Prolonged Herpes Simplex Virus Latency In Vitro after Treatment

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1986, p /86/4589-5$2./ Copyright D 1986, American Society for Microbiology Vol. 29, No. 4 Prolonged Herpes Simplex Virus Latency In Vitro after Treatment of Infected Cells with Acyclovir and Human Leukocyte Interferon ADRIENNE C. SCHECK,' BRIAN WIGDAHL,' ERIK DE CLERCQ,2 AND FRED RAPP'* Department of Microbiology and Cancer Research Center, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 1733,1 and Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3 Louvain, Belgium2 Received 23 September 1985/Accepted 3 January 1986 We previously demonstrated that herpes simplex virus type 1 (HSV-1) can be established in a latent form in vitro by the treatment of HSV-infected human cells with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) in combination with human leukocyte interferon (IFN-a). We now report that the substitution of BVDU with 9-[(2-hydoxyethoxy)methyl]guanine (acyclovir; ACV) during a combined treatment with IFN-a inhibited HSV-1 replication and established in vitro virus latency that could be maintained for a longer period after inhibitor removal and a continued incubation at 37 C. B]y contrast, the treatment of HSV-l-infected cells with combined IFN-a and 9-(1,3-dihydroxy-2-propoxymethyl)guanine, a congener of ACV, failed to establish in vitro virus latency. Furthermore, none of these inhibitors used alone was sufficient to establish in vitro virus latency. The use of nucleoside analogs differing from BVDU in their modes of action has enabled us to initiate studies designed to extend in vitro virus latency. We previously reported that (E)-5-(2-bromovinyl)-2'- deoxyuridine (BVDU) in combination with human leukocyte interferon (IFN-a) limits the expression of the herpes simplex virus (HSV) type 1 (HSV-1) genome and permits the establishment of a latent infection in vitro (25, 26). To examine the role and specificity of combined BVDU and IFN-a treatment on the establishment and maintenance of in vitro virus latency, nucleoside analogs with different mechanisms of action were combined with IFN-a to treat HSV- 1-infected cells. 9-[(2-Hydroxyethoxy)methyl]guanine (acyclovir; ACV) acts as a potent inhibitor of HSV replication (4, 5, 1, 23) and is phosphorylated in a manner similar to that of BVDU (3); however, the incorporation of ACV- 5'-triphosphate into virus DNA causes the termination of DNA chain elongation (14-16). In addition, ACV-5'- triphosphate acts as an irreversible inactivator of the HSV- 1-encoded DNA polymerase (7, 8). A congener of ACV, 9-(1,3-dihydroxy-2-propoxy-methyl)guanine (DHPG), also was used in this study. DHPG acts at different sites on the HSV-encoded DNA polymerase molecule and may inhibit virus replication by mechanisms additional to the inhibition of DNA polymerase (1, 2, 6). These nucleoside analogs were used in addition to BVDU to determine whether the establishment of in vitro virus latency was caused by an intrinsic property of BVDU or whether other nucleoside analogs were capable of establishing in vitro virus latency after high-multiplicity HSV-1 infection. The use of ACV in combination with IFN-a resulted in the establishment of in vitro virus latency and a longer latent period after the removal of inhibitors and an extended incubation at 37 C. of IFN-a (8 x 14 to 6.5 x 15 IU/mg of protein) per ml, infected with HSV-1 (2.5 PFU per cell; 92% of the cells were infected as given by the Poisson distribution), and treated daily for 7 days with the same inhibitor or inhibitor combination as previously described (25). The nucleoside analog concentration of 3 plm was selected to correlate the results obtained with previous studies concerning our in vitro HSV latency model in which the concentration of BVDU (3,uM), when combined with IFN-a, was 1-fold greater than the minimum required to establish HSV latency in vitro. At these inhibitor concentrations, there was no apparent cytotoxicity as determined by microscopic examination. After the termination of the combined IFN-a and nucleoside analog treatment, in vitro virus latency was maintained by increasing the incubation temperature (18) from 37 to 4.5 C. HSV-1 replication was activated in these latently infected cells by superinfectiig at the elevated temperature with human cytomegalovirus (HCMV; 1.Q PFU per cell) or by reducing the incubation temperature from 4.5 to 37 C. In addition, HSV-1 was activated by continued incubation at 37 C after the termination of the combined inhibitor treatment. Analysis of HSV-l-specific DNA by blot hybridization. DNA was extracted from latently infected cells, digested with the indicated restriction endonucleases, subjected to agarose gel electrophoresis (26), blot transferred (21, 22), and hybridized to 32P-radiolabeled DNA probes (13, 19) at 37C in the presence of 1% dextran sulfate, 57% formamide, and 5 x SSC (1 x SSC is.15 M sodium chloride plus.15 M sodium citrate) as described before (26). MATERIALS AND METHODS Establishment, maintenance, and reactivation of latent HSV-1 infection in vitro. To establish virus latency in vitro, human fetus lung fibroblast (HFL-F) cells (2 x 16 to 5 x 16 cells per flask) were pretreated for 24 h with 3,uM BVDU, ACV, or DHPG alone or in combinatiop with 125 to 2 IU * Corresponding author. 589 RESULTS The treatment of HSV-1-infected HFL-F cells with combined BVDU and IFN-a resulted in a greater than 7% cell survival compared with that of mock-infected, inhibitortreated controls, with infectious virus undetectable on day 7 (25). The treatment of HSV-1-infected HFL-F cells with ACV and IFN-a for 7 days resulted in the elimination of detectable infectious virus 1 to 2 days earlier (Table 1), and a marginally higher cell survival rate (data not shown) than

2 59 SCHECK ET AL. ANTIMICROB. AGENTS CHEMOTHER. Inhibitor TABLE 1. Effect of nucleoside analog and IFN-a treatment on establishment of latent HSV-1 infection in vitro Virus titer" (relative cell survival and cytopathic damage') at day postinfection: None 4. x x x 18 >2 x 18 NDc ND ND (+) (+) (++++) (++++) IFN-a 7. x x x x x 14 ND ND (+) ( ) (+ + ++) (+ + ++) BVDU 8. x x X 14 ND 9.1 x 13 ND UDd (+)(++) ( +++) (+++) (++++) (++ ++) (+ ++ +) ACV 4.8 x x x 13 ND 9.3 x 1' ND UD (-) (+) (++) (+) (+++ +) (++ ++) (+++ +) DHPG 8.3 x x x x 12 ND ND ND (-) (+) (+ +) (+++ ) BVDU and IFN-a ND 4.1 x x 13 ND 2.2 x 12 UD UD ACV and IFN-ot 4.2 x x x 13 ND UD ND UD DHPG and IFN-a 5.4 x x x x 12 UD UD UD (-) (-) (+) (++) (++++) (++++) (++++) a Virus titer is expressed as PFU per culture. b Relative cell survival and cytopathic damage were evaluated by microscopic analysis and compared with uninfected, inhibitor-treated control cell cultures. -No evidence of virus-induced cytopathic effect; , less than 5% cell survival. c ND, Not determined. d UD, Undetectable. that obtained with BVDU and IFN-a treatment. By contrast, the treatment of HSV-1-infected cells with BVDU alone, ACV alone, DHPG alone, IFN-a alone, or DHPG in combination with IFN-ot resulted in a less than 5% cell survival by 5 days postinfection (Table 1). To determine whether a 7-day combined ACV and IFN-a treatment of HSV-1-infected HFL-F cells also resulted in the maintenance of the virus genome in a form that could be activated, the infected cell population was maintained at 37 C after the termination of combined inhibitor treatment, and total infectious virus was quantitated daily from randomly selected flasks. Virus reactivation generally occurred at least 5 days earlier, and resulted in a greater production of total virus in cells treated with combined BVDU and IFN-a than in those cells treated with ACV and IFN-a (Fig. 1). In addition, as late as 23 days after the removal of inhibitors at 37 C, virus was undetectable in 3% of the cultures treated with ACV and IFN-a, whereas less than 4% of the cultures treated with BVDU and IFN-a were still negative on day 18. Additional experiments were performed in which titers of the supernatant virus were determined daily and the cultures were monitored for the appearance of virus-induced cytopathic effects. In all cases, virus reactivation eventually occurred. Furthermore, cell viability did not substantially change during the course of these experiments after the termination of the combined nucleoside analog and IFN-a treatment. Infectious center analyses (25) of HSV-1-infected cells treated with either inhibitor combination were performed at the time of the inhibitor removal and after a 1-day incubation at 4.5 C. No significant difference in the infectious center titer (ranging from.5 to 1.7%; data not shown) of latently infected cells was observed at either time. These data suggest that the delay in virus reactivation from latently infected cells treated with ACV and IFN-ot was not caused by a difference in the number of cells containing a virus genome that could be activated. To eliminate the possibility that the delay in virus reactivation was solely caused by a longer antiviral half-life in those cells treated with combined ACV and IFN-ac compared a) IL L o4[ o2~ ACV or BVDU I NO and IFN-a + I INHIBITORS 1 O o I ~~1I e I FIG. 1. Total virus production during establishment and reactivation of in vitro virus latency in HFL-F cells. HFL-F cells were pretreated for 24 h with BVDU and IFN-ax or ACV and IFN-a, infected with HSV-1 (2.5 PFU per cell), and treated with the same inhibitor combination for 7 days. At this time the inhibitors were removed and cells (approximately 1 x 16 to 2 x 16 cells per flask) were maintained at 37 C. Randomly selected cultures were assayed for total infectious virus at the times indicated by plaque assay in primary rabbit kidney cells as described (25). Symbols:, ACV and IFN-a;, BVDU and IFN-a; UD, undetectable (<5 PFU per culture).

3 VOL. 29, 1986 with the cells treated with BVDU and IFN-(x, cells were pretreated, mock-infected or virus-infected, and treated daily for 7 days with either inhibitor combination. On day 7, the inhibitors were removed, and the cells were infected at 37 C with.5 or.1 PFU of HSV-1 per cell. There was little, if any, difference in the rate of virus replication in these cells, suggesting that the delay in virus reactivation was not solely caused by a difference in the antiviral half-life of the inhibitor combinations (data not shown). To examine the state of the HSV-1 genome during the establishment and maintenance of in vitro virus latency, DNA blot hybridization studies were performed. Total cellular DNA was isolated from inhibitor-treated, HSV-1- infected cells on days 1, 3, 5, and 7 of the inhibitor treatment and 3 days after inhibitor removal and increase in incubation temperature from 37 to 4.5 C. The DNAs were digested with Hindlll, and DNA blot hybridization analysis was performed by using a 32P-radiolabeled genomic proportion mixture of recombinant plasmid DNAs containing greater than 95% of the long and short unique HSV-1 DNA sequences as a probe (9, 17, 2, 26). Regardless of whether BVDU and IFN-a or ACV and IFN-a was used to establish in vitro latency, most, if not all, of the unique regions of the long and short components of the HSV-1 genome were present in the latently infected cell population, compared with those of the virion DNA shown in the reconstruction experiment (Fig. 2A). Although there was some reduction in the amount of virus DNA from cells treated with either ACV and IFN-a or BVDU and IFN-a during the treatment interval and shortly after the removal of inhibitors and an increase in incubation temperature, a longer incubation at the elevated temperature did not result in a further reduction of virus DNA. In addition, there was a decrease in the relative amount of virus DNA present in those cells treated with ACV and IFN-a compared with those treated with BVDU and IFN-at during the establishment of virus latency. The inactivation of virus-encoded DNA polymerase in the presence of ACV-substituted DNA (8), in addition to the limited virus DNA replication occurring in the presence of BVDU and IFN-a during day 1 of the treatment interval, is consistent with the reduced amount of virus-specific DNA in ACV and IFN-a-treated cells compared with BVDU and IFN-a-treated cells. We previously demonstrated that the predominant form of the HSV-1 genome retained in HFL-F cells after combined BVDU and IFN-a treatment is a nonintegrated, linear, unit-length molecule (26). To determine whether the state of the virus genome in cells treated with ACV and IFN-a was the same as that observed in cells treated with BVDU and IFN-at, total cellular DNAs were digested with BamHI and DNA blot hybridization analysis was performed by using the 32P-radiolabeled BamHl S-P (K) fragment as a probe. As shown in Fig. 2B, an increase in the ratio of junction to terminal HSV-1 DNA fragments was seen in total DNA isolated from cells 24 h postinfection in the presence of BVDU and IFN-a, and was confirmed by densitometric analysis (data not shown). This is indicative of concatenate DNA, and suggests a limited amount of virus DNA synthesis in these cells. This alteration in the ratio of junction to terminal fragments is not apparent in DNA isolated from cells at a later time postinfection, or in DNA isolated from cells treated with ACV and IFN-ao. In addition, there was no apparent alteration in the size of the HSV-1 junction or terminal DNA fragments obtained by the digestion of total cellular DNA isolated from ACV and IFN-at- or BVDU and IFN-a-treated, HSV-1-infected HFL-F cells, compared with PROLONGED HSV LATENCY BY ACV AND IFN-ot 591 A B R _ e." I%,. r--- n kb Al 81 Al 81 Al 81 Al 81 Al BI 6.5- a o_ a.6-4- lt:w Vt - :- r *~ r :~ -4: R kb Al BI Al Bl Al BI Al BI Al BI RSiw *1W 4b FIG. 2. DNA blot hybridization analysis of DNA isolated from latently infected HFL-F cells. In vitro virus latency was established as described. Total cellular DNA was isolated from latently infected cells 1, 3, 5, 7, and Hindl,l 1 days postinfection, digested with (panel A) or BamHl (panel B), subjected to electrophoresis through a.5% agarose gel, blot transferred to aminophenylthioether paper, and hybridized to either a mixture of 32P-radiolabeled recombinant plasmid DNAs containing greater than 95% of the long and short unique HSV-1 DNA sequences (26; panel A) or a 32P-radiolabeled plasmid DNA containing the BamHI S-P (K) junction fragment (panel B); autoradiography was then performed. Sizes (kilobases) of the DNA fragments are indicated to the left of each panel. Lanes: Al, ACV and IFN-ox; BI, BVDU and IFN-ox; R, reconstruction experiment. the authentic junction and terminal DNA fragments obtained by the digestion of purified virion DNA in the reconstruction experiment. These results suggest that the predominant form of the virus genome maintained during in vitro latency established by combined ACV and IFN-at treatment was nonintegrated, linear, and unit-length, a form similar, if not identical to, the predominant form of the latent virus genome obtained by combined BVDU and IFN-at treatment. DISCUSSION We have previously shown that the nucleoside analog BVDU can be used in combination with IFN-at to establish in vitro virus latency in human fetal fibroblasts and sensory neurons (26-28). We now report that the substitution of the same concentration of ACV for BVDU during a combined treatment with IFN-at resulted in a longer latent interval in

4 592 SCHECK ET AL. vitro. This prolonged latent period was most probably not caused by (i) a major difference in the intracellular antiviral half-life of the inhibitor combinations, (ii) a significant difference in the number of cells containing a virus genome that could be activated, or (iii) a major difference in the predominant form of the virus genome maintained during in vitro latency. The extended latent interval observed at 37 C after the removal of ACV and IFN-a suggests a requirement for an alteration in the state of the virus genome or in the intracellular environment to allow virus reactivation to occur as it does after the removal of BVDU and IFN-a. As previously reported with combined BVDU and IFN-a treated, HSV-infected cells (25), in vitro latent infection was maintained for extended periods of time by increasing the temperature from 37 to 4.5 C after ACV and IFN-o treatment and reactivated by either superinfecting with HCMV at the elevated temperature or reducing the incubation temperature from 4.5 to 37 C (data not shown). Although prolonged incubation at 4.5 C blocked the production of infectious virus, it did allow an alteration either in the state of the virus genome, which was undetectable by DNA blot hybridization analysis, or in the intracellular environment such that on reduction of the incubation temperature from 4.5 to 37 C, the kinetics of virus reactivation were identical in cultures treated with ACV or BVDU in combination with IFN-a (data not shown). In addition, although a 3-day incubation at 4.5 C resulted in a diminution of the lag in the onset of virus replication after a reduction in the incubation temperature, superinfection with HCMV of latently infected cells maintained at 4.5 C for 3 days resulted in the rapid reactivation of HSV-1 in cells treated with either ACV and IFN-a or BVDU and IFN-a. This suggests that superinfection by HCMV may accelerate a process similar to that which occurs during prolonged incubation. HCMV infection stimulates the production of DNA synthesis enzymes (24) and may, therefore, stimulate DNA repair synthesis, the further production of HSV-1-encoded polymerase, or both, thus allowing the reactivation of latent HSV-1 from combined ACV and IFN-a-treated cells to occur at the same rate and time of onset as cells treated with combined BVDU and IFN-ot. The inability of combined DHPG and IFN-a treatment to establish virus latency, and the prolonged latent interval exhibited by those cells treated with ACV and IFN-a compared with those treated with BVDU and IFN-a was surprising in light of recent studies in the Epstein-Barr virus system by Lin and co-workers (11, 12). In this system, BVDU treatment results in a more prolonged suppression of virus replication than does ACV and, although both DHPG and ACV inhibit the synthesis of Epstein-Barr virus DNA, treatment with DHPG maintains the reduced genome copy for prolonged periods of time after the termination of inhibitor treatment. It is not known whether ACV-5'-triphosphate irreversibly binds to Epstein-Barr virus-induced DNA polymerase, as it does to HSV-1-induced polymerase (8). It is possible that the interaction between virus-encoded DNA polymerase and the triphosphate form of these nucleoside analogs may affect the ability of the nucleoside analog to induce virus latency in vitro. ACKNOWLEDGMENTS We thank R. Guyton, L. Pfenninger, and N. Saunders for excellent technical assistance, M. Reese for editorial assistance, and E. K. Neidigh and D. Wynn for typing the manuscript. HSV-1 recombinant clones were kindly provided by Dr. R. W. Hyman, The Pennsylvania ANTIMICROB. AGENTS CHEMOTHER. State University College of Medicine, Hershey, Pa. We are grateful to Dr. G. Elion (Burroughs Wellcome Co., Research Triangle Park, N.C.) for helpful discussions and for providing ACV and DHPG. IFN-a was generously provided by Life Sciences Inc., St. Petersburg, Fla. This work was supported by Public Health Service grants CA1845, CA9124, CA2753, and CA34479 awarded by the National Cancer Institute. LITERATURE CITED 1. Cheng, Y.-C., S. P. Grill, G. E. Dutschman, K. B. Frank, J.-F. Chiou, K. F. Bastow, and K. Nakayama Effects of 9-(1,3-dihydroxy-2-propoxymethyl)guanine, a new antiherpesvirus compound, on synthesis of macromolecules in herpes simplex virus-infected cells. Antimicrob. 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