Replication of herpes simplex virus type 1 in differentiated human promyelocytic HL-60 cells

VIROLOGY 170,468-476

(1989)

Replication

of Herpes Simplex Virus Type 1 in Differentiated Human Promyelocytic HL-60 Cells

CHAMSAI PIENTONG,* KLAUS WEISSHART,t JOACHIM E. KijHN,* CHARLES W. KNOPF,t AND RijDlGER W. BRAUN**’ *Institute of Medical Virology, University of Heidelberg, Im Neuenheimer Feld 324, and tlnstitute of Virus Research, German Cancer Research Center, lm Neuenheimer Feld 280, D-6900 Heidelberg, Federal Republic of Germany Received July 28, 1988; accepted January 3, 1989 The capability of herpes simplex virus type 1 (HSV-l), strain Angelotti (ANG), to replicate in human promyelocytic HL-60 cells treated with 1,2-tetradecanoyl-phorbol-1%acetate (TPA) and dimethyl sulfoxide (DMSO) was examined. Virus titrations and infectious center assays revealed that HSV-1 ANG replicated in nontreated HL-60 cells and in HL60 cells treated with TPA. An abortive infection was observed in DMSO-stimulated HL-60 cells. Viral DNA synthesis was detected in nontreated and TPA-treated cells, but not in DMSO-treated cells. Analysis of HSV-1 transcripts revealed that albeit the differences in pretreatment, HL-60 cells synthesized viral immediate-early (ICP4) and early (tk and pol) RNAs, whereas a late viral transcript (gC) was almost exclusively detected in nontreated and TPA-treated HL60 cells. In line with these observations were the results obtained from studies on viral protein synthesis. The immediate-early protein ICP4 was found in all three cell types. Early (pal), delayed-early (gf3), as well as late proteins (VP 5, gC) were identified in nontreated and TPA-treated cells, but only in reduced amounts in DMSO-treated cells. These data suggest a translational block of HSV replication in DMSO-treated HL-60 cells at the level of early gene expression. 0 1989 Academic Press, Inc.

this so-called “intrinsic resistance” toward a herpesviral infection developed by PMNL. The human promyelocytic cell line HL-60, as introduced by Collins et a/. (1977), provides a system to study the influence of cell differentiation on the susceptibility of cells for an HSV infection. This cell line was described to differentiate to macrophage-like cells upon treatment with 1,2-tetradecanoyl-phorbol-13-acetate (TPA) (Hubermann and Callaham, 1979). In contrast, treatment with dimethyl sulfoxide (DMSO) results in differentiation to PMNL-like cells (Collins et a/., 1978). These properties of HL-60 cells allow the investigation of the influence of cell differentiation on the replication of HSV. The experiments presented here provide evidence indicating that an abortive infection in DMSO-differentiated HL-60 cells was most likely caused by a translational block at the level of early gene expression.

INTRODUCTION It has been shown in several studies in recent years that herpes simplex virus (HSV) under specific conditions may replicate in different subsets of native human mononuclear leukocytes (MNL) and, depending on the stage of differentiation, also in several human mononuclear cell lines (Linnavuori and Hovi, 1983). Whereas HSV replication could not be achieved in freshly isolated macrophages (Plaeger-Marshall and Smith, 1978; Plaeger-Marshall et a/., 1981; Daniels et al., 1978), there is ample evidence that this restriction is overcome by aging the macrophages in culture (Daniels et a/., 1978; Domke-Opitz et a/., 1986). In general, macrophages appear to be permissive for infection by HSV as examplified in the mouse system (Lopez and Duras, 1979; Brticher et a/., 1984) and in man (DomkeOpitz et a/., 1986). Whereas the majority of human stem cell lines appears to be susceptible for HSV (Braun et a/., 1984; Epstein and Bertoglio, 1981), attempts to grow the virus in human polymorphonuclear leukocytes (PMNL) failed so far (Plaeger-Marshall and Smith, 1978; Braun et al., 1984; Smith et al., 1986). Our goal was therefore to investigate the nature of

‘To whom correspondence addressed.

0042-6822/89

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Copyright Q 1989 by Academic Press, Inc. All rights of reproductaon in any form reserved.

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MATERIALS

AND METHODS

Growth of cells and virus African green monkey kidney monolayer cells (Rita clone, RC-37; ltaldiagnostic Products, Rome) were cultivated in basal medium Eagle (BME) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin, and lOq/o heat-inactivated fetal calf serum (Biochrom, Berlin).

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468

REPLICATION

Virus stocks of standard HSV-1 ANG were prepared after propagation on these cells at a multiplicity of infection (m.o.i.) of 0.01 PFU per cell. Virus titrations, plaque assays, and infectious center assays were performed on RC-37 cells as previously described (Teute et a/., 1983; Braun et al., 1984). The human promyelocytic cell line HL-60 (Collins et a/., 1977) a gift from J. Gargus (Emory University, Atlanta, GA), was grown in suspension cultures in RPMI 1640 medium (Biochrom, Berlin), supplemented as given above. Treatment

of HL-60 cells with TPA and DMSO

HL-60 cells were grown at a density of 5 X 1O5 to 1 x 1O6cells/ml. Cells were refed with fresh medium and 1,2-o-tetradecanoyl-phorbol-13-acetate (TPA; Sigma, Munich) or DMSO (Merck, Darmstadt) was added at final concentrations of 1.6 X 1O-’ M and 1.5%, respectively, for 2 days, except as otherwise indicated. Infection

469

OF HSV-1 IN HL-60 CELLS

of HL-60 cells

Nontreated HL-60 cells and HL-60 cells treated with TPA or DMSO were infected with stocks of HSV-1 at an m.o.i. of 3 PFU/cell. After an adsorption period of 60 min at 37” cells were washed once in RPMI 1640 medium and were resuspended in the same medium containing lOq/o FCS and the appropriate concentrations of TPA and DMSO. Aliquots of infected cell cultures were taken at different times after infection for further analysis in plaque assays or infectious center assays as previously described (Teute et al., 1983; Braun et a/., 1984). DNA probes For analysis of viral DNA and RNA, the following specific subfragments of HSV-1 DNA were used: (1) the BamHI Y fragment (1.75 kb) cloned in M 13, which contains the 5’ part of the ICP4 gene (Mackem and Roizman, 1982a,b) and was kindly provided by C. H. Schroder, German Cancer Research Center, Heidelberg; (2) the BarnHI Q fragment (3.5 kb), comprising the complete viral tk gene (Mackem and Roizman, 1982a,b); (3) the Sal1 T fragment (3.7 kb) spanning the gC gene (Holland et al., 1984) (the latter two fragments were cloned into pBR322); (4) the Xhol fragment (1.2 kb) from position 1945-3119 of the HSV-1 ANG DNA polymerase gene cloned in M 13 (Knopf, 1986); and (5) the BamHl U fragment of HSV-1 ANG (2.5 kb) cloned in pAT153 (Knopf et a/., 1986). DNA analysis DNA of infected cells was prepared as previously described (Kohler et a/., 1988). After cleavage with the

respective restriction enzyme, the DNA was analyzed with an appropriate nick-translated DNA probe (Rigby eta/., 1977) by Southern blot hybridizations under stringent conditions (Kohler et al., 1988). RNA analysis For the analysis of immediate-early (IE) and early (E) RNA, HSV-infected cells were maintained for 7 and 12 hr, respectively, in RPMI 1640 medium containing 100 pg/ml cycloheximide (Serva, Heidelberg) or 100 pg/ml phosphonoacetic acid (PAA, Serva, Heidelberg), respectively. For analysis of viral late (L) RNA, cells were incubated for 18 hr after infection. Total cellular RNA was prepared according to Chirgwin et al. (1979). Poly(A)+ RNA was isolated according to Davis et al. (1986). Northern blot hybridizations and Sl nuclease mapping were performed as described (Davis et al., 1986). Probes used for Northern blots were either nick-translated or labeled by the “Oligo labeling” method as described by Feinberg and Vogelstein (1983; 1984). For Sl mapping the BarnHI U fragment was 5’end-labeled at the BamHl site, position 185 of the HSV-1 ANG pol gene (Knopf et al., 1986). Sera and antibodies The monoclonal antibody (MoAb) OKMl , used as a marker for HL-60 cell differentiation (Talle et a/., 1983; Hickstein et a/., 1987) was purchased from Ot-tho Diagnostics (Neckargemtind). The MoAb against ICP4 was a gift from E. Zweig (National Cancer Institute, Frederick, MD), and the monospecific anti-gB serum RS-14 was kindly provided by P. Spear (University of Chicago, Chicago, IL). A human serum sample previously shown to react with all major viral late proteins (Kuhn et a/., 1987) was used to detect viral late gene products by immunoprecipitation. The anti (HSV-1 ANG DNA polymerase)serum EX3 has recently been described (Weisshart and Knopf, 1988). lmmunoprecipitation

and immunoblot

Equal volumes of lysates from HSV-infected cells labeled with 14C-labeled amino acids were immunoprecipitated with a human immune serum against HSV as previously described by Kuhn et a/. (1987). Following incubation of infected cell lysates with antibody and protein A-Sepharose, Sepharose beads were washed and boiled in sample buffer and the proteins analyzed by SDS-PAGE. Viral IE proteins were collected from nuclear fractions of infected cells 6 hr p.i. as described by Metzler and Wilcox (1985). For E protein analysis cells were incubated in RPMI 1640 containing 100 @g/ml PAA for

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PIENTONG ET AL

12 hr. For analysis of viral late proteins, cells were harvested at different times p.i. and were washed in Tris-buffered saline (TBS; pH 8.0) containing 1 mM phenylmethylsulfonylfluoride (PMSF; Sigma, Munich). Proteins were extracted in sample buffer and were separated on SDS-polyacrylamide gels as previously described (Thomas and Kornberg, 1975). lmmunoblots were performed by electrophoretic transfer of PAGEseparated proteins on nitrocellulose sheets as described (Kiihn et al., 1987; Braun and Reiser, 1986; Towbin et a/., 1979). Blots were reacted with an appropriate peroxidase-labeled second antibody and were developed with 4-chloro-1 -naphthol.

RESULTS Replication of HSV-1 in native and differentiated HL-60 cells The incubation of HL-60 cells with TPA and DMSO for 2 or more days resulted in altered cell morphology according to the progressing differentiation of cells to monocytes/macrophages (TPA) or PMNL-like cells (DMSO) as previously shown (Collins et al., 1977, 1978; Hubermann and Callaham, 1979). After 2 days of differentiation more than 70% of TPA-treated cells and more than 85% of DMSO-treated cells showed a reactivity with the MoAb OKMl, which is not reactive with native HL-60 cells and indicative for HL-60 cell differentiation (Talle eta/., 1983; Hickstein eta/., 1987). The percentage of OKMl reactive cells increased to 95% and more after prolonged times of cultivation (data not shown). Furthermore, more than 50% of DMSOtreated cells showed positive reactions in a yeast particle ingestion assay (Collins et al., 1978). To study the influence of cell differentiation on the replication of HSV-1 ANG, HL-60 cells were incubated with different doses of TPA or DMSO for various times and were then infected with HSV-1 ANG at a standard m.o.i. of 3 PFU/cell. As shown in Fig. 1, pretreatment of HL-60 cells for 1 day with 1.6 X 1O-’ M TPA was sufficient to accelerate virus replication in comparison to nontreated cells. In contrast, HL-60 cells pretreated with 1.59/o DMSO for 2-8 days showed decreased virus titers and only limited virus replication 4-7 days after infection. Whereas virus titers in nontreated and TPA-treated HL-60 cell cultures reached lo6 PFU/ml, titers in DMSO-treated cultures never exceeded lo3 PFU/ml. When virus production was monitored for longer periods of time (25 days), it was observed that virus titers in DMSO-treated cultures never exceeded lo3 PFU/ml and after 14 days p.i. dropped below detectable levels. This effect appeared to be specific for

HL-60 cells, since such an influence of DMSO on virus replication was not observed in RC-37 cells. A restriction of virus replication in DMSO-treated HL60 cells was also observed in infectious center assays (ICAs), which were performed over time from 0 to 8 days p.i. In these assays, cocultivation of HSV-l-infected HL-60 cells with RC-37 indicator cells was terminated after 4 days, only allowing such HL-60 cells to form an infectious center which produces virus within the first 24-48 hr after cocultivation. As shown in Fig. 2, HSV-1 -infected TPA-treated cells already yielded 1O-20% ICs 2 days p.i., whereas nontreated cells did not reach such a level before Day 3 p.i. In contrast, DMSO-treated cells did not produce more than 0.05% ICs. Furthermore, infectious centers in DMSO-treated cells were not found before Day 5 p.i., which correlates to the slight titer increase observed by virus titrations.

Viral DNA synthesis To assess the level at which the replication of HSV1 was restricted in DMSO-induced cells, we analyzed the synthesis of viral DNA during infection in all three cell types by Southern blots using the cloned HSV-1 BamHl Q fragment as a probe. As shown in Fig. 3, viral DNA synthesis in nontreated HL-60 cells could be detected in traces as early as 32 hr after infection. TPAinduced cells, however, showed an even more rapid replication of viral DNA. In contrast, even 96 hr after infection, only limited viral DNA synthesis was observed in DMSO-induced HL-60 cells. Similar results were obtained when other viral DNA fragments, such as EcoRl A or Sal1 T, were used as probes.

Viral RNA synthesis To further comparatively analyze virus replication in the differently treated cells, the synthesis of individual viral transcripts was determined. Immediate-early, E, and L RNA was prepared and then analyzed by Nor-thern blot hybridization and Sl mapping as described under Materials and Methods. As shown in Fig. 4A, an RNA of approximately 4.2 kb, corresponding in size to the ICP4 transcript (Clements et a/., 1979; Watson et al., 1979) was detected in similar amounts in the virus-infected cells. Figures 4B and 5A show the results of a Northern blot anlaysis of E RNAs with the tk and pol gene specific DNA probes. In Fig. 4B a 1.4-kb RNA of the size expected for the tk transcript (Sharp et al., 1983) was found to be equally well expressed in each of the differently treated virusinfected cells. Also, the 4.5-kb pol RNA (Holland et al., 1984) was similarly expressed in all three cell types

REPLICATION 7

OF HSV-1 IN HL-60 CELLS

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FIG. 1. Replication of HSV-1 ANG in native, TPA-, and DMSO-treated HL-60 cells. Native HL-60 cells and HL-60 ceils treated with TPA (a) or DMSO (b) for 1, 2, 3, 4 and 2, 4, 6, 8 days, respectively, were infected with HSV-1 ANG at an m.o.i. of 3 PFU/cell. Virus titers were determined at various times p.i. by plaque assay. Virus in cell-free medium was used as a control for thermal inactivation.

(Fig. 5A). These data were further confirmed by Sl mapping (Fig. 5B). When viral late RNA was analyzed with a gC-specific DNA probe, a 2.7-kb RNA of the size previously reported for the gC RNA (Wagner, 1982; Frink et al., 1983) was detected. The gC RNA signal was considerably stronger in TPA-treated cells than that in nontreated HL-60 cells, whereas only reduced amounts of

this 2.7-kb RNA could be detected in DMSO-induced cells (Fig. 4C). Protein analysis From the transcript analysis we concluded that the viral late transcription was impaired in DMSO-treated HL-60 cells. To directly demonstrate the effects of this impairment on the synthesis of viral proteins, we stud-

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FIG. 2. Percentage of native, TPA-, or DMSO-treated HL-60 cells replicating HSV-1 ANG at different times p.i. Native HL-60 cells and HL-60 cells treated with TPA (a) or DMSO (b) for 1, 2, 3,4 and 2,4,6, 8 days, respectively, were infected with HSV-1 ANG at an m.o.i. of 3 PFU/cell. At time points indicated, samples of HSV infected HL-60 cells were harvested and tested in an infectious center assay by cocultivation with RC-37 indicator cells. The assay was terminated after 4 days and the number of plaques in cell cultures was determined.

REPLICATION

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FIG. 3. Southern blot hybridization of DNAfrom HSV-1 -infected HL60 cells with the BamHl Q fragment of HSV-1 ANG. At indicated times p.i. 10 pg of purified, cellular DNA from mock-infected and HSV-l-infected native, TPA-, and DMSO-treated HL-60 cells was cleaved with the restriction enzyme BamHl and was subjected to a 1% agarose slab gel. After Southern transfer, the DNA was hybridized with a “P-labeled HSV-DNA probe (HSV-1 BarnHI fragment Q). (1) Native HL-60 cells; (2)TPA-treated HL-60 cells; (3) DMSO-treated HL-60 cells.

ied the expression of selected viral proteins of the IE, E, and L class by immunoprecipitation and immunoblot analysis. As shown in Fig. 6, panel A, no differences could be detected, when the expression of the IE protein ICP4 was assessed in immunoblots using an ICP4specific MoAb. It should be noted that for this blot experiment, cells were not especially conditioned to enrich the IE proteins. This might explain the rather weak signal observed. 12

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FIG. 4. Northern blot analysis of HSV-1 from native, TPA-, and DMSO-treated HL-60 cells. Cytoplasmic RNA was isolated from cells under IE (A), E (B), and late (C) conditions and was hybridized in Northern blots to the viral DNA fragments BamHl Y (A), BarnHI Q (B), and SalI T(C). HSV-infected native HL-60 cells (1). TPA-treated cells (2) DMSO-treated cells (3). and mock-infected cells (4) were comparatively analyzed.

FIG. 5. Northern blot analysis (A) and Sl nuclease mapping (B) of the HSV-1 pol transcript. (A) Poly(A)+ RNA (2 ag) of DMSO-treated (1) TPA-treated (2) nontreated HSV-l-infected (3) and mock-infected (4) HL-60 cells was subjected to 1% agarose gel electrophoresis, blotted to nitrocellulose filters, and hybridized to the 1.2-kbXhol pol DNA fragment (8 X 1O* cpmlpg). (B) Total RNA (30 pg) of the cells as given above was hybridized to 5 X 1O4 cpm 5’ end-labeled BarnHI U fragment cleaved with Xhol (1.3 X 1O5 cpm/pg) and digested with nuclease Sl Protected DNA fragments were analyzed on 5% sequencing gels. pBR322 Hinfl DNA fragments were used as internal size markers as given in nucleotides. The bracket indicates the major start site of the pol transcript as previously shown (Yager and Coen, 1988).

With the rabbit anti (HSV-1 DNA polymerase)-serum EX3 directed against the C-terminus of the viral enzyme as recently described (Weisshart and Knopf, 1988) expression of the pol polypeptide of 140 kDa was detected in extracts of nontreated and TPA-treated cells, but not in DMSO-treated HL-60 cells (Fig. 6, panel B). The additional band of 52 kDa is most likely due to proteolytic cleavage and was routinely observed in virusinfected cells. Similarly, expression of the delayed-early viral protein gB was not detectable in DMSO-treated HL-60 cells by the immunoblot analysis with the monospecific anti-gB serum RS-14 (Fig. 6, panel C). In nontreated and TPA-treated HL-60 cells gB expression was observed from 32 hr p.i. The gB signal obtained, however, appeared to be more intense in TPA-treated cells. To demonstrate whether synthesis of late viral proteins took place at all in the DMSO-treated cells, additional immunoprecipitation experiments were performed. This technique by the selective enrichment of antibody reactive proteins achieves a greater sensitivity than the immunoblot. As shown in Fig. 7 the viral

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resulted in an acceleration of HSV-1 replication, whereas pretreatment of cells with DMSO resulted in an abortive infection. This was shown by an early increase of virus titers and infectious centers in TPAtreated cells and by an almost complete lack of virus replication in DMSO-treated cells. The slight titer increases observed in DMSO-treated cultures from the fourth day p.i. were far below the titers obtained in nontreated cultures and never exceeded 1O3PFU/ml. This low level of virus production was observed until 14 days p.i., whereafter titers dropped below detectable levels. We feel that this low level virus production may reflect the presence of a minor cell subset that does not respond to the DMSO treatment. This conclusion

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FIG. 6. lmmunoblot analysis of HSV-1 ANG-specific proteins in native, TPA-, and DMSO-treated HL-60 cells. Viral proteins were analyzed by immunoblots under IE (panel A), E (panel B), and Lconditions (panel C). Panel A, mock-infected (lane 1) and HEW-infected native (lane 2), TPA-treated (lane 3), and DMSO-treated (lane 4) HL-60 cells as probed with a MoAb against ICP4. Panel B, mock-infected (1) and HSV-infected (2) HL-60 cells. (A) native cells; (B) TPA-treated cells; (C) DMSO-treated cells; (R) RC-37 HSV-infected cell control. The blot was probed with a monospecific antibody from rabbit, directed against the HSV-1 pol protein. Panel C, mock-infected and HSV infected native (l), TPA-treated (2), and DMSO-treated (3) HL-60 cells probed with a monospecific anti-g8 antibody and analyzed at different times after infection as given in the upper margin.

proteins VP5, gB, and gC were successfully immunoprecipitated from each of the analyzed cell extracts by a human immune serum reactive with all major late viral proteins. Again, however, the signal obtained from the DMSO-treated HSV-infected HL-60 cells was significantly lower, indicating that both delayed-early and late protein synthesis were inhibited in these cells. DISCUSSION

Using the human promyelocytic cell line HL-60 we could show that differentiation of these cells with TPA

FIG. 7. lmmunoprecipitation of HSV-1 ANG late proteins from HSV1 infected native, TPA-, and DMSO-treated HL-60 cells. Native HL60 cells (1) and HL-60 cells pretreated with TPA (2) or DMSO (3) for 2 days were infected with HSV-1 ANG at an m.o.i. of 10 PFWcell and were pulse-labeled with r4C-labeled amino acids. After immunoprecipitation with a human immune serum against HSV-1, the proteins were subjected to a 10% SDS polyactylamide gel. Designation of viral proteins is given in the right margin.

REPLICATION

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OF HSV-1 IN HL-60 CELLS

is supported by the finding that not more than 85-90% of DMSO-treated cells were initially reactive with the MoAb OKM 1. Synthesis and expression of ICP4 RNA, which is a typical IE transcript, were found in similar amounts in nontreated, TPA-, and DMSO-treated HL-60 cells. Also, synthesis of two early transcripts, tk and pol, was observed to a similar extent in all three cell sources. As pol RNA in infected cells is a minor transcript compared to tk, we have performed additional Sl nuclease studies on pol RNA. The results of these studies were consistent with the findings of the Northern blot experiments (Figs. 4 and 5). Taken together, these results indicate a comparable level of E RNA synthesis in all three cell types. lmmunoblot experiments, however, showed expression of the pol protein in nontreated and TPA-treated cells, but not in DMSO-treated cells. The absence of the pol protein in the latter is likely to explain the impaired viral DNA synthesis in DMSO-treated cells as demonstrated in the Southern blot analysis (Fig. 3). When viral late transcripts were analyzed, the gC transcript was found to be most abundant in-TPAtreated cells. This again might reflect a more rapid replication of HSV-1 in TPA-treated cells in agreement with the results from virus titrations and ICAs (Figs. 1 and 2). In nontreated cells the gC signal was less intense and was greatly diminished in DMSO-treated cells. Weinheimer and McKnight (1987) have presented evidence that the transcription of E and L genes is coordinately regulated. Thus the reduced levels of gC transcription in DMSO-treated cell cultures rather correspond to a reduced pool of template DNA than to a direct restriction of gC RNA synthesis. In immunoprecipitation experiments a difference in late gene expression between TPA-treated and nontreated HL-60 cells was not observed. This might be explained by saturation of the antigen-binding capacity of antibodies in this assay. In addition, immunoprecipitation is likely to selectively enrich viral proteins from a small minority of producer cells, which might explain the finding of traces of VP5, gC, and gB in this assay under DMSO conditions. In immunoblot experiments with a monospecific gB antibody the kinetics of gB expression in TPA-treated and nontreated cells were comparable. The gB signal found in TPA-treated cells, however, appeared to be more intense compared to nontreated cells. This again agrees well with the finding of a more rapid virus replication in TPA-treated cells. Taken together, the results described here indicate an acceleration of virus replication by TPA treatment of HL-60 cells and an abortive infection in DMSOtreated cells. This abortive infection apparently is restricted at the translational level of E gene expression.

Interestingly the importance of a post-transcriptional control for pol expression has recently been documented by Yager and Coen (1988). It remains to be investigated whether such a proposed mechanism plays a significant role for the so-called “intrinsic resistance” developed by the HL-60 cells treated with DMSO. The HL-60 cell system, however, represents an ideal tool to further explore this restrictive cellular phenotype.

ACKNOWLEDGMENTS The authors express their gratitude to P. Spear, University of Chicago, for providing the monospecific anti-g8 serum and to M. Zweig, National Cancer Institute. Frederick, for the monoclonal antibody against ICP4. Furthermore, we are greatly obliged to C. H. Schrijder, German Cancer Research Center, Heidelberg, for providing the HSV1 ANG BarnHI Y DNA fragment. Ch. Pientong is a recipient of a stiAkademischer Austauschdienst” pend from the “Deutscher (DAAD).

REFERENCES BRAUN, R. W., and REISER,H. C. (1986). Replication of human cytomegalovirus in human peripheral blood T cells. J. Viral. 60, 29-36. BRAUN, R. W., TEUTE, H. K., KIRCHER,H., and MUNK, K. (1984). Replication of herpes simplex virus in human T lymphocytes; Characterization of the viral target cell. J. Immunol. 132, 914-919. BRUCHER,J., DOMKE, I., SCWR~DER,C. H., and KIRCHNER, H. (1984). Experimental infection of inbred mice with HSV. VI. Effect of interferon on in vitro virus replication in macrophages. Arch. Viral. 62, 83-93. CHIRGWIN,1. M., PRZYBYU, A. E., MACDONALD, R. J., and RUT~ER,W. J. (1979). Isolation of biological active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299. CLEMENTS, J. B., MC~.AUCHLAN,J., and MCGEOCH, D. J. (1979). Orientation of herpes simplex virus type 1 immediate early mRNA’s. Nucleic Acids Res. 7, 77-91, COLLINS, S. J., GALLO, R. C., and GALLAGHER,R. E. (1977). Continuous growth and differentiation of human myeloid leukemia cells in suspension culture. Nature (London) 270,347-349. COLLINS, S. J., RUSCETX, F. W., GALLAGHER, R. E., and GALLO, R. C. (1978). Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc. Nat/. Acad. Sci. USA 75, 2458-2462. DANIELS, C. A., KLEINERMAN,E. S., and SNYDERMAN, R. (1978). Abortive and productive infections of human mononuclear phagocytes by type 1 herpes simplex virus. Amer. J. Parhol. 91, 1 19-l 36. DAVIS, L. G., DIBNER, M. P., and BATTEY,J. F. (1986). “Basic Methods in Molecular Biology.” Elsevier, New York/Amsterdam/London. DOMKE-OPITZ, I., P. STRAUB, and H. KIRCHNER.(1986). Effect of interferon on replication of herpes simplex virus type 1 and 2 in human macrophages. J. Viral. 60, 37-42. EPSTEIN,A. L., and BERTOGLIO,J. H. (1981). Differential replication of herpes simplex virus-l in human myeloid cell lines representing various steps of differentiation. Hum. Lymphocyte Differ. 1, 217222. FEINBERG,A. P., and VOGELSTEIN,B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6- 13. FEINBERG,A. P., and VOGELSTEIN.B. (1984). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137, 266-267.

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