Virus Research, 15 (1990) 149-162 Elsevier
149
VIRUS 00553
Sequence and organization of the genomic termini of equine herpesvirus type 1 Ramana Department
R. Yalamanchili
and Dennis
J. O’Callaghan
of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport, LA 71130-3932, U.S.A. (Accepted 10 November 1989)
Summary The nucleotide sequence and organization of the genomic termini and of the junction of the long (L) and short (S) regions of the equine herpesvirus type 1 genome were determined. Sequencing of the XbaI-Q fragment (1441 nucleotides) revealed that the left terminus contains sets of inverted repeat and direct repeat sequences. The terminal sequence is described as DRl-UC-DR4 (18, 60, and 16 nucleotides, respectively) because of its homology to these elements of the ‘a’ sequence of herpes simplex virus. Located at each terminus of the S region as part of the inverted repeats is a 54 nucleotide sequence with homology to the Ub element of the HSV ‘a’ sequence. Thus, these data suggest that fusion of the EHV-1 genomic termini during replication will generate a sequence equivalent to Ub-DRl-Uc-DR4, which is known to be an ideal cleavage/packaging signal in herpesviral DNAs. Eighty-seven nucleotides of the L region left terminus sequence are repeated in an inverted fashion at nucleotide 892; also a 32 basepair portion, DRl-UC (18 and 14 basepairs respectively), is reiterated 20 times in an inverted fashion as part of a 54 basepair tandem repeat located at the other L region terminus (L-S junction). It is not known whether these small inverted repeats at the L termini mediate isomerization of the L region at a very low level. The organization of the terminal sequences of the EHV-1 genome and the similarity of these sequences to the cleavage/ packaging elements of other herpesviruses are discussed. Herpesvirus genomic structure; Herpesvirus vage/packaging signal; Herpesvirus reiterated
genomic sequence
termini;
Herpesvirus
clea-
Correspondence to: D.J. O’Callaghan, Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport, LA 71130-3932, U.S.A.
0168-1702/90/$03.50
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
150
Introduction Equine herpesvirus type 1 is a double-stranded DNA virus with a genomic size of 142 kilobasepairs (kbp) (Soehner et al., 1965; 0’ Callaghan et al., 1972, 1981, 1983). The EHV-1 genome is comprised of a fixed, long component (U,; 110 kbp) covalently linked to an invertible short component (S; 32 kbp). The short component has a unique sequence (Us; 6 kbp) flanked by inverted repeats (IRS; 13 kbp each). The inverted repeats allow the short component to invert relative to the L component, resulting in the formation of two isomeric DNA molecules (Henry et al., 1981; O’Callaghan et al., 1981, 1983, 1984; Whalley et al., 1981; Ruyechan et al., 1982). Parts of the inverted repeat sequences (0.78-0.79 and 0.83-0.87 map units of the internal inverted repeat; 0.91-0.95 and 0.99-1.0 map units of the terminal inverted repeat) and the left terminus sequences (0.00-0.04 map units) of the standard (STD) EHV-1 DNA molecule are conserved in the genome of EHV-1 defective interfering particles (DIP) (Baumann et al., 1984, 1986a, 1986b, 1987). The DIP genome must contain two cis-acting sequences for its propagation: (1) an origin of replication, and (2) a cleavage-packaging signal (CPS). An origin of replication is located within the S region inverted repeat sequences which are conserved in the DIP genome (Baumann et al., 1989). The terminal sequences are expected to contain the cleavage-packaging signal, because these sequences are located at the termini where cleavage of the concatameric DNA into unit length genomes occurs. The sequences essential for cleavage and packaging of herpesviral genomes have been shown to be present at the junction of the long region-short region (L-S junction) and at the termini in the case of herpes simplex virus (HSV); and at the termini in the case of varicella-zoster virus (VZV) and pseudorabies virus (PrV) (Mocarski and Roizman, 1981, 1982; Davison and Wilkie, 1981; Davison, 1984; Harper et al., 1986). The HSV CPS is described as the ‘a’ sequence, which is present in a single copy at the S terminus and in one to several copies at the L-S junction and the L terminus. The ‘a’ sequence of the HSV DNA varies in size from 220 to 500 bp, contains two unique sequences (Ub and UC) and several direct repeats (DR), and can be written as DRl-Ub-(DR2),-(DR4)“-UC-DRl. DRl is shared by adjacent copies of the ‘a’ sequence and is the site of cleavage (Mocarski and Roizman, 1982). In contrast, in the case of the genomes of VZV (Davison, 1984), PrV (Harper et al., 1986), and EHV-1 (Robinson et al., 1981), the sequences at the left terminus are not homologous to the sequences at the right terminus. The terminal sequences of VZV and PrV do not contain direct repeat sequences such as DRl, DR2, and DR4 of HSV. The terminal sequences of HSV and VZV genomes have a single unpaired nucleotide at each 3’ terminus, allowing the fusion of the termini and resulting in a sequence identical to that at the L-S junction. In the case of PrV, the L terminus is blunt-ended, whereas the S terminus has a two-nucleotide overhang (GG). Analysis of the sequence at the junction of the left and right termini of PrV DNA molecules revealed that the termini are joined by blunt end ligation of one strand and filling the two-nucleotide gap on the other strand. In this paper, we describe the sequence and organization of the L-S junction and the termini of the STD EHV-1 genome. The data reveal that the L terminus
151
contains sets of inverted repeat and direct repeat sequences, that a 32 nucleotide sequence is repeated in an inverted orientation at both termini of the L region, and that the termini of the L and S regions are not homologous. The organization of the sequences at the termini indicate that a sequence similar to the CPS of other herpesviruses would be generated in concatameric DNA during replication.
Materials and Methods Cells
and viruses
The Kentucky A strain of EHV-1 was propagated in L-M 929 suspension cell cultures as described previously (Q’Callaghan et al., 1968; Perdue et al., 1974). STD EHV-1 was propagated at a low multiplicity of infection (0.05 plaque forming units/cell) to minimize the generation of defective interfering particles.
Virus purification and isolation of viral DNA Viral DNA was isolated from purified vii-ions. Briefly, at 72 h after infection, cells and cell debris were removed by centrifugation, and the virus in the supernatant was precipitated by 7% polyethylene glycol and 2.3% NaCl. The virus was then purified by centrifugation through 5-30% dextran gradients several times as described previously (Perdue et al., 1974). The purified virus was resuspended in TE buffer (10 mM Tris-HCl, pH 7.4; 1 mM EDTA) and digested with proteinase K at a concentration of 100 pg/ml in the presence of 0.5% SDS at 37°C for 2 h. The viral DNA was purified by phenol/chloroform extraction followed by ethanol precipitation as described previously (Baumann et al., 1984).
Cloning of viral DNA The cloning of the EHV-1 genome was described previously (Robinson et al., 1981; Baumann et al., 1984, 1986a, 1986b, 1987; Grundy et al., 1989). The left terminal fragment XbaI-Q and its subfragments were recloned into M13mp18 and M13mp19 (Yanisch-Perron et al., 1985), using established protocols (Maniatis et al., 1982).
Nucieotide sequencing The dideoxy chain termination method (Sanger et al., 1977), employing the Klenow polymerase (New England Nuclear, Boston, MA) or Sequenase (United States Biochemical Corp., Cleveland, OH) was used. Deaza-GTP-containing nucleotide mixes (American Bionetics, Hayward, CA) were used with Klenow polymerase to minimize compressions (Barr et al., 1986), and nucleotide sequences of both strands were determined to avoid errors. The computer facilities of BIONET (Kristofferson, 1987) and the IBI/Pustell DNA/protein sequence analysis
152
software (International the sequences.
Biotechnologies,
Inc., New Haven, CT) were used to analyze
Results The left terminus sequences of EHV-1 The genome of EHV-1 DIPS is comprised of reiterations of subgenomic sequences from the left terminus of the L region and inverted repeats of the S region of the STD EHV-1 genome. Sequences within the central portion of the inverted repeats of the S region have recently been demonstrated to code for an origin of replication (Baumann et al., 1989). The other cis-acting function essential for DNA
L s >>>>. . . . . . . . . IRl
RI
(87)
(17)9
...............
HindfII
SIMI
.
149
VIRUS 00553
Sequence and organization of the genomic termini of equine herpesvirus type 1 Ramana Department
R. Yalamanchili
and Dennis
J. O’Callaghan
of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport, LA 71130-3932, U.S.A. (Accepted 10 November 1989)
Summary The nucleotide sequence and organization of the genomic termini and of the junction of the long (L) and short (S) regions of the equine herpesvirus type 1 genome were determined. Sequencing of the XbaI-Q fragment (1441 nucleotides) revealed that the left terminus contains sets of inverted repeat and direct repeat sequences. The terminal sequence is described as DRl-UC-DR4 (18, 60, and 16 nucleotides, respectively) because of its homology to these elements of the ‘a’ sequence of herpes simplex virus. Located at each terminus of the S region as part of the inverted repeats is a 54 nucleotide sequence with homology to the Ub element of the HSV ‘a’ sequence. Thus, these data suggest that fusion of the EHV-1 genomic termini during replication will generate a sequence equivalent to Ub-DRl-Uc-DR4, which is known to be an ideal cleavage/packaging signal in herpesviral DNAs. Eighty-seven nucleotides of the L region left terminus sequence are repeated in an inverted fashion at nucleotide 892; also a 32 basepair portion, DRl-UC (18 and 14 basepairs respectively), is reiterated 20 times in an inverted fashion as part of a 54 basepair tandem repeat located at the other L region terminus (L-S junction). It is not known whether these small inverted repeats at the L termini mediate isomerization of the L region at a very low level. The organization of the terminal sequences of the EHV-1 genome and the similarity of these sequences to the cleavage/ packaging elements of other herpesviruses are discussed. Herpesvirus genomic structure; Herpesvirus vage/packaging signal; Herpesvirus reiterated
genomic sequence
termini;
Herpesvirus
clea-
Correspondence to: D.J. O’Callaghan, Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport, LA 71130-3932, U.S.A.
0168-1702/90/$03.50
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
150
Introduction Equine herpesvirus type 1 is a double-stranded DNA virus with a genomic size of 142 kilobasepairs (kbp) (Soehner et al., 1965; 0’ Callaghan et al., 1972, 1981, 1983). The EHV-1 genome is comprised of a fixed, long component (U,; 110 kbp) covalently linked to an invertible short component (S; 32 kbp). The short component has a unique sequence (Us; 6 kbp) flanked by inverted repeats (IRS; 13 kbp each). The inverted repeats allow the short component to invert relative to the L component, resulting in the formation of two isomeric DNA molecules (Henry et al., 1981; O’Callaghan et al., 1981, 1983, 1984; Whalley et al., 1981; Ruyechan et al., 1982). Parts of the inverted repeat sequences (0.78-0.79 and 0.83-0.87 map units of the internal inverted repeat; 0.91-0.95 and 0.99-1.0 map units of the terminal inverted repeat) and the left terminus sequences (0.00-0.04 map units) of the standard (STD) EHV-1 DNA molecule are conserved in the genome of EHV-1 defective interfering particles (DIP) (Baumann et al., 1984, 1986a, 1986b, 1987). The DIP genome must contain two cis-acting sequences for its propagation: (1) an origin of replication, and (2) a cleavage-packaging signal (CPS). An origin of replication is located within the S region inverted repeat sequences which are conserved in the DIP genome (Baumann et al., 1989). The terminal sequences are expected to contain the cleavage-packaging signal, because these sequences are located at the termini where cleavage of the concatameric DNA into unit length genomes occurs. The sequences essential for cleavage and packaging of herpesviral genomes have been shown to be present at the junction of the long region-short region (L-S junction) and at the termini in the case of herpes simplex virus (HSV); and at the termini in the case of varicella-zoster virus (VZV) and pseudorabies virus (PrV) (Mocarski and Roizman, 1981, 1982; Davison and Wilkie, 1981; Davison, 1984; Harper et al., 1986). The HSV CPS is described as the ‘a’ sequence, which is present in a single copy at the S terminus and in one to several copies at the L-S junction and the L terminus. The ‘a’ sequence of the HSV DNA varies in size from 220 to 500 bp, contains two unique sequences (Ub and UC) and several direct repeats (DR), and can be written as DRl-Ub-(DR2),-(DR4)“-UC-DRl. DRl is shared by adjacent copies of the ‘a’ sequence and is the site of cleavage (Mocarski and Roizman, 1982). In contrast, in the case of the genomes of VZV (Davison, 1984), PrV (Harper et al., 1986), and EHV-1 (Robinson et al., 1981), the sequences at the left terminus are not homologous to the sequences at the right terminus. The terminal sequences of VZV and PrV do not contain direct repeat sequences such as DRl, DR2, and DR4 of HSV. The terminal sequences of HSV and VZV genomes have a single unpaired nucleotide at each 3’ terminus, allowing the fusion of the termini and resulting in a sequence identical to that at the L-S junction. In the case of PrV, the L terminus is blunt-ended, whereas the S terminus has a two-nucleotide overhang (GG). Analysis of the sequence at the junction of the left and right termini of PrV DNA molecules revealed that the termini are joined by blunt end ligation of one strand and filling the two-nucleotide gap on the other strand. In this paper, we describe the sequence and organization of the L-S junction and the termini of the STD EHV-1 genome. The data reveal that the L terminus
151
contains sets of inverted repeat and direct repeat sequences, that a 32 nucleotide sequence is repeated in an inverted orientation at both termini of the L region, and that the termini of the L and S regions are not homologous. The organization of the sequences at the termini indicate that a sequence similar to the CPS of other herpesviruses would be generated in concatameric DNA during replication.
Materials and Methods Cells
and viruses
The Kentucky A strain of EHV-1 was propagated in L-M 929 suspension cell cultures as described previously (Q’Callaghan et al., 1968; Perdue et al., 1974). STD EHV-1 was propagated at a low multiplicity of infection (0.05 plaque forming units/cell) to minimize the generation of defective interfering particles.
Virus purification and isolation of viral DNA Viral DNA was isolated from purified vii-ions. Briefly, at 72 h after infection, cells and cell debris were removed by centrifugation, and the virus in the supernatant was precipitated by 7% polyethylene glycol and 2.3% NaCl. The virus was then purified by centrifugation through 5-30% dextran gradients several times as described previously (Perdue et al., 1974). The purified virus was resuspended in TE buffer (10 mM Tris-HCl, pH 7.4; 1 mM EDTA) and digested with proteinase K at a concentration of 100 pg/ml in the presence of 0.5% SDS at 37°C for 2 h. The viral DNA was purified by phenol/chloroform extraction followed by ethanol precipitation as described previously (Baumann et al., 1984).
Cloning of viral DNA The cloning of the EHV-1 genome was described previously (Robinson et al., 1981; Baumann et al., 1984, 1986a, 1986b, 1987; Grundy et al., 1989). The left terminal fragment XbaI-Q and its subfragments were recloned into M13mp18 and M13mp19 (Yanisch-Perron et al., 1985), using established protocols (Maniatis et al., 1982).
Nucieotide sequencing The dideoxy chain termination method (Sanger et al., 1977), employing the Klenow polymerase (New England Nuclear, Boston, MA) or Sequenase (United States Biochemical Corp., Cleveland, OH) was used. Deaza-GTP-containing nucleotide mixes (American Bionetics, Hayward, CA) were used with Klenow polymerase to minimize compressions (Barr et al., 1986), and nucleotide sequences of both strands were determined to avoid errors. The computer facilities of BIONET (Kristofferson, 1987) and the IBI/Pustell DNA/protein sequence analysis
152
software (International the sequences.
Biotechnologies,
Inc., New Haven, CT) were used to analyze
Results The left terminus sequences of EHV-1 The genome of EHV-1 DIPS is comprised of reiterations of subgenomic sequences from the left terminus of the L region and inverted repeats of the S region of the STD EHV-1 genome. Sequences within the central portion of the inverted repeats of the S region have recently been demonstrated to code for an origin of replication (Baumann et al., 1989). The other cis-acting function essential for DNA
L s >>>>. . . . . . . . . IRl
RI
(87)
(17)9
...............
HindfII
SIMI
.
