 have no homologs in HSV-1 or VZV (C. A. Breeden and D. J. O’Callaghan, in preparation; Holden eta/., in preparation), and their role in infection remains to be ascertained. In this report, we present the DNA sequence of the IR5 gene and show that IR5 is a homolog of US10 of HSV-1, ORF64 of VZV, and ORF3 of EHV-4. RNA mapping analyses revealed that IR5 is transcribed to yield a single 0.9-kb mRNA that is not a member of a 3’coterminal family of mRNAs. Furthermore, analyses of the IR5 ORF revealed a zinc finger motif which was found to be perfectly conserved in the IR5 homolog US1 0 of HSV-1 and ORF3 of EHV-4 and partially conserved in ORF64 of VZV (Cullinane et a/., 1988; Davison and Scott, 1986; McGeoch et al., 1986). These results will be the basis for future work to determine the function(s) of the IR5 protein during EHV-1 replication. MATERIALS Ceils, virus, cloning,

AND METHODS

and sequencing

The Kentucky A strain of EHV-1 was passaged in L-M cells as described previously (O’Callaghan et a/., 1968). The generation of EHV-1 clones has been described elsewhere (Baumann eta/., 1986, 1989; Henry et a/., 1981; O’Callaghan et al., 1981, 1984; Robinson et al., 1981). A series of overlapping subclones of the PCS-1 and PCS-2 region (Fig. l), generated by restriction endonuclease and exonuclease Ill digestion, were sequenced using the dideoxy chain termination method of Sanger et al. (1977) as described previously (Grundy et al., 1989; Colle et al., 1992). For accuracy both DNA strands were sequenced. DNA sequences were compiled and analyzed using IBl/Pustell DNA/ Protein sequence analysis software and PC/Gene software by IntelliGenetics (International Biotechnologies, Inc., New Haven, CT). Isolation

of poly(A) RNA

To isolate immediate-early mRNA, 1 X 1O8 L-M cells were pretreated with 100 pg of cycloheximide (Sigma Chemical Co., St. Louis, MO) per milliliter for 1 hr. The L-M cells were infected with EHV-1 at a multiplicity of infection of 15 PFU per cell, and the cells were harvested at 4 hr postinfection. The cells were maintained in the presence of cycloheximide throughout the infection. To isolate early mRNA, 1 x 10’ cells were treated with 100 pg of phosphonoacetic acid (PAA: Abbot Laboratories, Chicago, IL) per milliliter at 1 hr prior to infection. Infected cells were maintained in the presence of

phosphonoacetic acid and were harvested at 6 hr postinfection. These concentrations of cycloheximide and PAA have been shown to inhibit protein synthesis and DNA synthesis by 98 and 96%, respectively (Caughman et al., 1985). To isolate late mRNA, infected cells were harvested at 8 hr postinfection in the absence of metabolic inhibitors. Purification of poly(A) RNA was accomplished as described previously (Harty and O’Callaghan, 1991). Northern

blot analysis

Northern blot analysis was performed using 5.0 pg of poly(A) RNA fractionated on a 1.2% formaldehyde agarose gel as described by Maniatis et al. (1982). The RNA was blotted onto GeneScreen Plus filters (New England Nuclear) by the method of Southern (1975). EHV-1 subclones were radiolabeled with [(u-32P]dNTPs as described by Rigby et a/. (1978) and hybridized to RNA on the filters as described previously (Gray et al., 1987a,b; Harty et al., 1989; Harty and O’Callaghan, 1991). The filters were washed extensively and exposed to X-OMAT film (Kodak, Rochester, NY) with an intensifying screen at -70°C. Sl Nuclease

analysis

Sl nuclease analysis was carried out essentially as described elsewhere (Berk and Sharp, 1977; Harty et al., 1989; Weaver and Weissman, 1979). Briefly, EHV-1 subclones were digested with the appropriate restriction endonuclease, dephosphorylated with calf intestinal alkaline phosphatase (Promega, Madison, WI), and 5’ end labeled using T4 polynucleotide kinase (BRL, Gaithersburg, MD) and [T-~~P]ATP (NEN, Boston, MA). For 3’ end labeling, subclones were cut with the appropriate restriction endonuclease and end labeled using the Klenow fragment and [a-32P]dNTP (ICN, Costa Mesa, CA). The probes were hybridized to 3.0 pg of poly(A)-selected RNA in 25 ~1 of Sl hybridization buffer { 40 mM pipes [piperazine-/I/,/V’-bis(2ethanesulfonic acid)] [pH 6.41, 400 mM NaCI, 1 .O mM EDTA, 80% formamide) at 65°C for 14-16 hr. The DNA-RNA hybrids were digested in 300 ~1of Sl digestion buffer [0.250 M NaCI, 30 mM sodium acetate (pH 4.6) 1 .O mM ZnCI2, 60 units of Sl nuclease (BRL)] at 37°C for 30 min. The protected fragments were analyzed on 6% polyacrylamide-urea sequencing gels and detected by autoradiography. RESULTS We have reported previously the DNA sequence and transcriptional mapping data for three genes mapping within the IR sequence of EHV-1: (i) IRl , the only imme-

706

HOLDEN

ET AL.

UL

(A)

y

(W

I 9500

9700

I 10100

9900

Mu I

I 10300

I 10500

I 10700

I 10900 nt

PCS-2

I I

stu I uuw’ I I

Sal I I

pcS2Sl

pcs2s2

IR5 mRNA * IFl5oRF 31 1

138 150

236

FIG. 1. Diagram of the EHV-1 genome. (A) The genome is organized into the unique long (UJ, internal repeat (IRS), unique short (U,), and terminal repeat (TRs) sequences. The locations of the genes IRl, IR2, IR3, IR4, IR5, IR6, and origin of replication (Ori) are shown. (B) Relevant IR5 subclones and their restriction sites are shown. Nucleotide numbers indicate the distances from the L-S junction, nucleotide 1 being the first nt within the S region. The location and direction of transcription of the IR5 specific transcript are indicated with an arrow. The IR5 ORF, represented by the box, is 236 amino acids, and a potential zinc finger is indicated by the closed box.

diate-early gene of EHV-1 (Gray eta/., 1987a,b; Grundy et al., 1989; Harty et a/., 1989; Harty and O’Callaghan, 1991); (ii) IR2, an early gene that is embedded within the IE gene (Hartyand O’Callaghan, 1991); and (iii) IR4, an early gene that exhibits homology to the immediateearly gene ICP22 of HSV-1 (Holden et al., 1992). We report here the DNA sequence and transcriptional mapping data for the IR5 gene of EHV-1. IR5 maps within each inverted repeat, contains a potential zinc finger, and exhibits homology to US10 of HSV-1, ORF64 of VZV, and ORF3 of EHV-4. Figure 1 illustrates the location of IR5 with respect to other IR genes of EHV-1. Also shown are the subclones used to generate a series of overlapping M 13-based clones for DNA sequencing. The position of the potential zinc finger lies between amino acids 138-l 50 of the potential IR5 protein. The DNA sequence (Fig. 2) of the IR5 gene region (1 157 nucleotides) has an overall guanine plus cytosine content of 68%, somewhat greater than the genomic guanine plus cytosine content of 57% (Soehner et a/., 1965; O’Callaghan et a/., 1983). The sequence contains an open reading frame of 236 amino acids located within nucleotides 9932 to 10642 of the IR segment. In addition to the first ATG located at nucleo-

tides 9932-9934, a second in-frame initiation codon is positioned at nucleotides 9971-9973. A comparison of the two initiation codons to Kozak’s consensus sequence (Kozak, 1989) revealed that the first ATG is in the more favorable context having a guanine at the f4 position and a guanine at the critical -3 position. Initiation of translation from the first ATG would result in a polypeptide of 236 amino acids with a predicted molecular weight of 24,793 Da. Interestingly, a sequence of 13 amino acids cAYWcCLGHAFAC mapping to amino acids 138-l 50 (nucleotides 10,344-l 0,380), conforms to the C-C-H-C zinc finger motif (Berg, 1986, 1990; Summers, 1991). In addition to the presence of cysteine and histidine residues at positions 1, 5, 9, and 13, the aromatic tyrosine and ttyptophan residues at positions 3 and 4, the glycine at position 8, and the hydrophobic alanine residue at position 10 are commonly found to be conserved in the C-C-H-C zinc finger (Summers, 1991). A comparison of the zinc finger of IR5 to that of Berg’s zinc finger consensus sequence (Berg, 1986) is illustrated in Fig. 3B. A computer database search revealed that IR5 exhibits significant homology to the gene products of US10 of HSV-1 (McGeoch et al., 1985, 1986), ORF64 of VZV (Davison and Scott, 1986), and ORF3 of EHV-4

707

THE IR5 GENE OF EHV-1 GGCTGGGGGAGCGGTAGCGAAAAAA CGGTTGTTGTTGTTTAGCGTTGCTCATCCACGCGACTCGGGGCGA 9672 GGTCGGGGGAAAGCGTGAATGACAGCGCGC~T~CAACA >>>>>>

9742

CCCACGCAGAGGGAGGGAGAGCTATGGGAAGGGGTGG~T~GGG~~AGG~CATCTATAGCTACCTA

9812

AACCAGCCAGCAGGCGTGTGTGTGTTCCCGCGATTCCACGG

9882

CCGCGCGCAATCAGTGCGCCCGATCTCCCGGCCACTGAACC Tq ->

======= MDGAYGH

9952 7

GTCCACAACGGCTCCCCGATGGCCGTCGACGGCGAGGAGTCCGGAGCGGGGACGGGGACGGGGGCGGGCG 10022 VHNGSPMAVDGEESGAGTGTGAG 30 CGGACGGGCTATACCCGACCGCACGGACACACCGCA ADGLYPTSTDTAAHAVSLPRSVGD

10092 54

CTTTGCCGCGGTCGTGCGCGCCGTGTCGGCGGAGGAGCGCC FAAVVRAVSAEAADALRSGAGPP

10162 77

GCGGAGGCCTGGCCGCGCGTGTACCGCATGTTCTGCGACATGTTTGGTCGCTACGCGGCCAGCCCCATGC 10232 AEAWPRVYRMFCDMFGRYAASPM 100 10302 CCGTCTTCCACTCGGCGGACCCGCTGCGCCGCC~~CGT~GGCTGTACCTCGTGGATCTCGGCGCGGCGCC PVFHSADP LRRAVGLYLVDLGAAP 124 GGTGGAGACCCACGCCGAGCTCAGCGGCCGCC~T~TCTTCTGC~GTACTGGTGCTGCCTGGGACACGCG10372 VETHAELSGRMLFCAYWCCLGHA 147 TTCGCCTGCTCGCGCCCGCAGATGTACGAGCGCGCGTGTGT~GC~TTTTTCGAGACCCGGCTC~ATCG 10442 FACSRPQMYERACARFFETRLGI 170

GCTGTTCCCCCGCCACGCAGCCGCCGCGGCGTACCTGCGCGCCCGCGGCCGC~GCTCCCTCTCCAGCTG 10582 LFPRHAAAAAYLRARGRKLPLQL 217 CCCTCGGCCCATCGGACCGCCAAAACGGTGGCCGGTGGCCGTGACCGGCC~TCGAT~CTTTTG~TATACT10652 PSAHRTAKTVAVTGQS I N F ###### 236 CACTATATACTAAACCCC~TTCCGCGAGTCTGCCCCCTGTTTGTGTTTCCGTCTCTCTATC~TTTCCCC ** ** *********** ** Tct -I

10722

CACCAATACCTCAACTATCGAGCGGGCGTGGGGACCC

10759

FIG. 2. Nucleotide sequence and predicted amino acid sequence of the IR5 ORF. The nucleotide numbers represent the distance from the L-S junction, nucleotide 1 being the first nucleotide in the S region. The amino acid sequence of IR5 is shown by single letter code beneath the DNA sequence. The potential zinc finger of IR5 is underlined. Potential cis-acting elements are shown with the following symbols: Tci, transcription initiation site (cap site); Tc,, polyadenylation site (transcription termination site); >, CAAT box; =, TATA box, #, polyadenylation signal, and *, G/C cluster. The precise transcription start site (shown by -) is at nucleotide 9891. The precise transcription termination site (Tct) is at nucleotide 10671, as shown by the vertical line (-1).

(Cullinane et a/., 1988) (Fig. 3A). These analyses revealed that IR5 is 190/oidentical to US10 of HSV-1 with regard to amino acid residues, whereas the percentages of identity with ORF64 of VZV and ORF3 of EHV-4 are 25 and 740/o,respectively. Interestingly, the highest level of homology observed among these four herpesviral proteins encompasses the zinc finger region. Finally, DNA sequence analysis of the IR5 gene identified several potential cis-acting elements flanking the IR5 ORF. A potential TATA box (Benoist et a/., 1980; Buchner, 1990) is located 61 nucleotides upstream of the IR5 ORF at nucleotides 9864-9870 (Fig. 2). A CCAATT sequence (Benoist et al., 1980;

Buchner, 1990; Coen et al., 1986) is located 147 nucleotides upstream of the TATA box at nucleotides 9709-9714 (Fig. 2). Lastly, the sequence (AATATA) mapping downstream of the IR5 ORF at nucleotides 10,645-l 0,650 (Fig. 2) exhibits excellent homology to a consensus polyadenylation sequence (Nordstorm et al., 1985). The poly(A) signal is followed by a region rich in G/T residues (G/T cluster; Birnstiel et a/., 1985; Weiss et a/., 1991; Zarkowar and Wickens, 1987). Northern

blot analysis

Earlier work has demonstrated that the inverted repeat segment is transcriptionally active during a cyto-

HOLDEN

708

ET AL.

(A) EHV-1 HSV-1 EHV-4 vzv

IR5 US10 ORF3 ORF64

MDGAYGHVHN--------------------------GSPMAMGEESGAG 24 --GAYA-----------RAR------TAVRV-----GSP-THTHLRQDPG 161 MAHAIPRPAEEIPLVPGRARSVRLGSTLPRVMDCAYGSPMAVDGDVRTGG 50 MN-----------LCGSR-------------------------------G 8 * * * *** #

EHV-1 HSV-1 EHV-4 vzv

IR5 US10 ORF3 ORF64

TGTGAGADGLYPTSTDTAAHAVSLPRSVGDFAAVVRAVSAEAADALRSGA 74 DEPTSDDSGLYPLDARALAHLVMLPADHRAFF-W----EVSRMCAANVR 208 DC--GGGEGLYPTSTDTAAHAVSLPRSVGEFASAVRAMSADAADALRRGA 98 EHPGGEYAGLYCTRHDTPAHQALMNDAERYFAAALCAISTEAYEAFIHSP 58 * * ** ** ## * ** #Rx** Y* ***

EHV-1 HSV-1 EHV-4 vzv

IR5 US10 ORF3 ORF64

122 G--PPASAWPRVYRMFCDMFGRYAASPMPVFFHSADPLRRAVGLYLVDLGA D--PPPPATGAMLGRHARLVHTQWLRANQETSPLWPWRTMINFI-TTMA 255 G--PPPEIWPRAYRMFCELFGRYAVSPMPVFHSADPLRRAVGRYLVDLGA 146 SERPCASLWGRAKDAFGRMCGELAADRQRP-PSVPPIRRAVLSLLREQCM107 *** * * * * ** # y*j+* * * #*

EHV-1 HSV-1 EHV-4 vzv

IR5 US.10 ORF3 ORF64

APVETHAELSGRMLFCAYWCCLGHA----------FACSRPQMYERA--PRVQTHAHMHDLLMACAFWCCLTHA----------STCSYAGLYSTH--APVETHAKLSTRLIFCAHWCCLGHA----------FGCSRQAMYERE--PDPQSHLELSERLILMAYWCCLGHAGLPTIGLSPDNKCIRGGI * *It**** ** *#*###y*##. #** t *

EHV-1 HSV-1 EHV-4 vzv

IR5 US10 ORF3 ORF64

CARFFETRLGIGETPPADAERYWAALLNMAGAEPELFPRHAAAAAYLRAR 209 312 CLHLFGAF-GCGDPALTPPLC CARFFEARLGIGETPPADSERYWVALLDMAGADPELFPRHAAAAAYLRTR 233 CHRLFDAYLGCGS-------------LGV--------PR-----TYERS 180 * * * ** # * #** *c #

EHV-1 IR5 EHV-4 ORF3

GRKLPLQLPSAHRTAKTVAVTGQSINF GRKLPLPLPP-QAGSATVSVASQSINF

159 302 183 157

236 259

(B) C - X2-4 - C - X2-15 - C/H - X2-4 - C/H EHV-1 IR5 HSV-1 US10 EHV-4 ORF3

271 162

c - X3-C-X3C - X3-C-X3CX3-C-X3-

vzv

123

M - X3 - C - X3 - H - X14 - C

ORF64

138

H-X3 H-X3 H-X3

-C -C -C

FIG. 3. (A) Alignment of the predicted amino acid sequence of gene IR5 of EHV-1 to those of US1 0 of HSV-1 (McGeoch eta/., 1985), ORF64 of VZV (Davison and Scott, 1986) and ORF3 of EHV-4 (Cullinane ef a/., 1988). Alignment was carried out using the CLUSTAL program of PC/GENE software (see Materials and Methods). Gaps introduced (shown as dashes) allow for the different lengths of the proteins and generate maximum alignment of conserved residues. The # sign represents amino acids that are conserved among all four proteins. The asterisk (‘) represents amino acids that are conserved among three proteins. (B) Comparison of the zinc finger motif of the four proteins to the consensus zinc finger motif (Berg, 1986). The cysteines and histidines are indicated by C and H, respectively. The X represents any amino acid.

lytic infection (Gray et al., 1987a, 1987b; Harty et a/., 1991; Har-ty and O’Callaghan, 1991; Holden et a/., 1992; Robertson et a/., 1988). To address the nature of the IR5 specific mRNA, Northern blot analyses were performed using poly(A) RNA isolated at various times postinfection. Probe pCS2S2, a clone specific for the IR5 gene (Fig. l), hybridized to a 0.9-kb mRNA that was readily detected at 4 hr postinfection (Fig. 4). However, upon overexposure of the blot, the IR5 mRNA could be detected at 2 hr postinfection (data not shown). To determine the kinetic class of IR5, Northern blot analyses were performed using poly(A) RNA isolated under immediate-early, early, and late conditions. Probe pCS2S2 was hybridized to mRNA isolated from EHV-1

infected L-M cells maintained in the absence or presence of the appropriate metabolic inhibitor (Fig. 5). The 32P-labeled pCS2S2 hybridized to a 0.9-kb mRNA under both early and late conditions (Fig. 5, lanes 3 and 4). However, the presence of phosphonoacetic acid at early times greatly reduced the synthesis of the IR5 mRNA. Probe pCS2S2 failed to hybridize to mRNA isolated from mock-infected cells or immediate-early mRNA (Fig. 5, lanes 1 and 2, respectively). To address the direction of transcription, single-stranded subclones of PCS-2 (M 13mpl8 and M 13mpl9) were used in Northern blot analysis and revealed that the IR5 ORF is located on the same strand as IR4 and is transcribed in the opposite direction of the IRl and IR2 genes (data

709

THE IR5 GENE OF EHV-1

7

113 l,!i

hrr

,J.

-

0.9 kb

FIG. 4. Northern blot analysis of the poly(A) RNA showing the time course of synthesis of the IR5 transcript. Poly(A) RNA was isolated from EHV-l-infected L-M cells from 1 to 15 hr postinfection. The probe used was PCS2S2 (see Fig. 1). M, mRNA isolated form mockinfected cells (see Materials and Methods for details). All blots were exposed for 15 hr under identical conditions.

and a G/T cluster, elements commonly found at the 3’ terminus of eukaroytic genes, suggested that this AATATA sequence may be the functional poly(A) signal. Sl nuclease analysis was employed to map the 3’terminus of the IR5 mRNA. Clone PCS-2 was 3’ end labeled at the Mlul site (Fig. l), and the 32P-labeled probe was hybridized to late poly(A) RNA. Following digestion with Sl nuclease, a single fragment of 298 nucleotides was observed (Fig. 7, lane 7). These results mapped the 3’terminus of the IR5 mRNA to lie 19 to 21 nucleotides downstream of the polyadenylation signal, a position that corresponds precisely to the CA dinucleotide at nucleotides 10,670-l 0,671. The Sl mapping data and Northern blot analyses of IR5 mRNA indicated that the IR5 ORF is transcribed as a 0.9-kb mRNA that potentially encodes a 24.8-kDa protein. DISCUSSION

not shown). The above findings indicate that the IR5 mRNA is approximately 0.9 kb in size and is expressed as a y-l transcript. Sl Nuclease analysis To position the 5’terminus of the IR5 mRNA, Sl nuclease analysis was employed. First, Sl nuclease analysis was performed using clone PCS-2 5’ labeled at the Ddel site (Fig. 1). Since the IR5 mRNA is more readily detected at late times, late mRNA was utilized in the Sl nuclease experiments. Following hybridization of the 5’ labeled PCS-2 (Ddel) probe with late poly(A) RNA, the DNA-RNA hybrids were digested with Sl nuclease and analyzed on 69’0 polyacrylamide-urea sequencing gels. The use of probe PCS-2 (Ddel) resulted in the protection of a fragment of approximately 430 nucleotides (Fig. 66, lane 3). These results mapped the transcription initiation site of IR5 to lie approximately 20-25 nucleotides downstream of the TATA box (see Fig. 2). To confirm these results, a second smaller probe (PCS2 labeled at the Stul site) was utilized. The use of PCS-2 (Stul) resulted in the protection of a 273 nucleotide fragment (Fig. 6A, lane 6) which positioned the 5’ initiation site to lie approximately 21 nucleotides downstream of the TATA (Fig. 2). Therefore, both probes mapped the transcription initiation site to approximately the same position upstream of the IR5 ORF and revealed that transcription is initiated at 21 to 23 nucleotides downstream of the TATA box. Nucleotide sequencing identified a near consensus polyadenylation signal sequence (AATATA) downstream of the IR5 ORF (Fig. 2). The fact that the polyadenylation signal sequence was followed by a CA dinucleotide sequence (nucleotides 10,670-l 0,671)

Ongoing work in our laboratory has identified six genes within the IR sequence of the EHV-1 genome (see Introduction). In contrast, IR sequences of HSV-1 and VZV have been shown to possess only one and three genes, respectively (Davison and Scott, 1986; McGeoch et al., 1986). In this study, we report the DNA sequence and transcriptional characterization of IR5, a homolog of US10 of HSV-1, ORF64 of VZV, and ORF3 of EHV-4, that maps within the IR sequences of EHV-1. The IR5 gene expresses a single 0.9-kb mRNA that is first detected at 2 hr postinfection. The synthesis of the IR5 mRNA is greatly reduced in the presence of phosphonoacetic acid, suggesting that IR5 expression is enhanced by DNA replication. The IR5 mRNA has the potential to encode a protein of 24.8 kDa that

PCSZSZ

M I

IE I

E I

L I

-

12

0.9

kb

34

FIG. 5. Northern blot analysis of EHV-1 transcripts isolated at IE, E, and L times and probed with PCS2S2 (Fig. 1). IE, E, and L poly(A) RNAs were isolated at 4, 6, and 8 hr postinfection, respectively. The IE MRNA was isolated from cells treated with 100 pglml of cycloheximide. The E MRNA was isolated from cells treated with 100 pg/ml of phosphonoacetic acid. The mock (M) and late mRNA was isolated in the absence of virus and metabolic inhibitors, respectively.

710

HOLDEN

ET AL. B

1078872-

b

603-

-430

-310

:p73 281271-

123 1 PCS-2

234567 5’

5’

mRNA

mRNA

+3’

-3

sr stu

I

dde I

FIG. 6. Sl nuclease analysis to map the 5’terminus of the IR5specific mRNA. (A) Clone PCS-2 (see Fig. 1) was 5’labeled at its Stul site (*). The 5’ end labeled probe was hybridized to late poly(A) RNA (lane 6). The Sl resistant fragments were fractionated on a 6% polyacrylamide-urea sequencing gel. Lanes 1 1 2, 3, and 4 contain DNA fragments for sequencing M 13mpl8 as molecular weight markers. Lanes 5 and 6 represent mock and late poly(A) RNA, respectively. Lane 7 contains Haelll-digested @X-l 74-RF DNA as additional molecular weight markers. (B) Clone PCS-2 was 5’end labeled at the Ddel site (Fig. 1) and hybridized to late poly(A) RNA as depicted below the gel. Lanes 2 and 3 represent mock and late poly(A) RNA, respectively. Lane 1 contains the molecular weight markers. Sizes are in nucleotides.

contains a zinc finger motif. It will be of interest to determine whether this zinc finger motif functions to allow the IR5 protein to interact with DNA. US1 0 homologs of HSV-1 have now been identified in VZV (ORF64), EHV-4 (ORF3), and EHV-1 (IR5). Lee et al. (1982) were the first to demonstrate that US10 mapped within the US segment of the HSV-1 genome and encoded a 33-kDa protein. In contrast to US1 0 of HSV-1, the US1 0 homologs (ORF64,ORF3, and IR5) of the other three alphaherpesviruses map within the IR sequences and are colinear (Cullinane eta/., 1988; Davison and Scott, 1986). Rixon and McGeoch (1984) demonstrated that US1 0 is expressed as a late mRNA and belongs to a family of 3’ coterminal transcripts. Unlike US1 0, the IR5 ORF neither possesses tandemly repeated amino acid sequences nor overlaps an ORF homologous to HSV-1 US1 1. A computer alignment of US1 0 homologs revealed a low degree of amino acid conservation and identified histidines and cysteines conserved at specific locations within the ORFs that form the structural motif (C-C-H-C) of a zinc finger. Within the EHV-1 IR5 ORF, the first cysteine residue of the zinc finger motif is located at amino acid 138 (Fig. 3). The zinc finger motif is perfectly conserved in the

US1 0 protein of HSV-1 and the ORF3 protein of EHV-4 (Fig. 3). However, the VZV homolog lacks the first cysteine residue of the motif in a position comparable to those of the other proteins since a methionine lies at amino acid 123 of ORF64. Although the US10 gene has been sequenced and its mRNA characterized, the nature of this protein remains undefined. It has been speculated that US10 of HSV-1 is a virion structural protein (Lee et al., 1982); however, the US10 gene is not essential for HSV-1 replication in cell culture (Longnecker and Roizman, 1986). The size of the IR5 predicted protein, the presence of the zinc finger motif, and the fact that it is rich in proline residues suggest that IR5 protein may correspond to nucleocapsid protein IVa which is present only in mature, DNA-containing capsid species (“H” capsids) and is thought to be associated with the viral genome (O’Callaghan and Randall, 1976; Perdue et al., 1974, 1975, 1976). The noteworthy features of the IR5 sequence include the presence of several potential c&acting elements. The IR5 promoter is composed of a TATA box and a CAAT box and a potential binding site for the EHV-1 IE protein lies 540 nucleotides upstream of the TATA box. In support of our findings that IR5 is ex-

711

THE IR5 GENE OF EHV-1

b

-370

-*298

-227

-I81

5

1234

5’

6

7

mRNA

*3’ PCS-2

downstream of a potential TATA box, and similar experiments identified a transcription termination site downstream of the IR5 ORF. There is no published information on the transcription of ORF64 of VZV or ORF3 of EHV-4. Thus, it will be of interest to ascertain whether these two US10 homologs are transcribed within a family of overlapping mRNAs or in a manner similar to that of the IR5 gene. Finally, data presented here and elsewhere (see Introduction) show that the organization of the IR segments of the genome of the Kentucky A stain of EHV-1 is significantly different from those of other alphaherpesviruses. Although these data confirm that many of the genes mapping within the S region of alphaherpesviruses are similar, gene arrangement varies within the S region and some viruses such as EHV-1 possess genes not present in HSV-1 (Harty and O’Callaghan, 1991; C. A. Breeden and D. J. O’Callaghan, in preparation; Holden et a/., in preparation; Colle et a/., 1992). It is anticipated that the data presented in this paper will provide the basis for future experimentation on the functional role of IR5 in EHV-1 lytic infection.

iv& I FIG. 7. Sl nuclease analysis to map the 3’terminus of the IR5-specific mRNA. Probe PCS-2 was 3’ labeled at the n/llul site (Fig. 1). The 3’ end labeled probe was hybridized to mock and late poly(A) RNA (lanes 6 and 7, respectively) as depicted below the gel. The Sl resistant fragment was analyzed on a 6% polyacrylamide-urea sequencing gel. Lanes 1,2,3, and 4 contain the DNA sequence of M 13mpl8 representing molecular weight markers. Lane 7 represents a Haell digest of pBR322 DNA used as additional molecularweight markers. The molecular weight markers and the protected fragments are given in base pairs.

pressed as a late gene, it is interesting to note our recent observation that both the EHV-1 IE protein and the product of the UL3 gene, an ICP27 homolog, are required for efficient transactivation of the IR5 promoter in transient expression assays (Smith et a/., 1992). Downstream of the TATA box is an ORF of 236 amino acids with several in-frame ATGs, and a polyadenylation signal and G/T cluster were identified downstream of the IR5 ORF. Lastly, positioned between IR5 and IR6 starting at nt 10,899 is a 27-bp sequence tandemly repeated 14 times, which is similar to the repeat downstream of ORF3 of EHV-4 (Cullinane et al., 1988). Transcriptional analyses of IR5 revealed that the 0.9kb transcript appears abundantly during late stages of a lytic infection, a characteristic shared by the US10 mRNA (Rixon and McGeoch, 1984). However, US10 belongs to a 3’ coterminal family of mRNAs, whereas Northern blot analysis of IR5 identified a single mRNA species. Sl nuclease mapping identified a single transcription initiation site approximately 21 nucleotides

ACKNOWLEDGMENTS We are grateful to Suzanne Zavecz. Bridget Higginbotham, and Scarlett Flowers for excellent technical assistance. Support for this investigation was obtalned from Public Health Research Grant Al 22001 from the Natlonal Institutes of Health, a Grayson-Jockey Club Research Foundation Inc. research grant, and Grant 89-37266-4735 from the U.S. Department of Agriculture Animal Molecular Biology Program.

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