The genomes of simian varicella virus and varicella zoster virus are colinear

IGus Research, 26 (1992) 255-266

255

Elsevier Science Publishers B.V. All rights reserved 0168-1702/92/$05.0092/$05.00

VIRUS 00844

The genomes of simian varicella virus and varicella zoster virus are colinear Carla Y. Pumphrey and Wayne L. Gray Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

(Received 10 July 1992; revision received 20 August 1992, accepted 3 September 1992)

Summary

Simian varicella virus (SW) causes an exanthematous disease in non-human primates which is clinically similar to varicella zoster virus (VZV) infection of humans. In this study, the genetic relatedness of SVV and VZV was confirmed and the location of SW DNA sequences homologous to VZV restriction endonuclease (RE) fragments and viral genes was determined. VZV DNA RE fragments representing 98.3% of the VZV genome were 32P-labeled and hybridized to RE digested, immobilized SW DNA. Homologous sequences were located throughout the viral DNAs in similar map positions, indicating a colinear relationship between the VZV and SW genomes. 32P-labeled VZV glycoprotein (gp I, II, III, and IV) and gene 62 DNA probes also hybridized to SW DNA in a colinear manner. The results suggest that the location of specific SW genes may be predicted from the known map positions of homologous VZV genes. This study provides further support for SVV infection of non-human primates as a model for VZV infection of humans.

Simian varicella virus; Varicella zoster virus; DNA homology

Correspondence to: W.L. Gray, Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. Fax: (1) (501) 686-5359.

256

Introduction

Simian varicella virus (SVV) causes a naturally occurring disease in non-human primates which clinically and pathogenically resembles human varicella zoster virus (VZV) infections (Oakes and d’Offay, 1988). Outbreaks of a mild form of simian varicella in several species of macaque monkeys (Macaca sp.1 were characterized by fever, lethargy, vesicular skin rash, and low morbidity and mortality (Blakely et al. 1973). In contrast, SW epizootics in African vet-vet (Cercopithicus aethiops) and patas monkeys (Erythrocebus patus) were associated with vesicular skin rash, disseminated infection, and high morbidity and mortality (Clarkson et al., 1967; Allen et al., 1974; Riopelle et al., 1971). VZV and SW are antigenically related as demonstrated by complement fixation, viral neutralization, immunofluorescence, and immunoprecipitation assays (Blakely et al., 1973; Felsenfeld and Schmidt, 1975, 1977; Allen, et al., 1974; Fletcher and Gray, 1992). Monkeys immunized with VZV were resistant to simian varicella upon subsequent challenge with SW (Felsenfeld and Schmidt, 1979). In addition, Soike et al. (1987) found that monkeys immunized with specific VZV glycoproteins were partially protected from infection upon challenge with SVV. The SW genome is similar to the VZV genome in both size and structure (Gray et al., 1992; Dumas et al., 1981; Ecker and Hyman, 1982; Straus et al., 1981). The SVV genome is approximately 121 kilobase pairs (kbp) in size and consists of a long component (L, = 100 kbp) and a short component (S, = 20 kbp). The S component is composed of a unique sequence (Us, = 5.3 kbp) which is bracketed by inverted repeat sequences (IRS and TRs, = 7.2 kbp). The inversion of the S component relative to the L component allows the genome to exist in two isomeric forms. The genomes of SVV and VZV share 70-75% DNA homology (Gray and Oakes, 1984). However, the distribution of this homology has not been established. The purpose of the present study was to determine whether the SW and VZV genomes are colinear. The results indicate that homologous SVV and VZV DNA restriction endonuclease (RE) fragments are located in similar genomic map positions. In addition, SW homologues to VZV glycoprotein genes (gp I, II, III, and IV) and the putative VZV immediate early gene 62 were identified. The SVV and VZV homologues were located in colinear map positions.

Materials

and Methods

Cell culture and rk-uses

African Green monkey kidney (Vero) cells were cultured in Eagle’s minimal essential medium (EMEM) supplemented with 5% newborn calf serum and gentamicin (50 pg/ml). The Delta herpesvirus (DHV) was propagated in Vero cells by mixing infected and uninfected cells at a ratio of 1: 4. This SW strain was originally isolated from the blood of an infected patas monkey during the 1973

257

simian varicella epizootic at the Delta Regional Covington, Louisiana (Ayres, 1971).

Primate

Center

(DRPC)

in

Cloned VZV RE fragments and genes

VZV RE fragments cloned into pBR322 and pACYC184 plasmids were provided by Dr. Richard Hyman (Ecker and Hyman, 1982). The VZV Hind111 J, an L component terminus, was derived by Hind111 digestion of the cloned VZV EcoRI C. However, the Hind111 J and the adjacent Hind111 K co-migrated on agarose gels. Therefore, the Hind111 J/K band was used as a hybridization probe. Plasmids containing open reading frames (ORFs) of the VZV glycoprotein genes gpll (pKIP39) and gpII1 (pKIP57) were provided by Dr. John Hay (State University of New York at Buffalo). The plasmids containing the VZV glycoprotein genes gp I (pW68.1A) and gp IV (in pGEM4) and VZV gene 62 (pN148) ORFs were provided by Dr. Don Gilden (University of Colorado School of Medicine). Purification of viral nucleocapsids and isolation of viral DNA

SW nucleocapsids were purified by the method of Straus et al. (1981). The nucleocapsid pellet was resuspended in 3 ml TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5) with 1% SDS and 200 ,ug/ml proteinase K and incubated at 50°C for 3 h. The viral DNA was extracted with phenol, phenol-chloroform, and chloroform, and precipitated with 0.1 vol. 3 M sodium acetate and 2 ~01s. of ethanol. The DNA was pelleted by centrifugation at 10,000 X g for 30 min at 4°C and resuspended in TE buffer. Electrophoresis and Southern blot transfers

SW DNA was digested with BarnHI, BglII, or EcoRI RE. Digested DNAs were fractionated by gel electrophoresis through 0.5% agarose containing Tris borate buffer (TBE, 0.89 M Tris, 0.89 M boric acid, 0.002 M EDTA) and 0.5 pg/ml ethidium bromide. The DNAs were alkali denatured, neutralized, and transferred to Gene Screen Plus nylon membranes (DuPont, Wilmington DE) by the capillary blot method as described by the manufacturer. DNA Hybridizations

Viral DNAs were 32P-labeled using a random primers DNA labeling system (Bethesda Research Laboratories). Gene Screen blots of RE digested SW DNAs were hybridized with radiolabeled VZV DNA probes. Standard stringency hybridizations were performed at 42°C in hybridization buffer (50% formamide, 1% SDS, 10% dextran sulfate, 1 M NaCl, and 100 pg/ml salmon sperm DNA) and denatured VZV DNA probe (2.0 X lo6 counts per minute). Reduced stringency hybridizations were performed at 37°C in hybridization buffer containing 40%

258

formamide and 6.0 X 10h counts per minute denatured VZV DNA probe. The blots were washed in 2 Y SSC (0.3 M sodium chloride, 0.3 M sodium citrate) and 1% SDS at 65°C (standard conditions) or 56.6”C (reduced stringency). Autoradiography was performed using Kodak X-omat AR film and intensifying screens at - 70°C for variable time periods. The standard and reduced stringency hybridization conditions corresponded to Tm - 22°C and Tm - 34°C respectively, as calculated by the equation Tm = 81.5 + 16.6 (log Ml + 0.41 (% G + C) - 0.72 (% formamide) where Tm is the melting temperature of the DNA, M is the monovalent salt molarity, and (% G + Cl is the percentage of guanosine plus cytosine in the DNA (Schildkraut and Lifson, 1965; Howley et al., 1979). The (% G + C) is 46% for VZV DNA (Ludwig et al., 1972; Davison and Scott, 1986) and = 46% for SVV DNA (W.L. Gray, unpublished observation).

Results Hybridization of cloned VZV HindIII fragments to SW DNA

To determine whether the VZV and SW genomes are colinear, thirteen VZV Hind111 RE fragments representing 98.3% of the VZV genome were “*P-labeled and hybridized to immobilized SW DNA. VZV RE fragment and specific gene probes did not hybridize to SW DNA under standard hybridization conditions (data not shown). However, previous studies demonstrated DNA homology between the SW and VZV genomes using reduced stringency hybridization conditions (Gray and Oakes, 1984). This method has been shown to be optimal for detecting DNA homology between heterologous, but genetically related DNAs (Howley et al., 1979; Law et al., 1979). The stringency of hybridization may be reduced by decreasing the formamide concentration in the hybridization buffer (Howley et al., 1979). Therefore, these experiments were performed under reduced stringency conditions (40% formamide, 37°C). For reference, RE maps of the VZV and SVV genomes are provided in Fig. 1. Each of the VZV DNA fragments from the L component hybridized to SVV DNA RE fragments derived from the L component of the SW genome. The VZV DNA probes hybridized to SVV DNA RE fragments located at colinear genomic map positions (Figs. 2 and 3, Table 1). For example, the VZV Hind111 A (map units 0.10-0.32) hybridized to the SVV EcoRI A and H; the SVV Bglll B, E, P, and R; and the SW BamHI A, F, J, and K. The SW DNA sequences which are homologous to the VZV Hind111 A fragment were located between map units 0.10-0.30. VZV DNA probes derived from the S component of the genome hybridized to sequences located in the SW DNA S component (Fig. 3, Table 1). The VZV Hind111 H (map units 0.81-0.84) consists of L component and viral internal repeat (IRS) sequences (Fig. 1). The sequences in the IRS are also present (map units 0.97-1.00) in the terminal repeat (TRs). The VZV Hind111 H hybridized to the

-1

Y

-

1

t

Q

m

c/

E

Bg

Ba

B. 0a

Hindtlt J/K

Bg

Hind K

E BgBa

Hindlil A

I

Hind

E 8gBa

E BgBa

HindIll E

E Bg Ba

Ba

Hindlll II

E Bg

Hindlll t3

E Bg Ba

Fig. 2. Hybridization of VZV L component probes to SW DNA digested with EcoRl (El, BgllI (Bg), or BumHI (Ba) RE. (Af SW DNA RE fragmt were fractionated by agarose electrophoresis and transferred to filters. A photographic negative of an ethidium bromide stained gel is shown. “2P-labeled Hind111 VZV DNA probes were hybridized to the SW DNA under reduced stringency conditions and the results were determined autoradiography. The VZV Hind111 DNA probe used is indicated at the bottom of each autoradiogram. SVV DNA fragments hybridized by each probe designated. The VZV Hind111 J/K probe included the VZV DNA terminal Hind111 J and a ~-migrating Hind111 K. Hybridization of VZV MndIII J/F the SVV B&Ii N was detected upon longer exposure of the autoradiogram.

A’

261 E

Bg Ba

Hindlll L

E Bg Ba

E Bg Ba

E Bg Ba

Hfndlll F

Hlndlll G

Hindlll H

Bg Ba

E

Hindlll C

E BgBa

Hlndlll M

Fig. 3. Hybridization of VZV DNA L and S component probes to SW DNA digested with EcoRI (E), BgfII (Bg), and BarnHI (Ba) RE. The methods are described in Fig. 2. The VZV Hind111 probe used and the SW DNA fragments hybridized are indicated for each autoradiogram. The faint BumHI bands detected by the VZV Hind111 H are partial digestion products. Hybridization of VZV Hind111 H to the SW Bgf II C could be detected upon longer exposure of the autoradiogram.

SVV EcoRI E; Bg111 C and I; and BumHI D. The SW sequences hybridized corresponded to map units 0.80-0.86 which includes L component and IRS sequences. The VZV Hind111 H also hybridized to the SVV EcoRI L, BglII K, and BarnHI G. The hybridized SW sequences.included in these RE fragments were located between map units 0.97-1.00 and contain SVV TRs sequences.

1.0 -

.oo-

<.oo-

coz *do-

--

rl

.E

.30-

.20-

1

.lO -

0.0 -

0.0

.10

20

JO

.a0

.m

.oo

SVV DNA

20

.a0

.oo

1.0

Fig. 4. Diagram indicating the regions of homology between the SW and VZV genomes. The boxes indicate the locations of homologous sequences as determined by DNA hybridization (Figs. 2 and 3, Table 1). The SW sequences homologous to the VZV Hind111 J were deduced by comparing the results of the VZV Hind111 J\K and K hybridizations to SW DNA. The VZV genome (left vertical axis), the VZV Hind111 RE map (right vertical axis), and the SW genome (horizontal axis) are shown.

262 TABLE 1 VZV NindIII RE fragments hybridize to genomic SVV DNA vzv probe ’

Location ’

J/K

0.00-0.07

SW Fragments hybridized Eco RI

BgiII

BumHI

SVV region of homology ’

NDd

J

A

0.01-0.10

A

0.04-0.09

A

0.07-0.20

A F J K H

0. IO-O.30

I. N

K

0.04-0.07

G

I

0.07-0.10

A

0.10-0.32

A G A H

E

0.32-0.40

F 0.P

D

0.40-0.50

B

0.50-0.64

L

0.64-0.67

F

0.68-0.75

B N B J Q D M D K E K

L N B N B E P R G M

0.34-0.40

Q

G

0.75-0.81

H

0.81-0.84

E L

A A F 0 F

c

0.41-0.55 0.44-0.62

0.65-0.67 0.67-0.76

Ii C

0.75-0.83

C

0.80-0.86 0.97-1.00

C

0.86-0.97

c

I K D

M

0.84-0.86 0.97-0.99

E L

I K

a b ’ d

B M B E N c

L P D G

0.90-0.94 0.83-0.86 0.97-1.00

Cloned Hind111 RE fragments. values expressed in map units. the map units given represent the maximum SW DNA sequences hybridized. not determined.

The results of the hybridization experiments are summarized in Table 1. Each of the VZV DNA probes hybridized to SW DNA confirming the genetic relatedness between the VZV and SW genomes. In addition, the homologous SVV and VZV DNA sequences were located in similar genomic map positions. The sum of these results indicated a colinear relationship between the VZV and SW genornes (Fig. 4).

263 E

Ba

B

E

Ba

E

Ba

Ba

E

E

B

BB

C

Ba

E

C

9P 111

BP tt

BP1

IE 62

9P IV

Fig. 5. Hybridization of specific VZV glycoprotein genes (gp I, 11, III, IV) and VZV gene 62 to genomic SW DNA digested with BernHI (Ba) and EcoRI (E) RE. SW DNA RE fragments were fractionated by agarose electrophoresis and transferred to filters. 32P-labeled VZV gene probes were hybridized to the SW DNA under reduced stringency conditions and the results were determined by autoradiography. The VZV gene probe used and the SW DNA fragments hybridized are indicated for each autoradiogram.

Hybridization of specific VZV genes to SW DNA

The finding that the VZV and SW genomes are colinear indicated that SW and VZV gene homologues may exist and be located in colinear map positions. Therefore, VZV genes of known map positions were used as probes to determine the presence and location of potential SW gene homologues. Specifically, 32Plabeled VZV gp1, II, III, and IV genes and gene 62 were hybridized to SW DNA. Fig. 1 shows the positions of the VZV genes. The results are shown in Fig. 5 and summarized in Table 2. VZV gp1 and VZV gpIV, which are located primarily within the Us component of the VZV genome, hybridized to SVV DNA sequences

TABLE 2 VZV genes hybridize to genomic SW DNA vzv probe a

Location b

gPI gPI1 izPII1 &TPIV

0.93-0.95 0.46-0.48 0.53-0.55 0.91-0.93 0.83-0.86 0.97-1.00

62

SW fragments hybridized EcoRI

BumHI

SW region of homology c

C B B C E L

L B B L D G

0.90-0.94 0.44-0.5s 0.44-0.55 0.90-0.94 0.81-0.86 0.97-1.00

a Cloned VZV gene probes. b values expressed in map units. ’ the map units given represent the maximum SW DNA sequences hybridized.

264

located in the S component between map units 0.90-0.94. VZV gpII and VZV gpII1 hybridized to SVV DNA sequences between map units 0.44-0.55. These SVV sequences are comparable to the map positions of the VZV glycoprotein II and III genes. The VZV gene 62, located in the internal (IRS) and terminal (TRs) repeat sequences of the VZV genome, hybridized to SW DNA sequences located within the repeat sequences of the SVV genome (map units 0.81-0.86 and 0.97-1.00). These results demonstrated that SW DNA sequences homologous to VZV glycoprotein genes and gene 62 exist and are located in colinear map positions.

Discussion

The results of this study confirm the previously described genetic relatedness of SVV and VZV (Gray and Oakes, 1984). The homologous SW and VZV DNA sequences were located throughout the viral genomes and in similar genomic map positions, indicating a colinear relationship between the viral genomes. VZV and several other alphaherpesviruses, including HSV-1, HSV-2, equine herpesvirus type 1 (EHV-l), and pseudorabies virus (PRV) possess homologous DNA sequences and colinear genomes (Davison and Wilkie, 1983). The colinear reIationship between the alphahe~esvirus genomes suggests that these viruses have diverged from a common ancestral herpesvirus. SW DNA sequences homologous to VZV gene 62 and VZV glycoprotein genes (gpl, II, III, IV) were identified. The VZV gene 62 is a putative immediate early gene and is homologous to the HSV-1 immediate early gene Vmw175 (Davison and Scott, 1986). The VZV gene 62 product, like the HSV-1 Vmw175 gene product (ICP4), functions as a transactivator of viral transcription and may be a potent regulator of viral gene expression (Everett, 1984; Cabirac et al., 1990; Disney and Everett, 3990). Previous studies have demonstrated that VZV and SW proteins and glycoproteins possess extensive cross-reactivity (Fletcher and Gray, 1992). SW possesses at least 6 glycoproteins ranging in size from 46 to 115 kDa. Each of the SW glycoproteins were immunoprecipitated by VZV immune sera. Conversely, each of the VZV gly~proteins appeared to be immunoprecipitated by SVV immune sera. These studies suggest that SW and VZV possess homologous glycoprotein genes which encode antigenically related glycoproteins. The identification of SW sequences which are homologous and colinear with VZV gene 62 and glycoprotein genes suggests that the location of specific SVV genes may be predicted based upon the known map positions of homologous VZV genes. The initial mapping and characterization of SW genes will therefore be facilitated, since the entire VZV genome has been sequenced and 71 ORFs have been identified (Davison and Scott, 1986). Based on the antigenic and genetic relatedness between SW and VZV and the clinical similarities between human and simian varicella, SVV infection of non-human primates has been used as a model for studying VZV pathogenesis and for evaluating the effectiveness of antiviral agents (Arvin et al., 1983; Soike et al.,

265

1987, 1990). Recently, SW has been demonstrated to establish latent infection of dorsal root ganglia in infected monkeys indicating that the simian varicella model may be useful to investigate herpesvirus latency (Mahalingam et al., 1991). The demonstration of colinearity between the SVV and VZV genomes provides further support for simian varicella as a model for human VZV infections. Acknowledgements

We thank Ms. Nanette Gusick for her excellent technical assistance. This investigation was supported by Public Health Service Grant AI 26070 from the National Institutes of Health, by Grant lN167 from the American Cancer Society, by U.A.M.S. Institutional Biomedical Research Support Grant RR 05350-26, by an award from the U.A.M.S. Caduceus Club, and by a grant from the U.A.M.S. Graduate Student Research Fund (C.Y.P.). We thank Drs. J. Menna and M. Roop for critical review of the manuscript. References Allen, W.P., Felsenfeld, A.D., Wolf, R.H. and Smetana, H.F. (1974) Recent studies on the isolation and characterization of delta herpesvirus. Lab. Anim. Sci. 24, 222-228. Arvin, A.M., Martin, D.P., Gard, E.A. and Merigan, T.C. (1983) Interferon prophylaxis against simian varicella in Erythrocebus patas monkeys. J. Infect. Dis. 147, 149-154. Ayres, J.P. (1971) Studies of the delta herpesvirus isolated from the patas monkey (Etyrhrocebus patas). Lab. Anim. Sci. 21,685-695. Blakely, G.A., Lourie, B., Morton, W.G., Evans, H.H. and Kaufmann, A.F. (1973) A varicella-like disease in macaque monkeys. J. Infect. Dis. 127, 617-625. Cabirac, G.F., Mahalingam, R., Wellish, M. and Gilden, D. H. (19901 Trans-activation of viral tk promoters by proteins encoded by varicella zoster virus open reading frames 61 and 62. Virus Res. 15, 57-68. Clarkson, M.J., Thorpe, E., and McCarthy, K. (1967) A virus disease of captive vervet monkeys Cercopifhicus aethiops caused by a new herpes virus. Arch. Gesamte Virusforsch. 22, 219-234. Davison, A.J. and Scott, J.E. (1986) The complete DNA sequence of varicella-zoster virus. J. Gen. Virol. 67, 1759-1816. Davison, A.J. and Wilkie, N.M. (19831 Location and orientation of homologous sequences in the genome of five herpesviruses. J. Gen. Virol. 64, 1927-1942. Disney, G.H. and Everett, R.D. (1990) A herpes simplex virus type 1 recombinant with both copies of the Vmw175 coding sequences replaced by the homologous varicella-zoster virus open reading frame. J. Gen. Viral. 71, 2681-2689. Dumas, A.M., Geelen, J.L.M.C., Weststrate, M.W., Wertheim, P., and Van Der Noordaa, J. (1981) Xbal, PsrI, and EglII restriction endonuclease maps of the two orientations of the varicella-zoster virus genome. J. Virol. 39, 390-400. Ecker, J.R. and Hyman, R.W. (19821 Varicella zoster virus DNA exists as two isomers. Proc. Natl. Acad. Sci. USA 79, 156-160. Everett, R.D. (19841 Transactivation of transcription by herpes virus products: requirement for two HSV-1 immediate early polypeptides for maximal activity. EMBO J. 3, 3135-3141. Felsenfeld, A.D. and Schmidt, N.J. (1975) Immunological relationship between delta herpesvirus of patas monkeys and varicella-zoster virus of humans. Infect. Immun. 12, 261-266. Felsenfeld, A.D. and Schmidt, N.J. (1977) Antigenic relationships among several simian varicella-like viruses and varicella-zoster virus. Infect. Immun. 15, 807-812.

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