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Persistence of herpes simplex virus type-2 genome in a human leukemic cell line
Biochimica et Biophysica Acta, 740 (1983) 271-281 Elsevier
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BBA 91231
PERSISTENCE OF HERPES SIMPLEX VIRUS TYPE-2 GENOME IN A HUMAN LEUKEMIC CELL LINE NILAMBAR BISWAL, UNSIL KO, STEVEN AKMAN, DOUGLAS D. ROSS, AVROM POLLAK and EUGENE CIMINO
University of Maryland Cancer Center, Bressler Research Laboratories, 655 W. Baltimore Street, Baltimore, MD 21201 (U.S.A.) (Received March 16th, 1983)
Key words: Virus-cell interaction; Virus infection," DNA synthesis," DNA polymerase; Herpes simplex virus; (Human leukemic cell)
To study the nature of virus-cell interaction in persistently infected cells we have examined production of infectious virus, synthesis of viral DNA and DNA polymerase in a human leukemic cell line K562. It was found that only one of three 1(562 cell lines was permissive for limited growth of HSV-2 and infectious virus was released in a cyclical fashion. Intranuclear inclusions with electron-dense fibrils and particles resembling viral structures were observed in the virus-infected but not control K562 cells. Viral DNA synthesis could not be detected by centrifugation in CsCI density gradients; but was readily identified by Southern blot hybridization of virus-infected intraeellular DNA with purified viral DNA. Viral DNA polymerase was synthesized by infected cells during active infectious virus production. In one of the two K562 cell lines that did not produce infectious virus, a few DNA fragments from infected cells were found to hybridize with purified viral DNA. These results suggest that variable lengths of HSV-2 genome can be harbored and propagated by different human leukemic K562 cells.
Introduction
During recent years a variety of cultured cell lines of hematopoietic origin have been shown to support persistent growth of HSV [1-8]. Of particular importance are cultured human lymphoid and myeloid cell lines where HSV can continue to replicate in a dynamic state for prolonged periods after initial infection, thus providing in vitro model systems to study the mechanism of HSV persistence in hematopoietic cells. Rianldo et al. [8] have reported that human T, B and myeloid cell lines persistently infected with HSV-1 grow like non-infected cultured cells but with oscillating
Abbreviations: HSV, herpes simplex virus; EACA, c-aminocaprioic acid; Endo R, restriction endonuclease. 016%4781/83/$03.00 © 1983 Elsevier Science Publishers B.V.
cycles of crisis (increased virus production with variable degree of cytopathic effect). Katz et al. [6] have shown that null and B lymphoid lines arrested at pre B or at stages of I and II of the B differentiation maturation pathway also support persistent HSV growth. Menezes and Bourkas [7] have shown that depending upon the level of cell differentiation, various lymphoblastoid cell lines were permissive to HSV infection. These virus-infected cells developed HSV-specific Fc receptors and produced viral specific antigens and infectious virus. Recently it has been possible to regulate the expression of HSV gene functions in persistently infected cells under defined conditions. For example, treatment of the persistently infected human T lymphoblastoid cell line, CEM, with concanavalin A [9], HSV-1 antiserum or high temperature [10]
272 suppressed HSV-1 production and expression of viral antigens. However, treatment of these same cells with phytohemagglutinin [9] or lower temperature [10] reactivated infectious virus production. Viral DNA was found to be highly methylated in the concanavalin A-treated nonproducer cells, but not in the productively infected or phytohemagglutinin-treated cells [11]. These results have suggested, but not proven, that methylation of viral D N A may be a prerequisite for the maintenance of the latent state of HSV-1. Related to these studies are observations from other laboratories where various other types of cultured mammalian cells such as neuronal cells [12,13], mouse L cells [14], chinese hamster ovary cells [15], human embryonic lung (HEL) cells [16-19] and human cervical epithelial cells [20] have been used to establish HSV persistence in vitro. Replication of HSV-2 or HSV-1 can be repressed by treatment of these virus-infected cells with various agents such as high incubation temperatures and cytosine arabinoside [16-18], adenine arabinoside [20] or with (E)-5-(2bromovinyl)-2'-deoxyuridine, in combination with human leukocyte interferon-a. [19]. HSV genome can be reactivated by superinfecting the infected cells with human cytomegalovirus [16,17] or by reducing the incubation temperature [19]. Adler et al. [13] have shown that rat neuronal cell line B103 can be conditioned to harbor HSV-I genome for long periods of time and to express viral specific proteins such as D N A polymerase, thymidine kinase, and other proteins in the absence of active viral DNA synthesis. We have initiated studies to analyze the mechanism by which HSV-2 persists and the consequence of that persistence in a human leukemic cell line (K562). This cell line was originally derived from the pleural effusion of a patient with chronic myeloid leukemia in blast crisis [21]. K562 cells can be induced to differentiate to express phenotypic markers for erythroid lineage [22] and display spontaneous and induced globin synthesis [23]. In this communication we describe the successful establishment of persistent infection of HSV-2 in a particular K562 cell line, the physical state of the virus particles and viral DNA and the expression of viral D N A polymerase within these cells.
Materials and Methods Virus. Herpes simplex virus type 2, strain 333 (HSV-2) was used throughout this study. The source, propagation and assay protocols for this virus have been published before [24,25]. Cells. Suspension cultures of human leukemic K562 cells were obtained from three different sources: from Drs. R. Hoffman of New Haven, CT, B.B. Lozzio of Knoxville, TN and D. Groth of Atlanta, GA and were designated by us as K562 (Clegg), K562 (Loz) and K562 (Groth) respectively. These cell lines in different labs have been found to evolve tangible phenotypic and genotypic changes [26] and are well characterized at this Cancer Center by Ross et al. [26] for their heterogeneity, karyotyping, sensitivity to growth inhibitors and hemin. Thus, for a comparative study these individual cultures were cloned in 0.3% agar and the cloned cultures were subsequently maintained in RPMI 1640 medium (Grand Island Biologicals Co.) supplemented with heat-inactivated 10% fetal calf serum, 2 mM glutamine, 0.225% sodium bicarbonate, 100 units/ml of penicillin and 100/~g/ml of streptomycin at 37C under 5% CO 2 atmosphere. The suspension cultures were passed in sterile bottles every 4th day at an initial concentration of (2-3). 105 cells/ml. Cultures of fibroblastic HEp-2 cells and Vero cells were maintained and propagated in minimum essential medium as described before [24,27]. H S V-2 growth curves in K562 and HEp-2 cells.
Suspension cultures of cloned K562 cells were concentrated to approx. 2.5. 10 7 cells/ml by centrifugation and infected with HSV-2 at a multiplicity of infection of about 10. After adsorption for 90 min at 37C, the cells were washed twice with phosphate-buffered saline to remove the unadsorbed virus, 100 ml of growth medium was added and the infection continued at 37°C. At indicated times, duplicate samples of 10 ml each were sonicated for 1 min in a Branson sonic oscillator at 10 kHz and centrifuged at 1500 × g for 10 min to clarify the supernatant. This supernatant which contained both the intra- as well as extracellular virus particles was assayed for infectious HSV-2 in monolayers of Vero cells in 60 mm plastic petri plates [24,25]. Growth of HSV-2 in permissive HEp-2 cell monolayers was measured
273 by a technique described previously [23,24]. Kinetics of cell growth. Virus-infected or control K562 cells were incubated in sterile culture flasks at an initial concentration of 2.105 cells/ml. Cell counts were performed daily in a Coulter counter (Coulter Electronics) and the number of viable cells was determined by the trypan blue exclusion test. Electron microscopy. For ultrastructural studies, infected K562 cells at the 4th passage level were washed and fixed for 2 h at room temperature in a 4% formaldehyde/1% gluteraldehyde phosphatebuffered mixture [28]. After postfixation in osmium tetroxide, the samples were processed and thin-sectioned for transmission electron microscopy (transmission EM) and examined in a JEOL 100 cx electron microscope.
Centrifugation of intracellular DNA in CsCI density gradients. HSV-2 infected or mock-infected K562 cells were labeled with [3H]dT (5 /tCi/ml) for 24 h. As a positive control, monolayers of HEp-2 cells were infected with HSV-2 at a multiplicity of infection of 10 plaque forming units/ml and incubated for 16 h in the presence of [3H]dT (5/~Ci/ml). The cells were harvested, washed twice in Tris-sodium chloride buffer (0.01 M Tris-HC1 (pH 8.0) 0.1 M NaC1 and 0.005 M EDTA), lysed with sodium dodecyl sulfate and treated with protease K as described before [29,30]. The median density of the solution was adjusted to 1.705 g / c m 3 with CsC1 crystals and the mixture was centrifuged to equilibrium for 3 days in a Spinco SW 50.1 rotor. After measuring the refractive indices of some representative fractions from the gradient, trichloroacetic acid = precipitable radioactivity of each fraction was determined. Labeling of viral DNA with 32p by 'nick translation" The 56S HSV-2 DNA was isolated from purified virus by a method described previously [24,31]. This DNA was labeled in vitro with 32p by a slight modification of 'nick translation' method described by Rigby et al. [32]. About 1 /~g of HSV-2 DNA was incubated at 15°C in a 0.1 ml reaction mixture containing 100 /xCi each of [a32p]dATP, d G T P , d C T P and T T P (spec. radioact. = 3000 Ci/mmol, Amersham Corp.), 50 mM Tris-HC1 (pH 7.8), 5 mM MgC12, 10 mM 2-mercaptoethanol, 0.5 ng DNAase and 2 units of E. coli DNA polymerase I. After 60 min, the
reaction was terminated with 10 mM EDTA and 0.5% SDS, diluted to 0.4 ml with Tris-sodium chloride buffer and deproteinized with an equal volume of phenol/chloroform (1 : 1) mixture. The entire mixture was shaken for 10 rain, centrifuged at 12000 × g for 5 min and the labeled DNA in the aqueous layer was separated from the unincorporated nucleotides by passing through a column of Sephadex G-50. In this way the viral DNA could be labeled to (2-4). 108 cpm//~g and was used for hybridization purposes later.
"Southern' blot transfer of restricted DNA fragments. DNA from HSV-2 infected K562 cells were isolated by deproteinization with p h e n o l / chloroform and isoamyl alcohol as described before [24,29]. The purified DNAs were digested overnight with EcoRI and BamHI restriction enzymes according to the specifications of the supplier (Bethesda Research Laboratories). The cleaved DNA fragments were separated by electrophoresis for 16 to 20 h at 2 V / c m in 0.8% agarose slab gels, stained with ethidium bromide and photographed with a Polaroid camera using type 55 positive/negative film [33]. The DNA fragments in the agarose gels were then denatured for 30 rain with 0.5 M N a O H and 1.5 M NaC1, neutralized to p H 7.2 with 0.5 M Tris-HC1 (pH 7.2) and 3.0 M NaCI. The denatured DNA fragments were then transferred to sheets of nitrocellulose membranes as described by Southern [34]. After drying, the filters were baked under vacuum for 2 h at 80°C and stored at 4°C under vacuum until hybridization. Hybridization of viral DNA to cellular DNA on filters. Purified viral DNA labeled with 32p by nick translation described above was hybridized to the denatured cellular DNA fragments fixed on nitrocellulose membranes as follows: radioactive viral DNA was sonicated, denatured at 117°C in sealed ampoules and quickly chilled in an ice bath. Nitrocellulose membranes containing the cellular D N A fragments were incubated in Denhardts' medium [35] for 4 to 6 h to reduce nonspecific binding of viral DNA to the charged membranes. These membranes were then exposed to denatured radioactive viral DNA in the presence of 6-SSC (SSC = 0.15 M NaC1 and 0.015 M sodium citrate) containing 0.02% each of Ficoll, bovine serum albumin and polyvinylpyrolidone. D N A - D N A
274
hybridization was allowed for 60 h at 62°C in a rotatory drum. The membranes were then washed 3-4-times with large volumes of 0.005 M Tris (pH 9.2) until the radioactivity of the washed fluid was lower than 100 cpm per 0.2 ml of wash. The membranes were dried, exposed to Kodak Blue Brand X-ray films for an appropriate time (ranging from few hours to 4 days) and the autoradiograms were developed as described previously [33].
Preparation of crude cell extracts for enzyme assay. Approx. 2 × 10 7 HSV-2 infected or mock infected K562 (Clegg) cells at passage levels of 1, 4, 6 and 12 (low levels of virus production, except passage 4, see Table I) were washed twice in phosphate-buffered saline and frozen as cell pellet at - 9 0 ° C until all the samples were collected. At the time of assay the frozen samples were thawed, suspended in 1 ml of Tris-buffer (0.05 M Tris-HC1 (pH 7.8), 5 mM dithiothreitol and 1 mM ~-aminocaproic acid). The cell suspension was sonicated in an ice bath for 1 min at 9 K C / s and an equal volume of cold solution of 4 M KC1, 10 mM EDTA, 5 0 0 / l g / m l of bovine serum albumin and 2 mM c-aminocaproic acid was slowly added. The resulting precipitate was removed by centrifugation at 10000 rev./min and supernatant was dialyzed extensively against buffer A (0.05 M TrisHCI (pH 7.8), 1 mM EACA, 2 mM dithiothreitol, 0.2% NP-40 and 20% glycerol). The dialysate was clarified by centrifugation at 15 000 rev./min, for 30 rain and the supernatant as crude cell extract was assayed for D N A polymerase. Assay for HSV-2-induced DNA polymerase. HSV-2-induced D N A polymerase in the crude cell extract was assayed by a slight modification of method described by Weissbach et al. [36]. The reaction mixture (total volume of 100 /~1) contained 0.01 M Tris-HC1 (pH 7.8), 0.15 M (NH4)SO4, 1 mM dithiothreitol, 2.5 mM MgC12, 5 /xg of activated calf thymus DNA, 20 ~tg bovine serum albumin, 0.05 m M [ 3 H ] T T P (600 cpm/pmol), 0.2 mM each of dCTP, dATP and d G T P and 25 /xl of cell extract as the source of HSV-2 D N A polymerase. Duplicate samples at indicated time points were withdrawn and the reaction was terminated by adding an equal volume of cold 10% trichloroacetic acid containing 0.1 M sodium pyrophosphate. The precipitate was filtered on Whatman G F / C filters, washed sequentially
with cold 5% trichloroacetic adid and cold distilled water and the radioactivity on dried filters was measured in a liquid scintillation spectrometer. Results
Restriction of HSV-2 growth in K562 cells To determine if cultures of various K562 cell lines supported the replication of HSV-2, cloned cultures of three K562 cell lines (Clegg, Loz and Groth) were infected with HSV-2 at a multiplicity of infection of 10 plaque forming units/cell. After adsorption for 90 min at 37C, the cells were washed and duplicate samples were assayed for infectious HSV-2 as described in Materials and Methods section. Results presented in Fig. 1 show that none of the three K562 cell lines supported production of new infectious HSV-2 progenies over a period of 48 h after initiation of infection. The virus particles adsorbed to all the three cell lines, however,
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Fig. 1. Growth of HSV-2 in leukemic K562 cells and HEp-2 cells. Various cultures of K562 Clegg, Loz and Groth cells were concentrated and infected with HSV-2 at a multiplicity of infection of approx. 10 (see Materials and Methods). After 90 rain of adsorption at 37C, the cells were washed twice and incubation was continued. At indicated times, duplicate samples were assayed for infectious virus as described in Materials and Methods. Growth of HSV-2 in HEp-2 cells (O e): K56(Clegg) cells (© .... ©); K562(Groth) cells (zx zx): K562(Loz) cells (n O).
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since infectious virus, whose infectivity titer gradually decreased, could be recovered up to 48 h after infection. The supernatant fluid alone from such cultures did not demonstrate infectious virus particles under similar conditions of infection. In contrast, monolayers of HEp-2 cells supported HSV-2 growth and new infectious progeny virions were produced with a typical growth curve. Thus, these results demonstrate that all three K562 cell lines tested were nonpermissive for HSV-2 (333) growth although the virus adsorbed to the cells. However, Rinaldo et al. [8] have demonstrated that K562 cells supported a productive replication of HSV-1, although the titer of progeny virions was at a lower level. To test the possibility that prolonged passage of the K562 cell lines will produce new infectious HSV-2 at a later time, we continued passing the infected cells and tested them for virus production as well as viable cell growth as described below.
Cyclical appearance of HSV-2 in K562 (Clegg) cells The infected cells continued to grow like uninfected control cells up to the 4th passage (16 days after infection) with a doubling time of approx. 24 h. The number of viable cells, as determined by the Trypan blue exclusion assay, ranged from 90-95% of the total. During this time of continued growth, the cells usually attained a density of 3.106 cells/ml in 4 days from an initial concentration of (2-3). 105 cells/ml. Thus, for routine propagation and to maintain a cell viability of greater than 90%, all the cells (infected and noninfected) were suspended in fresh growth medium every fourth day at a concentration of about 2.5 • 105 cells/ml. After the first passage no infectious virus could be detected in the cell homogenates up to the 4th serial passage in 16 days (Table I). At the 4th passage level the infected K562 (Clegg) cells also ceased to replicate for about 8 days and the viability of the cells was reduced to 70%. During this 8-day period of quiescence, feeding the cells with fresh growth medium did not induce replication of infected cells and no infectious virus could be detected. The cells then spontaneously recovered from the 8-day quiescence and started to grow again with the same efficiency as the control cells. The number of viable cells increased to greater than 90% and a low level of virus capable
TABLE I I N F E C T I O N O F K562 (CLEGG) CELLS BY HSV-2 Every fourth day the cells were diluted to about 2.105 cells/ml with fresh medium and the incubation continued. Virus titer shown is from cells just before the 1st passage on 4th day. PFU, plaque-forming units. Days after infection 0
Passage number -
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Virus titer (PFU/ml)
2.5.105
5.7.105
1
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1.2.105
2 4a 12 16 20 24
1.1 • 106 1.2.106 2.5- 106 2.2" 106 1.7" 105 1.6- 105
9.0.104 4.104 0 0 0 3' 103 (small plaques) 3- 103 (small plaques) 1- 103 (small plaques) 0 0 0 7.102 (pin-point plaques) 0
1 3 4 4 4
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2.5.106
32
6
3 • 106
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7 8 10 12
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of producing small plaques in Vero cells were observed (Table I). Upon continued passage the cells again stopped producing any detectable levels of virions up to the l l t h passage. At the 12th passage, however, the cells produced virions capable of producing only extremely small plaques (readily visible under microscope). This observation of restricted but cyclical virus production was limited to K562 (Clegg) cells. Other two cell lines K562 (Loz) and K562 (Groth) failed to produce any virus particles after the 1st passage. For this reason subsequent experiments were conducted only with the K562 (Clegg) cells. Since a limited number of infectious virions continued to be produced by the infected K562 (Clegg) cells in a cyclical fashion, we designed experiments to examine the intracellular DNA of K562 (Clegg) cells for the presence of viral DNA.
Electron microscopic examination of infected K562 (Clegg) cells Approx. 50% of cells examined from infected
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cultures showed cytopathologic effects when compared to cells from uninfected cultures. The most conspicuous morphological variations were nuclear fragmentation and cytoplasmic vaculoization (Fig. 2a). Associated with nuclear fragmentation was a high degree of heterochromatin condensation and margination. In a small percentage (2-4%) of infected cells we observed intranuclear inclusions measuring 0.4-1.5 ~m in diameter. These inclusions were of two types. In one type the inclusion consisted of an undulating mass of 15-24 nm thick fibrils intimately associated with the nucleolus (Fig. 2A and B), whereas the more frequently observed second type consisted of parallel arrays of electron-dense material 36-60 nm in diameter. In cross-section (Fig. 2C), the electron-dense material can be seen to be completely surrounded by electron lucent particles which bear a close morphological similarity to viral capsomeres. Thus these results demonstrate ultrastructural modifications in the infected cells especially when such cells are in their quiescent state at the 4th passage level.
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DNA analysis by CsCI buoyant density measurement. K562 cells infected with HSV-2 at the sec-
Fig. 3. CsC1 buoyant densities of DNA samples from K562 (Clegg) cells infected with HSV-2. Persistently infected K562 (Clegg) cells or productively infected HEp-2 cells were labeled with [3H]dT and the DNA from these cells was centrifuged to equilibrium in CsCI as described in Materials and Methods. At the end of centrifugation, fractions were collected from the top by an Isco fraction collector and the trichloroacetic acid-precipitable radioactivity of each fraction was determined (see Materials and Methods). Radioactivity in the DNA from (A) mock infected K562(Clegg) cells, (B) productively infected HEp-2 cells, (C) persistently infected K562 (Clegg) cells at 4th passage or (D) at 6th passage level.
ond, 4th and 6th passage levels were chosen for this experiment. Infected cells at the 4th passage level did not show any infectious virus production while at the 6th passage the cells produced low levels of infectious virions. The cells were labeled with [3H]dT, intraceUular DNA was isolated and centrifuged to equilibrium in CsC1 density gradients as described in Materials and Methods. Under such experimental conditions, it is possible to monitor the synthesis of both cellular and viral D N A since the buoyant density of HSV-2 DNA P
CsC1 = 1.727 g / c m 3) is much greater and readily distinguishable from that of the cellular DNA p CsC1 = 1.699 g / c m 3) [24,27,29]. Results presented in Fig. 3 show that uninfected K562 (Clegg) cells had only one peak of radioactivity (Fig. 3A) which banded at a density of about 1.700 g / c m 3. Productively infected HEp-2 cells however, showed two peaks of radioactivity banding at the expected positions of the viral DNA and cellular DNA (Fig.
Analysis of intracellular DNA of HSV-2-infected K562 (Clegg) cells To examine the possibility that HSV-2 DNA may have become resident in the infected K562 (Clegg) cells, two experimental approaches were taken.
Fig. 2. Electron micrographs of K562 cells infected with HSV-2. (A) A K562 cell from HSV-2-infected culture at the 4th passage level. Note nuclear fragmentation, marginated heterochromatin in fragment on left, nucleolus associated inclusion in larger fragment, and vacuolization of cytoplasm. (B) Higher magnification of inclusion seen in A. (C) Second type of inclusion described in text. Note capsomere-like particles surrounding electron-dense structures.
278
Fig. 4. Analysis of viral D N A sequences in K562 cells by Southern blot transfer and D N A - D N A hybridization. Pure 56S HSV-2 D N A was labeled in vitro with 32p to (2-4). 108 c p m / / t g by nick translation and hybridized to restricted fragments of intracellular D N A s fixed on nitrocellulose membranes by methods described in Materials and Methods section. (A) D N A s from purified virions, HSV-2-infected K562 cells or from productively-infected HEp-2 cells were cleaved with restriction endonucleases, electrophoresed in 0.8% agarose gels, stained with ethidium bromide and photographed as described in Materials and Methods. Electrophoretic mobilities of fragments generated by cleavage of D N A s from (i) purified virions by E c o R I (lane 1) or B a m H I (lane 2), (ii) infected K562 cells by BarnHI (lane 3) and (iii) infected HEp-2 cells by B a m H I are presented. (B) After electrophoresis, the restricted D N A fragments in agarose gels were denatured, transferred to nitrocellulose membranes, and hybridized to radioactive viral D N A as described in Materials and Methods. The autoradiograms of the D N A fragments that hybridized to radioactive viral D N A are represented in lanes 5-11. Lanes 5 and 6: Endo R. B a m H l cleaved D N A fragments from HSV-2 infected K562 (Clegg) cells at 4th and 6th, passage respectively. X-ray films were exposed for 4 days. Lane 7: Endo R. B a m H I cleaved D N A from HSV-2-infected HEp-2 cells 16 h after infection. X-ray films were exposed for 8 h. Lanes 8 and 9: Endo R. B a m H I and EcoRI cleaved D N A fragments from purified viral DNA. X-ray films were exposed for 2 h. Lane 10: Endo R. EcoRI cleaved D N A fragments from HSV-2-infected K562 (Loz) cells at 4th passage. Electrophoresis was conducted for 22 h and X-ray films were exposed for 4 days. Lane 11: Endo R. E c o R l
3B). On the other hand, only one peak of radioactivity corresponding to the buoyant density of cellular DNA but not to the viral DNA was noticed in the infected K562 (Clegg) cells whether the D N A was isolated from the 4th passage (no virus production) (Fig. 3C) or from the 6th passage (Fig. 3D) (small plaque-forming low levels of virus production Table I). Similar analysis of [3H]dT-labeled D N A from the 4th passage of either K562 (Loz) or K562 (Groth) revealed only one peak of radioactivity corresponding to the cellular DNA but not to the viral DNA. These results suggest that either the infected cells contained no appreciable amounts of viral DNA or that viral DNA was integrated into the cellular DNA and could not be resolved by the method of centrifugation in CsC1 density gradients. To investigate whether the viral D N A was intergrated, the cellular DNA was analysed for the presence of viral DNA by D N A - D N A hybridization on nitrocellulose membranes by a method described by Southern [34]. Southern blot analysis of viral DNA sequences in cellular DNA. Intracellular DNAs from HSV-2 infected K562 cells at passage levels of 4 and 6 (as described earlier) were cleaved with endo R. B a m H I and the resulting DNA fragments were separated by electrophoresis in 0.8% agarose gels. After staining with ethidium bromide, the DNA fragments were denatured, transferred to nitrocellulose membranes and hybridized with 32p-labeled viral DNA probe (see Materials and Methods). For comparison, DNA from either purified virions or from productively infected HEp-2 cells was similarly processed and hybridized with the viral DNA probe. A photograph of the ethidium bromide stained DNA fragments is presented in Fig. 4A. Restricted DNA fragments generated by cleavage of pure viral DNA with either EcoRI (Fig. 4al) or B a m H I (Fig. 4A2) were resolved without any background smear in 0.8% agarose gels and as expected, hybridized to homologous 32p-labeled viral DNA (Fig. 4B9 and B8). Under
cleaved D N A fragments from control noninfected K562 after hybridization with nick-translated radioactive viral D N A . Electrophoresis and exposure of X-ray film as described for lane 10 above.
279 similar conditions, cleavage of intracellular DNA from productively infected HEp-2 cells with endo R. BamHI yielded distinct visible DNA bands on a smear of cellular DNA fragments (Fig. 4A4). 32p-labeled viral DNA probe hybridized specifically (Fig. 4B7) with the visible DNA bands whose electrophoretic mobilities correspond with those of the BamHI fragments from purified viral DNA. Restricted fragments generated by cleavage of intracellular DNA from HSV-infected K562 cells by either BamHI or EcoRI migrated as smears in agarose tracks. A representative photograph of such a gel containing BamHI DNA fragments from the 4th passage of HSV-2 infected K562 cells is presented in Fig. 4A3. Upon hybridization with radioactively-labeled viral DNA probe, however, a number of DNA fragments with distinct electrophoretic mobilities were observed. The patterns of electrophoresis of most of these DNA bands, whether isolated from the intracellular DNA after 4th (Fig. 4B5) or 6th (Fig. 4B6) passages were very similar and correspond to those of the BamHI D N A fragments from purified virions (Fig. 4B8) or from productively infected HEp-2 cells. However, the hybridization pattern of the DNA from HSV-2 infected K562 (Groth) or from K562 (Loz) cells with HSV-2 DNA was entirely different. As shown in Fig. 4B, track 10, hybridization of EcoRI cleaved DNA fragments from the representative K562 (Loz) ceils at the corresponding 4th passage level with radioactive HSV-2 DNA demonstrated that a number of DNA fragments have been deleted out or some are beyond the level of detection. Under similar conditions of hybridization, only one endo R. EcoRI DNA fragment from uninfected control K562 (Clegg) cells hybridized with HSV-2 D N A (Fig. 4B6). These results may indicate that certain cloned K562 cells are capable of harboring HSV-2 DNA for a long period of time or that uninfected K562 cells may contain a D N A sequence complementary to small portion of HSV-2 DNA. Further experiments involving the measurements of reassociation kinetics are in progress to quantitatively determine the equivalents of HSV-2 genome in various clones of K562 cells. HSV-2-induced DNA polymerase in infected K562 (Clegg) cells Having established that the viral DNA se-
quences can persist in the infected K562 (Clegg) cells for an extended period of time we wanted to investigate if viral D N A polymerase is synthesized in such cells. Results presented in Fig. 5 show that the kinetics of incorporation of [3H]TMP into activated calf thymus DNA by extracts from HSV2-infected K562 cells at various passage levels. Higher amounts of [3H]TMP was incorporated by extracts of infected cells at passage levels of 1, 6, and 12 than by extracts from mock infected K562 (Clegg) cells or from extracts of the infected K562 (Clegg) cells at the 4th passage. It should be pointed out that these results were obtained when DNA polymerase was assayed in the presence of high (0.15 M) concentration of (NH4)2SO 4. Although this concentration is known to be inhibitory to most cellular DNA polymerases it is possible that a cellular DNA polymerase resistant to high salt may have been overproduced. To rule out this possibility and to demonstrate that only viral DNA
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TIME IN MINUTES
Fig. 5. Kineticsof [3H]dTMPincorporationinto activatedcalf thymus DNA by HSV-2-inducedDNA polymerasein virus-infected K562 (Clegg)cell extract. DNA polymerasewas assayed in duplicate samples by a method describedpreviously[36] (see Materials and Methods). [3H]TMP incorporation by extracts from K562 (Clegg) cells infected with HSV-2 at passage of 1 (11 II); 4 (O O); 6 (A A); 12 (e O); or from mock-infectedK562(Clegg)cells (zx A).
280 polymerase was being assayed in these experiments, cell extracts were pretreated with the immunoglobulin fraction from hyperimmune anti HSV-2 rabbit serum and tested for DNA polymerase activity. It was found that HSV-2 specific immunoglobulin inhibited [3H]TMP incorporation by the extracts from virus infected cells to levels similar to those observed for the mock infected cell extracts. These results demonstrate that viral DNA polymerase was synthesized in the HSV-2 infected K562 (Clegg) cells especially at the time of infectious virus production. Discussion In this report we describe experiments that indicate persistence and replication 6f HSV-2 DNA in a human leukemic K562 (Clegg) cell line over a period of several generations. These cells were quite inefficient in producing infectious virus however, and the virus titer never reached the titer of the initial inoculum (Fig. 1). The titer of the virus dropped significantly to an undetectable level after the first serial passage to appear again in a cyclical fashion at the 5th and 12th passage levels after infection (TAble I). During the periods of virus production, viral DNA could be detected by hybridization of viral DNA with Southern blots of infected cellular DNA but not by centrifugation in CsC1 density gradients. Electron microscopic analysis of the infected cells revealed intranuclear electron dense materials, some of which resembled viral capsomeres. Thus, these results taken together demonstrate that HSV-2 DNA entered and persisted in one K562 (Clegg) culture, out of three K562 cell cultures tested for persistent infection. The semipermissivenes of only one K562 cell culture for HSV-2 infection further substantiates the heterogeneity in the physiological states of various clones of K562 cells cultured in various laboratories. No infectious virus could be detected in the other two K562 cell cultures (Loz and Groth) included in these experiments and the titer of the initial inoculum seemed to decrease much more rapidly than the K562 (Clegg) cell line (Fig. 1). Viral DNA however, entered into the K562 (Loz) cells since a number of EcoRI cleaved DNA fragments within the infected cells had sequences homologous to a portion of the HSV-2 DNA (Fig.
4B10) when the cells were at their 4th passage. It is not clear whether the entire viral DNA integrated into the cell D N A or whether there was a gradual loss of integrated DNA after serial passage of the infected cells. It is known that HSV-DNA sequences are lost during serial passage of hamster cells transformed by ultraviolet irradiated HSV-2 [37,38]. It is also known that a significant portion of the viral D N A is lost and defective virions are generated upon serial passage of undiluted stocks of HSV [39,40]. Although the precise molecular mechanism of the generation of defective viral D N A is not known, it is possible that integrated viral DNA sequences may also be deleted during passage of persistently infected cells, perhaps by various abnormal or illegitimate recombinational events. Rinaldo et al. [8] have reported that a culture of K562 cells in their hands was more sensitive to HSV-1 infection and there was significant cell death. These results may indicate that interaction of K562 cells with HSV-1 may be different from that of HSV-2 or that, as discussed earlier, susceptibility of various K562 cell cultures to HSV-2 infection may differ significantly depending upon their physiological states. Adler et al. [13] have shown that a chemically transformed cell line, of neuronal origin, B103, was nonpermissive for KOS strain but not for HF, 17 and 33 strains of HSV-I. This nonpermissiveness of B103 neuronal cell line to KOS strain was, however, temperature and multiplicity dependent [41]. Although we have not attempted to induce the infected K562 cells to produce larger quantities of virus, the experiments described here indicate that K562 cells are capable of harboring HSV-2 genome for extended periods of time and of releasing detectable levels of virions from time to time. Further studies of the organization and expression of the viral DNA within these cells should shed light on the complex mechanism by which the genome of HSV can be regulated and controlled. References 1 Nahamias, A.J., Kibrick, S. and Rosan, R.C. (1964) J. Immunol. 93, 69-74 2 Robey, W.G., Graham, B.J., Harris, C.L., Madden, M.J., Pearson, G.R. and van de Woude, G.F. (1976) J. Gen. Virol. 32, 51-62
281 3 Westmoreland, D. (1978) J. Gen. Virol. 40, 559-575 4 Morahan, P.S. and Morse, S.S. (1979) in Virus-lymphocyte Interactions: Implication for Disease (Proffitt, M.R. ed.), pp. 17-35, Elsevier/North-Holland, NY 5 Plaeger-Marshall, S., Wilson, L.A. and Smith, J.A. (1982) Infect. Immuni. 35, 151-156 6 Katz, E., Mitrani-Rosenbaum, S. Margalith, E. and BenBassat, H. (1981) Intervirology 16, 33-42 7 Menzes, J. and Bourkas, A.E. (1980) J. Virol. 33, 115-122 8 Rinaldo, C.R., Jr., Richter, B.S., Black, P.H. and Hirsch, M.S. (1979) Infect. Immun. 25, 521-525 9 Hammer, S.M., Richter, B.S. and Hirsch, M.S. (1981) J. Immunol. 127, 144-148 10 Cummings, P.J., Lakomy, R.J. and Rinaldo, C.R. Jr. (1979) Infect. Immun. 25, 521-525 11 Youssoufian, H., Hammer, S.M., Hirsch, M.S. and Mulder, C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2207-2210 12 Vahlne, A. and Lycke, E. (1977) Proc. Soc. Exp. Biol. Med. 156, 82-87 13 Adler, R., Glorioso, J.C. and Levine, M. (1978) J. Gen. Virol. 39, 9-20 14 Nii, S. (1969) Biken, J. 12, 45-67 15 Hampar, B. and Burroughs, M.A.K. (1969) J. Natl. Cancer Inst. 43, 621-634 16 O'Neill, F.J., Goldberg, R.J. and Rapp, F. (1972) J. Gen. Virol. 14, 189-197 17 Nishiyama, Y. and Rapp, F. (1981) J. Gen. Virol.52, 113-119 18 Wigdahl, B.L., Isom, H.C. and Rapp, F. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6522-6526 19 Wigdahl, B.L., Scheck, A.C., De Clercq, E. and Rapp, F. (1982) Science 217, 1145-1146 20 Vesterinen, E., Leinikki, P. and Saksela, E. (1977) Acta Pathol. Microbiol. Scand. (B) 85, 289-295 21 Lozzio, C.B. and Lozzio, B.B. (1975) Blood 45, 321-334 22 Anderson, L.C., Jokiner, M., Galmberg, L.G. (1979) Nature 278, 364-365 23 Rutherford, T., Clegg, J.B., Higgs, D.R., Jones, R.W.,
24
25 26
27 28 29 30 31 32 33 34 35 36 37 38
39 40 41
Thompson, J. and Weatherall, D.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 348-352 Graham, B.J., Ludwig, H., Bronson, D.L., Benyesh-Melnick, M. and Biswal, N. (1972) Biochim. Biophys. Acta 259, 13-23 Bookout, J.B., Schaffter, P.A., Purifoy, D.J.M. and Biswal, N. (1979) Virology 93, 598-604 Dimery, I.W., Ross, D.D., Testa, J.R., Felsted, R.L., Gupta, S.K., Glover, C., Akman, A.K., Schimpff, S.C. and Bachur, N.R. (1982) Proc. Am. Assoc. Cancer Res. 73, 39, 1982 Campbell, W.F., Murray, B.K., Biswal, N. and BenyeshMelnick, M. (1974) J. Natl. Cancer Inst. 52, 757-761 McDowell, E.M. and Trump. B.F. (1976) Arch. Pathol. Lab. Med. 100, 405-414 Biswal, N., Murray, B.K. and Benyesh-Melnick, M. (1974) Virology 61, 87-99 Biswal, N. and Murray, B.K. (1974) Intervirol 4, 1-13 Hirsch, I., Cabral, G., Patterson, M. and Biswal, N. (1977) Virology 81, 48-61 Rigby, P.W.J., Dieckman, M., Rhodes, C. and Berg, P. (1977) J. Mol. Biol. 113, 237-251 Biswal, N., Sharma, S., Khan, N.C., Cabral, G. and Patterson, M. (1978) Virology 85, 568-586 Southern, E.M. (1975) J. Mol. Biol. 98. 503-517 Denhardt, D.T. (1966) Biochem. Biophys. Res. Commun. 23, 641-646 Weissbach, A., Hong, S.C., Aucker, J. and Muller, R. (1973) J. Biol. Chem. 248, 6270-6277 Frenkel, N., Locker, H., Cox, B., Roizman, B. and Rapp, F. (1976) J. Virol. 18, 885-893 Rapp, F. (1980) in Viruses in Naturally Occuring Cancer, (Essex, M., Todaro, G. and zur Haussen, eds.), pp. 63-80, Cold Spring Harbor Labs Bronson, D.L., Dressman, G.R., Biswal, N. and BenyeshMelnick, M. (1973) Intervirol 1, 141-153 Frenkel, N., Jacob, R.J., Honess, R.W., Hayward, G.S., Locker, H. and Roizman, B. (1975) J. Virol. 16, 153-167 Levine, M., Goldin, A.L. and Glorioso, J.C. (1980) J. Virol. 35, 203-210
271
BBA 91231
PERSISTENCE OF HERPES SIMPLEX VIRUS TYPE-2 GENOME IN A HUMAN LEUKEMIC CELL LINE NILAMBAR BISWAL, UNSIL KO, STEVEN AKMAN, DOUGLAS D. ROSS, AVROM POLLAK and EUGENE CIMINO
University of Maryland Cancer Center, Bressler Research Laboratories, 655 W. Baltimore Street, Baltimore, MD 21201 (U.S.A.) (Received March 16th, 1983)
Key words: Virus-cell interaction; Virus infection," DNA synthesis," DNA polymerase; Herpes simplex virus; (Human leukemic cell)
To study the nature of virus-cell interaction in persistently infected cells we have examined production of infectious virus, synthesis of viral DNA and DNA polymerase in a human leukemic cell line K562. It was found that only one of three 1(562 cell lines was permissive for limited growth of HSV-2 and infectious virus was released in a cyclical fashion. Intranuclear inclusions with electron-dense fibrils and particles resembling viral structures were observed in the virus-infected but not control K562 cells. Viral DNA synthesis could not be detected by centrifugation in CsCI density gradients; but was readily identified by Southern blot hybridization of virus-infected intraeellular DNA with purified viral DNA. Viral DNA polymerase was synthesized by infected cells during active infectious virus production. In one of the two K562 cell lines that did not produce infectious virus, a few DNA fragments from infected cells were found to hybridize with purified viral DNA. These results suggest that variable lengths of HSV-2 genome can be harbored and propagated by different human leukemic K562 cells.
Introduction
During recent years a variety of cultured cell lines of hematopoietic origin have been shown to support persistent growth of HSV [1-8]. Of particular importance are cultured human lymphoid and myeloid cell lines where HSV can continue to replicate in a dynamic state for prolonged periods after initial infection, thus providing in vitro model systems to study the mechanism of HSV persistence in hematopoietic cells. Rianldo et al. [8] have reported that human T, B and myeloid cell lines persistently infected with HSV-1 grow like non-infected cultured cells but with oscillating
Abbreviations: HSV, herpes simplex virus; EACA, c-aminocaprioic acid; Endo R, restriction endonuclease. 016%4781/83/$03.00 © 1983 Elsevier Science Publishers B.V.
cycles of crisis (increased virus production with variable degree of cytopathic effect). Katz et al. [6] have shown that null and B lymphoid lines arrested at pre B or at stages of I and II of the B differentiation maturation pathway also support persistent HSV growth. Menezes and Bourkas [7] have shown that depending upon the level of cell differentiation, various lymphoblastoid cell lines were permissive to HSV infection. These virus-infected cells developed HSV-specific Fc receptors and produced viral specific antigens and infectious virus. Recently it has been possible to regulate the expression of HSV gene functions in persistently infected cells under defined conditions. For example, treatment of the persistently infected human T lymphoblastoid cell line, CEM, with concanavalin A [9], HSV-1 antiserum or high temperature [10]
272 suppressed HSV-1 production and expression of viral antigens. However, treatment of these same cells with phytohemagglutinin [9] or lower temperature [10] reactivated infectious virus production. Viral DNA was found to be highly methylated in the concanavalin A-treated nonproducer cells, but not in the productively infected or phytohemagglutinin-treated cells [11]. These results have suggested, but not proven, that methylation of viral D N A may be a prerequisite for the maintenance of the latent state of HSV-1. Related to these studies are observations from other laboratories where various other types of cultured mammalian cells such as neuronal cells [12,13], mouse L cells [14], chinese hamster ovary cells [15], human embryonic lung (HEL) cells [16-19] and human cervical epithelial cells [20] have been used to establish HSV persistence in vitro. Replication of HSV-2 or HSV-1 can be repressed by treatment of these virus-infected cells with various agents such as high incubation temperatures and cytosine arabinoside [16-18], adenine arabinoside [20] or with (E)-5-(2bromovinyl)-2'-deoxyuridine, in combination with human leukocyte interferon-a. [19]. HSV genome can be reactivated by superinfecting the infected cells with human cytomegalovirus [16,17] or by reducing the incubation temperature [19]. Adler et al. [13] have shown that rat neuronal cell line B103 can be conditioned to harbor HSV-I genome for long periods of time and to express viral specific proteins such as D N A polymerase, thymidine kinase, and other proteins in the absence of active viral DNA synthesis. We have initiated studies to analyze the mechanism by which HSV-2 persists and the consequence of that persistence in a human leukemic cell line (K562). This cell line was originally derived from the pleural effusion of a patient with chronic myeloid leukemia in blast crisis [21]. K562 cells can be induced to differentiate to express phenotypic markers for erythroid lineage [22] and display spontaneous and induced globin synthesis [23]. In this communication we describe the successful establishment of persistent infection of HSV-2 in a particular K562 cell line, the physical state of the virus particles and viral DNA and the expression of viral D N A polymerase within these cells.
Materials and Methods Virus. Herpes simplex virus type 2, strain 333 (HSV-2) was used throughout this study. The source, propagation and assay protocols for this virus have been published before [24,25]. Cells. Suspension cultures of human leukemic K562 cells were obtained from three different sources: from Drs. R. Hoffman of New Haven, CT, B.B. Lozzio of Knoxville, TN and D. Groth of Atlanta, GA and were designated by us as K562 (Clegg), K562 (Loz) and K562 (Groth) respectively. These cell lines in different labs have been found to evolve tangible phenotypic and genotypic changes [26] and are well characterized at this Cancer Center by Ross et al. [26] for their heterogeneity, karyotyping, sensitivity to growth inhibitors and hemin. Thus, for a comparative study these individual cultures were cloned in 0.3% agar and the cloned cultures were subsequently maintained in RPMI 1640 medium (Grand Island Biologicals Co.) supplemented with heat-inactivated 10% fetal calf serum, 2 mM glutamine, 0.225% sodium bicarbonate, 100 units/ml of penicillin and 100/~g/ml of streptomycin at 37C under 5% CO 2 atmosphere. The suspension cultures were passed in sterile bottles every 4th day at an initial concentration of (2-3). 105 cells/ml. Cultures of fibroblastic HEp-2 cells and Vero cells were maintained and propagated in minimum essential medium as described before [24,27]. H S V-2 growth curves in K562 and HEp-2 cells.
Suspension cultures of cloned K562 cells were concentrated to approx. 2.5. 10 7 cells/ml by centrifugation and infected with HSV-2 at a multiplicity of infection of about 10. After adsorption for 90 min at 37C, the cells were washed twice with phosphate-buffered saline to remove the unadsorbed virus, 100 ml of growth medium was added and the infection continued at 37°C. At indicated times, duplicate samples of 10 ml each were sonicated for 1 min in a Branson sonic oscillator at 10 kHz and centrifuged at 1500 × g for 10 min to clarify the supernatant. This supernatant which contained both the intra- as well as extracellular virus particles was assayed for infectious HSV-2 in monolayers of Vero cells in 60 mm plastic petri plates [24,25]. Growth of HSV-2 in permissive HEp-2 cell monolayers was measured
273 by a technique described previously [23,24]. Kinetics of cell growth. Virus-infected or control K562 cells were incubated in sterile culture flasks at an initial concentration of 2.105 cells/ml. Cell counts were performed daily in a Coulter counter (Coulter Electronics) and the number of viable cells was determined by the trypan blue exclusion test. Electron microscopy. For ultrastructural studies, infected K562 cells at the 4th passage level were washed and fixed for 2 h at room temperature in a 4% formaldehyde/1% gluteraldehyde phosphatebuffered mixture [28]. After postfixation in osmium tetroxide, the samples were processed and thin-sectioned for transmission electron microscopy (transmission EM) and examined in a JEOL 100 cx electron microscope.
Centrifugation of intracellular DNA in CsCI density gradients. HSV-2 infected or mock-infected K562 cells were labeled with [3H]dT (5 /tCi/ml) for 24 h. As a positive control, monolayers of HEp-2 cells were infected with HSV-2 at a multiplicity of infection of 10 plaque forming units/ml and incubated for 16 h in the presence of [3H]dT (5/~Ci/ml). The cells were harvested, washed twice in Tris-sodium chloride buffer (0.01 M Tris-HC1 (pH 8.0) 0.1 M NaC1 and 0.005 M EDTA), lysed with sodium dodecyl sulfate and treated with protease K as described before [29,30]. The median density of the solution was adjusted to 1.705 g / c m 3 with CsC1 crystals and the mixture was centrifuged to equilibrium for 3 days in a Spinco SW 50.1 rotor. After measuring the refractive indices of some representative fractions from the gradient, trichloroacetic acid = precipitable radioactivity of each fraction was determined. Labeling of viral DNA with 32p by 'nick translation" The 56S HSV-2 DNA was isolated from purified virus by a method described previously [24,31]. This DNA was labeled in vitro with 32p by a slight modification of 'nick translation' method described by Rigby et al. [32]. About 1 /~g of HSV-2 DNA was incubated at 15°C in a 0.1 ml reaction mixture containing 100 /xCi each of [a32p]dATP, d G T P , d C T P and T T P (spec. radioact. = 3000 Ci/mmol, Amersham Corp.), 50 mM Tris-HC1 (pH 7.8), 5 mM MgC12, 10 mM 2-mercaptoethanol, 0.5 ng DNAase and 2 units of E. coli DNA polymerase I. After 60 min, the
reaction was terminated with 10 mM EDTA and 0.5% SDS, diluted to 0.4 ml with Tris-sodium chloride buffer and deproteinized with an equal volume of phenol/chloroform (1 : 1) mixture. The entire mixture was shaken for 10 rain, centrifuged at 12000 × g for 5 min and the labeled DNA in the aqueous layer was separated from the unincorporated nucleotides by passing through a column of Sephadex G-50. In this way the viral DNA could be labeled to (2-4). 108 cpm//~g and was used for hybridization purposes later.
"Southern' blot transfer of restricted DNA fragments. DNA from HSV-2 infected K562 cells were isolated by deproteinization with p h e n o l / chloroform and isoamyl alcohol as described before [24,29]. The purified DNAs were digested overnight with EcoRI and BamHI restriction enzymes according to the specifications of the supplier (Bethesda Research Laboratories). The cleaved DNA fragments were separated by electrophoresis for 16 to 20 h at 2 V / c m in 0.8% agarose slab gels, stained with ethidium bromide and photographed with a Polaroid camera using type 55 positive/negative film [33]. The DNA fragments in the agarose gels were then denatured for 30 rain with 0.5 M N a O H and 1.5 M NaC1, neutralized to p H 7.2 with 0.5 M Tris-HC1 (pH 7.2) and 3.0 M NaCI. The denatured DNA fragments were then transferred to sheets of nitrocellulose membranes as described by Southern [34]. After drying, the filters were baked under vacuum for 2 h at 80°C and stored at 4°C under vacuum until hybridization. Hybridization of viral DNA to cellular DNA on filters. Purified viral DNA labeled with 32p by nick translation described above was hybridized to the denatured cellular DNA fragments fixed on nitrocellulose membranes as follows: radioactive viral DNA was sonicated, denatured at 117°C in sealed ampoules and quickly chilled in an ice bath. Nitrocellulose membranes containing the cellular D N A fragments were incubated in Denhardts' medium [35] for 4 to 6 h to reduce nonspecific binding of viral DNA to the charged membranes. These membranes were then exposed to denatured radioactive viral DNA in the presence of 6-SSC (SSC = 0.15 M NaC1 and 0.015 M sodium citrate) containing 0.02% each of Ficoll, bovine serum albumin and polyvinylpyrolidone. D N A - D N A
274
hybridization was allowed for 60 h at 62°C in a rotatory drum. The membranes were then washed 3-4-times with large volumes of 0.005 M Tris (pH 9.2) until the radioactivity of the washed fluid was lower than 100 cpm per 0.2 ml of wash. The membranes were dried, exposed to Kodak Blue Brand X-ray films for an appropriate time (ranging from few hours to 4 days) and the autoradiograms were developed as described previously [33].
Preparation of crude cell extracts for enzyme assay. Approx. 2 × 10 7 HSV-2 infected or mock infected K562 (Clegg) cells at passage levels of 1, 4, 6 and 12 (low levels of virus production, except passage 4, see Table I) were washed twice in phosphate-buffered saline and frozen as cell pellet at - 9 0 ° C until all the samples were collected. At the time of assay the frozen samples were thawed, suspended in 1 ml of Tris-buffer (0.05 M Tris-HC1 (pH 7.8), 5 mM dithiothreitol and 1 mM ~-aminocaproic acid). The cell suspension was sonicated in an ice bath for 1 min at 9 K C / s and an equal volume of cold solution of 4 M KC1, 10 mM EDTA, 5 0 0 / l g / m l of bovine serum albumin and 2 mM c-aminocaproic acid was slowly added. The resulting precipitate was removed by centrifugation at 10000 rev./min and supernatant was dialyzed extensively against buffer A (0.05 M TrisHCI (pH 7.8), 1 mM EACA, 2 mM dithiothreitol, 0.2% NP-40 and 20% glycerol). The dialysate was clarified by centrifugation at 15 000 rev./min, for 30 rain and the supernatant as crude cell extract was assayed for D N A polymerase. Assay for HSV-2-induced DNA polymerase. HSV-2-induced D N A polymerase in the crude cell extract was assayed by a slight modification of method described by Weissbach et al. [36]. The reaction mixture (total volume of 100 /~1) contained 0.01 M Tris-HC1 (pH 7.8), 0.15 M (NH4)SO4, 1 mM dithiothreitol, 2.5 mM MgC12, 5 /xg of activated calf thymus DNA, 20 ~tg bovine serum albumin, 0.05 m M [ 3 H ] T T P (600 cpm/pmol), 0.2 mM each of dCTP, dATP and d G T P and 25 /xl of cell extract as the source of HSV-2 D N A polymerase. Duplicate samples at indicated time points were withdrawn and the reaction was terminated by adding an equal volume of cold 10% trichloroacetic acid containing 0.1 M sodium pyrophosphate. The precipitate was filtered on Whatman G F / C filters, washed sequentially
with cold 5% trichloroacetic adid and cold distilled water and the radioactivity on dried filters was measured in a liquid scintillation spectrometer. Results
Restriction of HSV-2 growth in K562 cells To determine if cultures of various K562 cell lines supported the replication of HSV-2, cloned cultures of three K562 cell lines (Clegg, Loz and Groth) were infected with HSV-2 at a multiplicity of infection of 10 plaque forming units/cell. After adsorption for 90 min at 37C, the cells were washed and duplicate samples were assayed for infectious HSV-2 as described in Materials and Methods section. Results presented in Fig. 1 show that none of the three K562 cell lines supported production of new infectious HSV-2 progenies over a period of 48 h after initiation of infection. The virus particles adsorbed to all the three cell lines, however,
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Fig. 1. Growth of HSV-2 in leukemic K562 cells and HEp-2 cells. Various cultures of K562 Clegg, Loz and Groth cells were concentrated and infected with HSV-2 at a multiplicity of infection of approx. 10 (see Materials and Methods). After 90 rain of adsorption at 37C, the cells were washed twice and incubation was continued. At indicated times, duplicate samples were assayed for infectious virus as described in Materials and Methods. Growth of HSV-2 in HEp-2 cells (O e): K56(Clegg) cells (© .... ©); K562(Groth) cells (zx zx): K562(Loz) cells (n O).
275
since infectious virus, whose infectivity titer gradually decreased, could be recovered up to 48 h after infection. The supernatant fluid alone from such cultures did not demonstrate infectious virus particles under similar conditions of infection. In contrast, monolayers of HEp-2 cells supported HSV-2 growth and new infectious progeny virions were produced with a typical growth curve. Thus, these results demonstrate that all three K562 cell lines tested were nonpermissive for HSV-2 (333) growth although the virus adsorbed to the cells. However, Rinaldo et al. [8] have demonstrated that K562 cells supported a productive replication of HSV-1, although the titer of progeny virions was at a lower level. To test the possibility that prolonged passage of the K562 cell lines will produce new infectious HSV-2 at a later time, we continued passing the infected cells and tested them for virus production as well as viable cell growth as described below.
Cyclical appearance of HSV-2 in K562 (Clegg) cells The infected cells continued to grow like uninfected control cells up to the 4th passage (16 days after infection) with a doubling time of approx. 24 h. The number of viable cells, as determined by the Trypan blue exclusion assay, ranged from 90-95% of the total. During this time of continued growth, the cells usually attained a density of 3.106 cells/ml in 4 days from an initial concentration of (2-3). 105 cells/ml. Thus, for routine propagation and to maintain a cell viability of greater than 90%, all the cells (infected and noninfected) were suspended in fresh growth medium every fourth day at a concentration of about 2.5 • 105 cells/ml. After the first passage no infectious virus could be detected in the cell homogenates up to the 4th serial passage in 16 days (Table I). At the 4th passage level the infected K562 (Clegg) cells also ceased to replicate for about 8 days and the viability of the cells was reduced to 70%. During this 8-day period of quiescence, feeding the cells with fresh growth medium did not induce replication of infected cells and no infectious virus could be detected. The cells then spontaneously recovered from the 8-day quiescence and started to grow again with the same efficiency as the control cells. The number of viable cells increased to greater than 90% and a low level of virus capable
TABLE I I N F E C T I O N O F K562 (CLEGG) CELLS BY HSV-2 Every fourth day the cells were diluted to about 2.105 cells/ml with fresh medium and the incubation continued. Virus titer shown is from cells just before the 1st passage on 4th day. PFU, plaque-forming units. Days after infection 0
Passage number -
Viable cells (per ml)
Virus titer (PFU/ml)
2.5.105
5.7.105
1
4.9.105
1.2.105
2 4a 12 16 20 24
1.1 • 106 1.2.106 2.5- 106 2.2" 106 1.7" 105 1.6- 105
9.0.104 4.104 0 0 0 3' 103 (small plaques) 3- 103 (small plaques) 1- 103 (small plaques) 0 0 0 7.102 (pin-point plaques) 0
1 3 4 4 4
28
2.5.106
32
6
3 • 106
36 40 48 56
7 8 10 12
1 - lO 6 2.2.106 2.1 • 105 2.2.106
64
14
3 • 106
of producing small plaques in Vero cells were observed (Table I). Upon continued passage the cells again stopped producing any detectable levels of virions up to the l l t h passage. At the 12th passage, however, the cells produced virions capable of producing only extremely small plaques (readily visible under microscope). This observation of restricted but cyclical virus production was limited to K562 (Clegg) cells. Other two cell lines K562 (Loz) and K562 (Groth) failed to produce any virus particles after the 1st passage. For this reason subsequent experiments were conducted only with the K562 (Clegg) cells. Since a limited number of infectious virions continued to be produced by the infected K562 (Clegg) cells in a cyclical fashion, we designed experiments to examine the intracellular DNA of K562 (Clegg) cells for the presence of viral DNA.
Electron microscopic examination of infected K562 (Clegg) cells Approx. 50% of cells examined from infected
216
277
cultures showed cytopathologic effects when compared to cells from uninfected cultures. The most conspicuous morphological variations were nuclear fragmentation and cytoplasmic vaculoization (Fig. 2a). Associated with nuclear fragmentation was a high degree of heterochromatin condensation and margination. In a small percentage (2-4%) of infected cells we observed intranuclear inclusions measuring 0.4-1.5 ~m in diameter. These inclusions were of two types. In one type the inclusion consisted of an undulating mass of 15-24 nm thick fibrils intimately associated with the nucleolus (Fig. 2A and B), whereas the more frequently observed second type consisted of parallel arrays of electron-dense material 36-60 nm in diameter. In cross-section (Fig. 2C), the electron-dense material can be seen to be completely surrounded by electron lucent particles which bear a close morphological similarity to viral capsomeres. Thus these results demonstrate ultrastructural modifications in the infected cells especially when such cells are in their quiescent state at the 4th passage level.
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DNA analysis by CsCI buoyant density measurement. K562 cells infected with HSV-2 at the sec-
Fig. 3. CsC1 buoyant densities of DNA samples from K562 (Clegg) cells infected with HSV-2. Persistently infected K562 (Clegg) cells or productively infected HEp-2 cells were labeled with [3H]dT and the DNA from these cells was centrifuged to equilibrium in CsCI as described in Materials and Methods. At the end of centrifugation, fractions were collected from the top by an Isco fraction collector and the trichloroacetic acid-precipitable radioactivity of each fraction was determined (see Materials and Methods). Radioactivity in the DNA from (A) mock infected K562(Clegg) cells, (B) productively infected HEp-2 cells, (C) persistently infected K562 (Clegg) cells at 4th passage or (D) at 6th passage level.
ond, 4th and 6th passage levels were chosen for this experiment. Infected cells at the 4th passage level did not show any infectious virus production while at the 6th passage the cells produced low levels of infectious virions. The cells were labeled with [3H]dT, intraceUular DNA was isolated and centrifuged to equilibrium in CsC1 density gradients as described in Materials and Methods. Under such experimental conditions, it is possible to monitor the synthesis of both cellular and viral D N A since the buoyant density of HSV-2 DNA P
CsC1 = 1.727 g / c m 3) is much greater and readily distinguishable from that of the cellular DNA p CsC1 = 1.699 g / c m 3) [24,27,29]. Results presented in Fig. 3 show that uninfected K562 (Clegg) cells had only one peak of radioactivity (Fig. 3A) which banded at a density of about 1.700 g / c m 3. Productively infected HEp-2 cells however, showed two peaks of radioactivity banding at the expected positions of the viral DNA and cellular DNA (Fig.
Analysis of intracellular DNA of HSV-2-infected K562 (Clegg) cells To examine the possibility that HSV-2 DNA may have become resident in the infected K562 (Clegg) cells, two experimental approaches were taken.
Fig. 2. Electron micrographs of K562 cells infected with HSV-2. (A) A K562 cell from HSV-2-infected culture at the 4th passage level. Note nuclear fragmentation, marginated heterochromatin in fragment on left, nucleolus associated inclusion in larger fragment, and vacuolization of cytoplasm. (B) Higher magnification of inclusion seen in A. (C) Second type of inclusion described in text. Note capsomere-like particles surrounding electron-dense structures.
278
Fig. 4. Analysis of viral D N A sequences in K562 cells by Southern blot transfer and D N A - D N A hybridization. Pure 56S HSV-2 D N A was labeled in vitro with 32p to (2-4). 108 c p m / / t g by nick translation and hybridized to restricted fragments of intracellular D N A s fixed on nitrocellulose membranes by methods described in Materials and Methods section. (A) D N A s from purified virions, HSV-2-infected K562 cells or from productively-infected HEp-2 cells were cleaved with restriction endonucleases, electrophoresed in 0.8% agarose gels, stained with ethidium bromide and photographed as described in Materials and Methods. Electrophoretic mobilities of fragments generated by cleavage of D N A s from (i) purified virions by E c o R I (lane 1) or B a m H I (lane 2), (ii) infected K562 cells by BarnHI (lane 3) and (iii) infected HEp-2 cells by B a m H I are presented. (B) After electrophoresis, the restricted D N A fragments in agarose gels were denatured, transferred to nitrocellulose membranes, and hybridized to radioactive viral D N A as described in Materials and Methods. The autoradiograms of the D N A fragments that hybridized to radioactive viral D N A are represented in lanes 5-11. Lanes 5 and 6: Endo R. B a m H l cleaved D N A fragments from HSV-2 infected K562 (Clegg) cells at 4th and 6th, passage respectively. X-ray films were exposed for 4 days. Lane 7: Endo R. B a m H I cleaved D N A from HSV-2-infected HEp-2 cells 16 h after infection. X-ray films were exposed for 8 h. Lanes 8 and 9: Endo R. B a m H I and EcoRI cleaved D N A fragments from purified viral DNA. X-ray films were exposed for 2 h. Lane 10: Endo R. EcoRI cleaved D N A fragments from HSV-2-infected K562 (Loz) cells at 4th passage. Electrophoresis was conducted for 22 h and X-ray films were exposed for 4 days. Lane 11: Endo R. E c o R l
3B). On the other hand, only one peak of radioactivity corresponding to the buoyant density of cellular DNA but not to the viral DNA was noticed in the infected K562 (Clegg) cells whether the D N A was isolated from the 4th passage (no virus production) (Fig. 3C) or from the 6th passage (Fig. 3D) (small plaque-forming low levels of virus production Table I). Similar analysis of [3H]dT-labeled D N A from the 4th passage of either K562 (Loz) or K562 (Groth) revealed only one peak of radioactivity corresponding to the cellular DNA but not to the viral DNA. These results suggest that either the infected cells contained no appreciable amounts of viral DNA or that viral DNA was integrated into the cellular DNA and could not be resolved by the method of centrifugation in CsC1 density gradients. To investigate whether the viral D N A was intergrated, the cellular DNA was analysed for the presence of viral DNA by D N A - D N A hybridization on nitrocellulose membranes by a method described by Southern [34]. Southern blot analysis of viral DNA sequences in cellular DNA. Intracellular DNAs from HSV-2 infected K562 cells at passage levels of 4 and 6 (as described earlier) were cleaved with endo R. B a m H I and the resulting DNA fragments were separated by electrophoresis in 0.8% agarose gels. After staining with ethidium bromide, the DNA fragments were denatured, transferred to nitrocellulose membranes and hybridized with 32p-labeled viral DNA probe (see Materials and Methods). For comparison, DNA from either purified virions or from productively infected HEp-2 cells was similarly processed and hybridized with the viral DNA probe. A photograph of the ethidium bromide stained DNA fragments is presented in Fig. 4A. Restricted DNA fragments generated by cleavage of pure viral DNA with either EcoRI (Fig. 4al) or B a m H I (Fig. 4A2) were resolved without any background smear in 0.8% agarose gels and as expected, hybridized to homologous 32p-labeled viral DNA (Fig. 4B9 and B8). Under
cleaved D N A fragments from control noninfected K562 after hybridization with nick-translated radioactive viral D N A . Electrophoresis and exposure of X-ray film as described for lane 10 above.
279 similar conditions, cleavage of intracellular DNA from productively infected HEp-2 cells with endo R. BamHI yielded distinct visible DNA bands on a smear of cellular DNA fragments (Fig. 4A4). 32p-labeled viral DNA probe hybridized specifically (Fig. 4B7) with the visible DNA bands whose electrophoretic mobilities correspond with those of the BamHI fragments from purified viral DNA. Restricted fragments generated by cleavage of intracellular DNA from HSV-infected K562 cells by either BamHI or EcoRI migrated as smears in agarose tracks. A representative photograph of such a gel containing BamHI DNA fragments from the 4th passage of HSV-2 infected K562 cells is presented in Fig. 4A3. Upon hybridization with radioactively-labeled viral DNA probe, however, a number of DNA fragments with distinct electrophoretic mobilities were observed. The patterns of electrophoresis of most of these DNA bands, whether isolated from the intracellular DNA after 4th (Fig. 4B5) or 6th (Fig. 4B6) passages were very similar and correspond to those of the BamHI D N A fragments from purified virions (Fig. 4B8) or from productively infected HEp-2 cells. However, the hybridization pattern of the DNA from HSV-2 infected K562 (Groth) or from K562 (Loz) cells with HSV-2 DNA was entirely different. As shown in Fig. 4B, track 10, hybridization of EcoRI cleaved DNA fragments from the representative K562 (Loz) ceils at the corresponding 4th passage level with radioactive HSV-2 DNA demonstrated that a number of DNA fragments have been deleted out or some are beyond the level of detection. Under similar conditions of hybridization, only one endo R. EcoRI DNA fragment from uninfected control K562 (Clegg) cells hybridized with HSV-2 D N A (Fig. 4B6). These results may indicate that certain cloned K562 cells are capable of harboring HSV-2 DNA for a long period of time or that uninfected K562 cells may contain a D N A sequence complementary to small portion of HSV-2 DNA. Further experiments involving the measurements of reassociation kinetics are in progress to quantitatively determine the equivalents of HSV-2 genome in various clones of K562 cells. HSV-2-induced DNA polymerase in infected K562 (Clegg) cells Having established that the viral DNA se-
quences can persist in the infected K562 (Clegg) cells for an extended period of time we wanted to investigate if viral D N A polymerase is synthesized in such cells. Results presented in Fig. 5 show that the kinetics of incorporation of [3H]TMP into activated calf thymus DNA by extracts from HSV2-infected K562 cells at various passage levels. Higher amounts of [3H]TMP was incorporated by extracts of infected cells at passage levels of 1, 6, and 12 than by extracts from mock infected K562 (Clegg) cells or from extracts of the infected K562 (Clegg) cells at the 4th passage. It should be pointed out that these results were obtained when DNA polymerase was assayed in the presence of high (0.15 M) concentration of (NH4)2SO 4. Although this concentration is known to be inhibitory to most cellular DNA polymerases it is possible that a cellular DNA polymerase resistant to high salt may have been overproduced. To rule out this possibility and to demonstrate that only viral DNA
l
I
I
b x Q. U v
>.. IVU
om
r-,
2
I
Io
I
20
3O
4O
TIME IN MINUTES
Fig. 5. Kineticsof [3H]dTMPincorporationinto activatedcalf thymus DNA by HSV-2-inducedDNA polymerasein virus-infected K562 (Clegg)cell extract. DNA polymerasewas assayed in duplicate samples by a method describedpreviously[36] (see Materials and Methods). [3H]TMP incorporation by extracts from K562 (Clegg) cells infected with HSV-2 at passage of 1 (11 II); 4 (O O); 6 (A A); 12 (e O); or from mock-infectedK562(Clegg)cells (zx A).
280 polymerase was being assayed in these experiments, cell extracts were pretreated with the immunoglobulin fraction from hyperimmune anti HSV-2 rabbit serum and tested for DNA polymerase activity. It was found that HSV-2 specific immunoglobulin inhibited [3H]TMP incorporation by the extracts from virus infected cells to levels similar to those observed for the mock infected cell extracts. These results demonstrate that viral DNA polymerase was synthesized in the HSV-2 infected K562 (Clegg) cells especially at the time of infectious virus production. Discussion In this report we describe experiments that indicate persistence and replication 6f HSV-2 DNA in a human leukemic K562 (Clegg) cell line over a period of several generations. These cells were quite inefficient in producing infectious virus however, and the virus titer never reached the titer of the initial inoculum (Fig. 1). The titer of the virus dropped significantly to an undetectable level after the first serial passage to appear again in a cyclical fashion at the 5th and 12th passage levels after infection (TAble I). During the periods of virus production, viral DNA could be detected by hybridization of viral DNA with Southern blots of infected cellular DNA but not by centrifugation in CsC1 density gradients. Electron microscopic analysis of the infected cells revealed intranuclear electron dense materials, some of which resembled viral capsomeres. Thus, these results taken together demonstrate that HSV-2 DNA entered and persisted in one K562 (Clegg) culture, out of three K562 cell cultures tested for persistent infection. The semipermissivenes of only one K562 cell culture for HSV-2 infection further substantiates the heterogeneity in the physiological states of various clones of K562 cells cultured in various laboratories. No infectious virus could be detected in the other two K562 cell cultures (Loz and Groth) included in these experiments and the titer of the initial inoculum seemed to decrease much more rapidly than the K562 (Clegg) cell line (Fig. 1). Viral DNA however, entered into the K562 (Loz) cells since a number of EcoRI cleaved DNA fragments within the infected cells had sequences homologous to a portion of the HSV-2 DNA (Fig.
4B10) when the cells were at their 4th passage. It is not clear whether the entire viral DNA integrated into the cell D N A or whether there was a gradual loss of integrated DNA after serial passage of the infected cells. It is known that HSV-DNA sequences are lost during serial passage of hamster cells transformed by ultraviolet irradiated HSV-2 [37,38]. It is also known that a significant portion of the viral D N A is lost and defective virions are generated upon serial passage of undiluted stocks of HSV [39,40]. Although the precise molecular mechanism of the generation of defective viral D N A is not known, it is possible that integrated viral DNA sequences may also be deleted during passage of persistently infected cells, perhaps by various abnormal or illegitimate recombinational events. Rinaldo et al. [8] have reported that a culture of K562 cells in their hands was more sensitive to HSV-1 infection and there was significant cell death. These results may indicate that interaction of K562 cells with HSV-1 may be different from that of HSV-2 or that, as discussed earlier, susceptibility of various K562 cell cultures to HSV-2 infection may differ significantly depending upon their physiological states. Adler et al. [13] have shown that a chemically transformed cell line, of neuronal origin, B103, was nonpermissive for KOS strain but not for HF, 17 and 33 strains of HSV-I. This nonpermissiveness of B103 neuronal cell line to KOS strain was, however, temperature and multiplicity dependent [41]. Although we have not attempted to induce the infected K562 cells to produce larger quantities of virus, the experiments described here indicate that K562 cells are capable of harboring HSV-2 genome for extended periods of time and of releasing detectable levels of virions from time to time. Further studies of the organization and expression of the viral DNA within these cells should shed light on the complex mechanism by which the genome of HSV can be regulated and controlled. References 1 Nahamias, A.J., Kibrick, S. and Rosan, R.C. (1964) J. Immunol. 93, 69-74 2 Robey, W.G., Graham, B.J., Harris, C.L., Madden, M.J., Pearson, G.R. and van de Woude, G.F. (1976) J. Gen. Virol. 32, 51-62
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