Activation of mouse retrovirus by herpes simplex virus type 1 cloned DNA fragments

VIROLOGY

103,228-231

(1980)

SHORT COMMUNICATIONS Activation

of Mouse Retrovirus by Herpes Simplex Type 1 Cloned DNA Fragments’

Virus

ANN L. BOYD,” LYNN ENQUIST,t GEORGE F. VANDE WOUDE,t AND BERGE HAMPAR$? *Carcinogenesis Intramural Program and SMicrobiology Section, Laboratory of Molecular Virology, National Cancer Institute, Frederick Cancer Research Center, Frederick, Maryland 21701, and i-Virus Tumor Biochemistry Section, Laboratory of Molecular Virology, National Cancer Institute, Bethesda, Maryland 20205 Accepted February

11, 1980

Cloned DNA fragments from herpes simplex virus (HSV) type 1 (strain Patton) were tested for activation of endogenous mouse retrovirus in BALB/3T3 cells. Activation within the L region of HSV-1 DNA was observed with the -3.4-kilobase pair (kbp) BamHI fragment which contains the virus thymidine kinase (TK) gene, and the -5.3-kbp EcoRI L fragment. Activation by the TK-containing BamHI fragment was abrogated by digestion with EcoRI. Activation within the S region of HSV-1 DNA was observed with the -15.2-kbp EcoRI H fragment and the -8.4-kbp EcoRIiHindIII H/G fragment. Assaying for retrovirus activation serves as an additional parameter for mapping biological functions within the HSV genome.

The activation and synthesis of endogenous mouse xenotropic (MUX) retrovirus with uv-irradiated herpes simplex virus (HSV) types 1 and 2 (1, 2) and with DNA isolated from various human and nonhuman herpesviruses, including HSV-1 and -2, have been reported (3). The latter findings affirmed the role of the herpesvirus genome in the activation process, and suggested that retrovirus activation could serve as a marker for mapping properties of specific DNA sequences within the HSV genome. The DNA of HSV is a linear, doublestranded molecule containing approximately 160 kilobase pairs (kbp), and is composed of two covalently linked components (L and S), each containing internal unique sequences and terminal redundant sequences(reviewed in Ref. (4 )>. The structure of the HSV DNA allows for inversion of the L and S components, resulting in four permutations of approximately equimolar concentration. ’ The U. S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. 2 To whom reprint requests should be addressed. 0042-6822/80/070228-04$02.00/O Copyright All rights

D 1980 by Academic Press, Inc. of reproduction in any form reserved.

228

Procedures have been described recently for cloning and amplifying in phage X specific HSV-1 DNA fragments generated by endonuclease restriction enzymes (5). We have used these cloned DNA fragments to identify regions of the HSV-1 (strain Patton) genome which activate endogenous mouse retrovirus. The BALB/3T3 cells (obtained from W. Brockman, University of Mighigan) were maintained in Eagle’s minimum essential supplemented with 10% heat-inactivated fetal calf serum. The feline-derived F81 cells (obtained from P. J. Fischinger, National Cancer Institute) were maintained as described (6 ). All cell lines were routinely tested by R. Del Giudice (Frederick Cancer Research Center) and found free of mycoplasma contamination. The source of cloned HSV-1 (strain Patton) DNA fragments was as described (5). The recently cloned EcoRI F (gift from K. Thompson, National Cancer Institute) and 0 fragments were included in the present studies. Procedures for identifying cloned HSV fragments have been described (5), and fragments were given letter designa-

229

SHORT COMMUNICATIONS

tions according to the nomenclature of Skare formation between 3T3 and F81 cells could account for the observed activation of MUX and Summers (7). HSV-1 (strain Patton) cloned DNA frag- retrovirus by specific HSV DNA fragments. ments were digested with the indicated BALB/3T3 cells prelabeled with 13H]thymiendonuclease restriction enzyme and tested dine in 100% of the cell nuclei (determined autoradiographically) were treated with for activation of MUX virus in BALB/3T3 cells (Table 1). Vaccinia virus DNA served mitomycin and overlaid with F81 cells as as a negative control (3) and HSV DNA described in the legend to Table 1. After isolated from strains 14-012 (type 1) and 12 hr the cells were exposed to 1000 hemag333 (type 2) served as positive controls glutinating units of B-propiolactone-in(3). The vector DNAs were negative for activated Sendai virus (9 ). Autoradiographic analysis carried out with cells fixed 3 hr activation when tested alone. Two fragments from the L region of the after treatment with Sendai virus indicated HSV-1 genome were positive for MUX virus that 7% of the labeled 3T3 cells had formed activation, the -5.3-kbpEcoR1 L fragment heterokaryons with unlabeled F81 cells, and the -3.4-kbp BumHI fragment con- while ~0.01% heterokaryon formation was taining the HSV thymidine kinase gene (8 ). observed in control cultures not exposed to Both fragments are within the unique se- Sendai virus. Activation of MUX retrovirus quences of the L region. Digestion of the in 3T3 cells was observed at a frequency of l-2 x lo-” in both Sendai virus-treated Ba,mHI TK-containing fragment withEcoR1 and untreated cultures, indicating that destroyed its ability to activate MUX virus. Similarly, the EcoRI N and G fragments heterokaryon formation was not sufficient, which are cleaved within the active TK in itself, to account for the retrovirus acregion were negative for activation. In the S tivation we observed with specific HSV region of HSV-1 DNA, the -15.2-kbp DNA fragments. These findings demonstrate that specific EcoRI H fragment was positive for activaregions within the HSV-1 genome contain tion as was the -8.4-kbp EcoRIIHindIII H/G fragment which lies within the H frag- DNA sequences capable of activating enment. The -1.9-kbp EcoRIIHindIII H/M dogenous MUX retrovirus (Fig. 1). Complete fragment within the EcoRI H fragment was mapping of the HSV-1 genome with respect to retrovirus activation must await availnegative for activation. The dose-response curves for activation by positive fragments ability of fragments not yet cloned, and it were similar to that described previously will be necessary to test fragments generated for HSV DNA (3) and was linear up to a by additional endonuclease enzymes. For concentration of 0.1-0.2 pg DNA. example, while the TK-containing BumHI Additional studies were carried out to ex- fragment, which overlaps the EccRI G and N fragments, was positive for activation, clude the possibility that heterokaryon

TK

I

L

H

Activation Positive HIG

I

I

Activation Negative A,A

AIL

I I

m

’

HlM

FIG. 1. MUX virus activation by HSV-1 (strain Patton)-cloned DNA fragments. Top: letter designation and map position of EcoRIIHindIII-digested fragments according to Skare and Summers (7). One of four possible permutations of the L and S regions is shown. Fragments positive (middle) and negative (bottom) for MUX virus activation are depicted according to the endonuclease restriction enzyme used for isolation. EcoRI fragments (solid bars), EcoRIiHindIII fragments (diagonal lines), BumHI fragment (horizontal lines).

-

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SHORT

COMMUNICATIONS TABLE

1

ACTIVATION OF MUX VIRUS WITH HSV-1 DNA CLONED FRAGMENTS” Activation:

Cloned DNA fragments” Positive fragments L (EcoRI) H (EcoRI) HG (EcoRIiHindIII) TK (BarnHI)

Negative fragments TK (BarnHI)-EcoRI

Exp. 1

Exp. 2

15,18

15,17

Exp. 3

foci/lo” Exp. 4

ceIIs/0.25-0.5 Exp. 5

Exp. 6

Exp. 7

13,14 10,ll

14,16 11,12

15,17

13,15

10,14 16,lO

3,2

6,6

18,16 13,12

kg DNA Exp. 8

Exp. 9

IO,12

21,18

2,4

1,0

14,16 9,12

16,14

digested’

1.2

TO D (EcoRI) M (EcoRI) N (EcoRI) F (EcoRI) I (EcoRI) G (EcoRI) 0 (EcoRI) GJ (EcoRliHindIII) AL (EcoRIiHindIII) AA (EcoRIiHindIII) HM (EcoRIiHindIII) Controls-positive” HSV-333 DNA HSV-14-012 DNA Controls-negative” h phage DNA pBR322 DNA Vaccinia DNA

2,1

291

5,6 2,3

5,5

12

272 2,3

4,5

3,l 491 Of1

1810

12,14

10,12

17,20

1,2

3,2

0,l

2,0

1,3

22 3,4

2,3 l,o

12,15

22,20 12,14

12,14

02

3,2 3,2

2,3

2,3

22

33,27

2,3 3,4

021 0,l

2,2

3,0

031 2,0

” The direct infectious center assay for MUX virus was a modification of described procedures (3). Replicate cultures of 3T3 cells (5 x 104) seeded 72 hr previously in loo-mm petri dishes were inoculated with 1 ml of a mixture containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 125 mM CaCl,, and 0.250.5 pg insert HSV DNA or appropriate test DNA. The DNA from vaccinia virus, HSV type 1 (strain 14.012), and HSV type 2 (strain 333) were prepared as described (3). Carrier salmon sperm DNA was added to bring the final concentration of each sample to 5 pg DNA. The DNA-containing mixtures were prepared 20 min before addition to the cells to allow precipitate formation. After 30 min adsorption at room temperature, the inoculated cells were flooded with growth medium containing dexamethasone (0.2 Fg ml-‘) and incubated for 6 hr at 37”. Representative cultures were treated with trypsin for determination of total cells present. The remaining cultures were refed, treated for 1 hr with mitomycin (10 pg ml-‘), the cells were washed three times with medium, refed, and incubated for 1 hr. The medium was removed and 1.5 x IO” F81 cells were added in medium containing Polybrene (2 kg ml-‘) and dexamethasone (0.2 pg ml-‘). The remainder of the procedure was as described (3). b Cloned fragments were digested with the indicated endonuclease restriction enzymes. Salmon spermcarrier DNA was added as necessary to bring the total DNA concentration to 5 pg. c The TK-containing BamHI fragment was tested following digestion with EcoRI. ” HSV-333, HSV-14-012, and vaccinia virus DNAs were isolated, sonicated, and tested as described previously (3). A phage DNA and plasmid pBR322 DNA were tested intact. Salmon sperm-carrier DNA was added to bring the total DNA concentration to 5 wg.

SHORT

231

COMMUNICATIONS

the G and N fragments were negative. Complete mapping will also require testing other HSV-1 strains since recent evidence indicates that HSV-1 strains may show intratypic variations in the endonuclease retriction enzyme patterns of their DNA (4, 10). The fact that we observed retrovirus activation with the BamHI fragment (map position 0.29-0.32) raises the possibility that activation is associated with the HSV TK gene located within this fragment (8). The BarnHI fragment obviously contains gene sequences other than TK (-1.3 kbp) which precludes any conclusion concerning a direct relationship between retrovirus activation and TK activity. It is of interest in this regard that digestion of the BasmHI fragment with EcoRI abrogates both the TK activity (8) and the activation potential of the DNA. The EcoRI L fragment (map position 0.46-0.49) from the L region also proved positive for retrovirus activation. Although resistance to phosphonoacetate (PAA’) maps within this region of the HSV-1 genome (II), there may be no correlation with retrovirus activation since EcoRI-digested DNA does not transfer the PAA’ phenotype (1~). Similarly, the activation-positive EcoRI H fragment (map position 0.87-0.97) and the EcoRIiHindIII H/G fragment (map position 0.92-0.97) from S region lie within the general area of the HSV-1 genome where coding sequences for glycoprotein D have been mapped (13), but the two activities may not be correlated. ‘The mechanism of retrovirus activation by HSV or its DNA is not presently known, and additional properties cannot be ascribed to those HSV DNA fragments which demonstrate activation potential. Previous findings (3) showed that activation of retrovirus required biologically active HSV DNA, since activation was not observed with crosslinked DNA. Information is available concerning gene organization within the HSV-1 genome (4, 8, 11, 14, 15), and at least one DNA region (Xba-1 F fragment, map position 0.29-0.45) has been shown capable of inducing morphologic transformation of hamster embryo cells (16). We show here that activation of mouse retrovirus can serve as an additional parameter for map-

ping biological functions within the HSV genome. ACKNOWLEDGMENT This research was supported in part by Contract NOl-CO-75380 with the National Cancer Institute, NIH, Bethesda, Maryland 20205. REFERENCES 1. HAMPAR, B., AARONSON, S. A., DERGE, ,J. G., CHAKRABARTY, M., SHOWALTER, S. D., and DUNN, C. Y., Proc. Nat. Acad. Sci. l:SA 73, 646-650 (1976). 19, 2. REED, C. L., and RAPP. F.. d. Viral. 1028-1033 (1976). 3. BOYD, A. L.. DERGE, J. G., and HAMPAR, B.. Proc. Nat. Acad. Sci. 1iSA 75, 4558-4562 (1978). i. ROIZMAP;, B., Cell 16, 481-494 (1979). 5. ENQUIST, L., MADDEN. M. J., SHIOP-STANSLY, P., and VANDE WOUDE. G. F.. Sciencr 203, 541-544 (1979). 6. HAMPAR, B., HATANAKA. M., AULAKH, G.. DERGE, J. G., LEE, L.. and SHOWALTER, S.. Virology 76, 876-881 (1977). 7 SKARE, J., and SUMMERS, W. C., Vimlogy 76, 581-696 (1977). 8. WIGLER, M., SILVERSTEIN, S., LEE, L. -S., PELLICER, A., CHENG, Y. -C., and AXEL, R., Cell 11, 223-232 (1977). 9. HAMPAR, B., RAND, K. H., LERNER, R. A., DEL VILLANO. B. C., JR., MCALLISTER, R. M., MARTOS, L. M., DERGE, J. G., LONG, C. W., and GILDEN, R. V., Virology 55, 453-463 (1973). 10. LONSDALE, D. M., MOIRA BROWN, S., SUBAKSHARPE, J. H., WARREN, K. G., and KOI’ROWSKI, H.. J. Gelz. Viral. 43, 151-171 (1979). 11. MORSE, L. S., PEREIRA, L., ROIZMAN, B.. and SCHAFFER, P. A., J. Viral. 26, 389-410 (1978). 12. KNIPE, D. M., RUYECHAN, W. T., and ROIZMAN, B., J. viral. 29, 698-704 (1979). 13. RUYECHAN, W. T., MORSE, L. S., KNIPE, D. M., and ROIZMAN, B., J. Viral. 29, 677-697 (1979). 14. MARSDEN, H. S., STOW, N. D., PRESTON, V. G., TIMBURY, M. C., and WILKIE, N. M., J. Viro2. 28, 624-642 (1978). 15. WILKIE, N. M., STOW, N. D., MARSDEN, H. S., PRESTON, V., CORTINI, R., TIMBURY, M. C., and SUBAK-SHARPE, J. H., In “Oncogenesis and Herpesviruses III” (eds. G. de-The, W. Henle, and F. Rapp, eds.), pp. 11-31. International Agency for Research on Cancer, Lyon, France, 1978. 16. CAMACHO, A., and SPEAR, P. G., Cell, 9931002 (1978).