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Characteristics of a Macrophage Culture Persistently Infected with Herpes Simplex Virus Type 1
Archives of Medical Research 30 (1999) 255–262
ORIGINAL ARTICLE
Characteristics of a Macrophage Culture Persistently Infected with Herpes Simplex Virus Type 1 Jaime Bustos*,** and Beatríz Gómez * *Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), México, D.F., Mexico **Departamento de Atención a la Salud, Universidad Autónoma Metropolitana (UAM)-Xochimilco, México, D.F., Mexico Received for publication October 19, 1998, accepted April 6, 1999 (98/133).
Background. Persistence of herpes simplex type 1 (HSV-1) has been reported in sensory neurons, corneal epithelium, and lymphocytes, although other cell types such as macrophages should also be considered as hosts for HSV-1 persistence. Here we report the establishment and characterization of HSV-1 persistence in an immortalized murine macrophage-like cell line (P388D1). Methods. The persistently HSV-1 infected culture (P388D1per) was obtained from surviving P388D1 macrophages infected with HSV-1 MP strain at multiplicity of 0.001. P388D1per was characterized by: extracellular production of viruses, cells expressing viral antigens, and cells releasing infectious viruses. Viral plaque size and cytophatic effect were determined in viruses (HSVA and HSVB) obtained from two different P388D1per passages. Host and viral proteins were detected in P388D1per and in P388D1 cells infected with HSV-1 by metabolic [35S]-methionine labeling assays. Results. P388D1per culture was characterised by cyclic production of infectious viruses from non-detectable to 106 TCID50/mL; from 1.0 to 15.0% cells expressing viral antigens and macrophages released infectious viruses from 0.008 to 12.5%. Differences in viral plaque size and cytopathic effect morphology between HSVA, HSVB and HSV-1 were observed. Similar patterns of viral proteins were observed in P388D1per and in P388D1 infected with HSV-1. Nonetheless, the characteristic interference effect of HSV-1 on host protein synthesis was not observed in P388D1per culture. Conclusions. An HSV-1 persistently infected immortalized macrophage culture was established and characterized. Virus produced during persistence showed phenotypic alterations with respect to the original virus. P388D1per cell protein synthesis was not affected by the presence of HSV-1. © 1999 IMSS. Published by Elsevier Science Inc. Key Words: P388D1, HSV-1, Persistence, Virus phenotypic alterations.
Introduction A remarkable feature of herpes simplex virus type 1 (HSV-1) is its ability to persist in the natural host (human) after primary infection in a latent or subdetectable form (1). Persistence of HSV-1 has been documented in patients with recurrent HSV-1 infections, herpes keratitis, and urogenital
Address reprint requests to: Dra. Beatríz Gómez, Departamento de Microbiología y Parasitología, Facultad de Medicina, UNAM. México, D.F. 04510, México. Tel.: (1525) 6-23-24-69; FAX: (1525) 6/23-23-86; E-mail: begomez @ servidor.unam.mx
tumors (2–4). Nonetheless, the sites where HSV-1 maintains itself in the human organism vary with the clinical manifestations. Until now, persistence of the virus has been reported in neurons, corneal epithelium, and lymphocytes (3–5), although other cell types such as macrophages should also be considered as hosts for HSV-1 persistence. Macrophages are widely distributed in the body, are important effector cells in host responses to virus infection, and participate in both innate and acquired immunity. Their presence in major organs, tissues, serous spaces and sites of inflammation suggests that they are the first immune cells encountered by the invading virus (6). In addition, microphages are among the cell types selected for the persistence of
0188-0128/99 $–see front matter. Copyright © 1999 IMSS. Published by Elsevier Science Inc. PII S0188-0128(99)00 0 2 1 - 4
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DNA and RNA (7); therefore, it is likely that HSV-1 can remain in these cells. Persistence of HSV-1 in human mononuclear cells has not been documented, although circumstantial evidence suggests that the virus can maintain itself in these cells. HSV-1 persistence has been reported ex vivo in a primary murine spleen macrophage culture obtained from mice resistant to infection (8) and in vitro in a promonocyte cell line (9,10), and in a macrophage-like cell line (11). In cells from patients with recurrent HSV-1 infections, higher concentration of HSV-1 DNA are present in the recrudescent phase than in the convalescent phase, although it is not clear if the increase in DNA results from replication of latent viruses or by exogenous infection (2). Moreover, mononuclear phagocytes (human and murine) are ex vivo–susceptible to HSV-1 infection with varied permissivity, which seems to be related to the particular differentiation state in which monocytes are restrictive (9,12,13). In this report, we readdress the issue of HSV-1 persistence in macrophages. Due to the intrinsic heterogeneity of macrophages from primary cultures as hosts for virus persistence, we selected a well-defined macrophage-like cell line, P388D1, which displays a number of macrophage characteristics and is considered as a mature macrophage (14). We describe here the establishment and characterization of a stable, productive, persistently infected macrophage culture that has been maintained for more than 2 years. Viral persistence was confirmed and monitored by measuring extracellular virus titer, presence of cell-viral antigen, and macrophage production of infectious viruses. In addition, the phenotype of viruses obtained from the persistently infected culture was determined together with the presence of viral and host proteins in the persistent culture and in HSV1-infected macrophages.
Materials and Methods Virus. Herpes simplex virus type 1 (HSV-1) macroplaque strain (MP), originally obtained from Dr. B. Roizman (15), was used because of its large plaque size. Viruses obtained from passages 47 and 60 of the persistently infected culture were named HSVA and HSVB, respectively. Viral propagation and titration were carried out by standard techniques in Vero cells (16). Cell cultures. P388D1 cells, a continuous DBA/2 mouse macrophage-like cell line (ATCC TIB 63) (17), courtesy of Dr. A. Tingle, University of British Columbia, Vancouver, Canada, were grown as a monolayer in RPMI 1640 medium (Gibco BRL, Inc., Gaithersburg, MD, USA) supplemented with 10% heat-inactivated (568C/30 min) fetal bovine serum (FBS) (Bioexport, México, D.F., México), antibiotics (100 ui/mL penicillin, 100 mg/mL streptomycin) (Sigma
Chemical Co., St. Louis, MO, USA), and 0.00001% b-mercaptoethanol (Sigma Chemical Co., complete medium). Vero cells originally from ATCC (CCL 81) were used and maintained in MEM medium (Gibco BRL) supplemented with antibiotics, 10 mg/mL amphotericin B (Sigma Chemical) and 5% heat-inactivated FBS. Establishment of persistence. Persistently infected cultures were obtained by the infection of macrophage monolayers using HSV-1 at multiplicity of infection (MOI) 0.001–0.1 after being washed with PBS. Non-adsorbed virus was removed after 1 h of adsorption at 378C in a 5%-CO2 humid atmosphere. Subsequently, RPMI 1640 medium was added, with antibiotics but without serum. The cultures were maintained for 7 days with a daily change of the medium. Thereafter, the cells were subcultured every 2–3 days at a split ratio of 1:2 with a daily change of complete medium. At each passage, the supernatant was collected for virus titration. Immunofluorescence assay. Cell-viral antigens were detected by direct immunofluorescence (IF). The Syva Microtrak HSV1/HSV2 Culture Identification/Typing Test Kit (Syva Co., Palo Alto, CA, USA) was used using the technique recommended by the manufacturer. The percentage of fluorescent cells was determined by observing three separate slides of the same passage. A minimum of 100 cells was observed and an average was calculated. Infectious center assay. Cells releasing infectious virus were detected by the infectious center assay. This was carried out on a monolayer of Vero cells grown on 24-well microtiter plates (Costar, Cambridge, MA, USA). Persistently infected cells were extensively washed (.20 times) with PBS to eliminate extracellular virus, and were later detached from the surface of the Petri dish with a pipette. Subsequently, a serial three-fold limiting dilution of the cell suspension was made with RPMI 1640 medium containing 1% FBS. One hundred microliters of each dilution were delivered per well. Thereafter, 200 mL of MEM medium with 1% FBS and 0.7% agar (Difco Laboratories, Detroit, MI, USA) were added and incubated for 5 days at 378C. The cultures were MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide: thiazolyl blue (Sigma Chemical) stained or fixed with 3% formaldehyde in PBS and stained with crystal violet. The plaques were counted and the percentage of cells releasing infectious virus was calculated from an average of three determinations of the same passage. Plaque size and cytopathic effect. Vero cell monolayers were infected with ten-fold serial dilutions of HSV-1, HSVA, or HSVB. Non-adsorbed virus was removed after 1 h of adsorption at 378C, and medium with 0.7% agar was added. Cultures were incubated for 5 days and fixed with 3% formaldehyde in PBS and stained with crystal violet. The cytopathic effect
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Figure 1. Extracellular virus titers of HSV-1-persistently infected macrophage-like culture (P388D1per).
(CPE) in Vero and P388D1 cells infected at a MOI of 1.0 was examined after 48 h of infection under the microscope. Preparation of cellular extracts. Persistent acute infected (MOI 1 at 48 h post-infection) or non-infected cells were extensively washed with PBS and then resuspended in 100 mL of lysis buffer (10 mM Tris-HCl pH 7.5; 140 mM NaCl; 5 mM EDTA; 1% Triton X-100 and 1% sodium deoxycholate) and centrifuged at 12,000 3 g for 5 min. The supernatants were kept at 2208C until used. Protein content was measured by the method of Lowry (18). Polyacrylamide gel electrophoresis of radioactively labeled cellular extracts. Radioactively labeled cellular extract analysis was performed essentially as described elsewhere (19). Control cells, acute infected cells (MOI of 1.0, 24 h postinfection), and persistent cell cultures were incubated for 2 h at 378C in a methionine-free MEM medium (Gibco BRL). Then, 100 mCi/mL of Tran35SLabel (ICN Pharmaceuticals, Inc., Costa Mesa, CA, USA) was added. After 4 h, the cellular extracts were prepared and separated by 10% SDS-PAGE electrophoresis (40 mg/well). The radioactive proteins were detected by autoradiography. For determination of apparent molecular weights (MW), protein markers (Bio-Rad Laboratories, Inc., Richmond, CA, USA) were included and autoradiograms were scanned in a Bio-Rad densitometer Model GS-670 (Bio-Rad Labora-
tories). Data were analyzed with the computer-aided Molecular Analyst program (Bio-Rad Laboratories).
Results Permissivity of P388D1 to HSV-1 infection. Permissivity of P388D1 was evaluated by infecting the cultures at MOI
Figure 2. Direct fluorescent antibody staining of P388D1 persistently infected cells.
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Table 1. Percentage of P388D1per cells capable of relasing infectious virus and expressing viral antigens
Passage 63 64 66 68 70 71 72 74 75
Extracellular virus titer (PFU/ml)
Cells releasinga infectious virus (%)
Cells expressingb viral antigens (%)
1.6 3 104 6.3 3 101 1.0 3 104 5.0 3 102 3.2 3 105 6.3 3 104 5.0 3 104 3.9 3 104 5.0 3 104
2.6 6 0.40 0.008 6 0.002 ND 0.05 6 0.03 11.0 6 0.50 5.6 6 0.45 9.0 6 0.30 7.9 6 0.72 12.5 6 0.64
ND ND 15.0 6 0.80 1.0 6 0.42 10.0 6 0.62 ND 7.8 6 0.38 4.5 6 0.30 ND
a
Determined by infectious center assay. b Determined by direct immuno fluorescence. ND: not determined.
from 0.001–1.0, determining infectious extracellular viruses and culture survival time. The highest titer value (105.6 TCID50/mL) and the shortest survival time (3 days) was obtained with MOI of 1; at a lower MOI, a decrease in virus titer with a concomitant increase of monolayer survival time was observed. Viral titers of 104.5, 104, and 103 TCID50/mL and survival times of 4, 8 and 12 days were obtained with MOI of 0.1, 0.01, and 0.001, respectively. However, at MOI of 1.0 viral, yields in macrophages were lower than in permissive cells (Vero). Titers of 105.6 vs. 109 TCID50/mL were obtained, respectively.
Establishment of persistently infected cultures. To obtain persistently infected cultures, P388D1 cells were infected at MOI from 0.001–0.1, surviving cells were additionally propagated, and viral persistence was confirmed. Cultures infected at MOI higher than 0.001 survived few passages; on the contrary, when the infection was at MOI of 0.001, a high percentage of the macrophages survived and were able to be further propagated. In these cultures, the presence of HSV-1 was confirmed; one (P388D1per) has been subcultured for more than 2 years (75 passages). Persistently infected cultures were highly reproducible by infecting at low MOI.
Figure 3. Plaque size on Vero cells of HSV-1 and virus isolated from persistently infected cells. Vero monolayers 5 days after inoculation with (A) HSV-1, (B) HSVA, and (C) HSVB.
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Confirmation of HSV-1 persistence in P388D1per. HSV-1 persistence was verified and monitored by determining extracellular virus production, presence of cell-viral antigen, and macrophage producing infectious viruses. The pattern of extracellular virus yield vs. passages given by P388D1per is shown in Figure 1. Throughout the passages of the culture, viruses with cyclic fluctuating titers from 106 TCID50/mL to no detectable infectious particles were produced. Macrophages in P338D1per propagated as effectively as in non-infected cells except for periods of crises, which were followed by the highest viral titers and characterized by extensive cytopathic effect, cell lysis, and drastic decrease in viable cells. When more than 200 cells survived, the culture could be propagated further. The first passages (1–20) were characterized by severe crises; thereafter, the crises became less severe. Nevertheless, another severe crisis occurred after passage 73. Percentage of cells with cell-viral antigen expression and infectious virus production. The presence of cell-viral antigens and the percentage of cells expressing in P388D1per passage were studied by IF (Figure 2), and the number of
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macrophages that produced infective viruses was analyzed by the infectious center assay. In nine passages that produced extracellular virus with titers fluctuating between 6.3 3 101 to 3.2 3 105 PFU/mL, the number of cells that expressed viral antigens and produced infectious viruses was determined (Table 1). Cells expressing viral antigens varied from 1.0% to 15.0% and macrophages releasing infective viruses, from 0.008% to 12.5%. Characteristics of viruses obtained from P388D1per. Viruses obtained from passages 47 (HSVA) and 60 (HSVB) were characterized by plaque size and cytophatic effect. The plaque sizes on Vero of HSVA and HSVB were smaller than those of the original virus. Their diameter varied from 1.0– 2.5 mm for HSVA, and from 0.5–1.0 mm for HSVB (Figures 3B and 3C). In contrast, the diameter of the plaques obtained with HSV-1 were from 2.0–5.0 mm (Figure 3A). The CPE of HSV-1, HSVA, and HSVB on Vero and P388D1 cells was examined. The CPE caused by the three viral strains on Vero cells differed, as shown in Figure 4. In Vero cells, original HSV-1 presented the characteristic syncytia of the MP strain in a permissive cell line (Figure 4A).
Figure 4. Cytopathic effect on Vero and P388D1 cells of HSV-1 and virus isolated from persistently infected cells. Vero monolayer infected with (A) original HSV-1, (B) HSVA, and (C) HSVB. Macrophage monolayer infected with (D) original HSV-1, (E) HSVA, and (F) HSVB.
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HSVA gave smaller syncytia with fewer fused and rounded cells (Figure 4B). In contrast, the CPE of HSVB was characterized by rounded cells and no visible or distinguishable fused cells (Figure 4C). No great differences in the cytophatic
effect presented by the different viruses were observed in P388D1 cells. HSV-1 produced some syncytia and rounded cells, while HSVA and HSVB presented only rounded cells without syncytia formation (Figures 4D to 4F). When P388D1 was infected with HSV-1, HSVA or HSVB, no significant differences in viral yield were observed. The resulting values were 4.5 3 106, 9 3 105, and 7.6 3 105 PFU/ mL. On the contrary, when the infection was carried out in Vero titers of 2 3 109, 1.4 3 107, and 1.9 3 107 PFU/mL were obtained respectively, implying that during persistence viral infectivity to the permissive host was reduced but remained unchanged to the cells where it persists. Pattern of infected cell proteins in P3881 per cultures and in cells infected with HSV-1. Protein synthesis of HSV-1 infected P388D1 cells and in P388D1per culture was determined by metabolic labeling with [35S]-methionine. Infected cell proteins (ICP) in a MW range from 25–151 kDa were observed, 12 in P388D1per culture (Figure 5, lane 2), and 14 in P388D1 infected with HSV-1 (Figure 5, lane 3). In addition, a higher reduction in the synthesis of cellular proteins was observed in HSV-1–infected cells than in noninfected cells and P388D1per (Figure 4, lanes 1–3). Discussion
Figure 5. Autoradiograms of the electrophoretic profiles of [35S]-methionine labeled cellular extracts of persistent infection and lytic infection with viruses isolated during persistence. Mock infection (lane 1); P388D11 HSV-1 (lane 2); P388D1per (lane 3). Molecular weight of protein standards is shown on the left.
Macrophages play an important role in the control of herpes virus infection (6) and might be the reservoir for HSV-1 persistence in humans. Therefore, the study of the HSV-1-macrophage interaction is important. Immortalized macrophage-like cell lines are a convenient system to study in vitro interactions between HSV-1 and macrophage, i.e., persistence. HSV-1 persistence in macrophage cell lines has been documented in vitro: in a promonocyte cell line, which produced infective viruses by differentiation (9), and in a macrophage-like cell line, a virus-productive culture which spontaneously shifted from productive to non-productive (11). The HSV-1 stable productive persistently infected culture described here was obtained in a murine (P388D1) cell line in which the viral yield was lower than in Vero monolayer. These P388D1 characteristics and the conditions used to establish the culture favored viral persistence, low MOI, and subculturing 7 days post-infection. HSV-1 persistence in cell lines can be increased by maintaining the infected culture without subculturing for longer periods (20). Therefore, it is likely that the virus, which persisted in the culture, emerged after various rounds of replication in macrophages. The pattern observed in our culture of a sharp rise in virus titer with a subsequent decrease in the number of viable cells (Figure 1) is frequently observed in HSV-1 persistently infected cell cultures (21–24). We observed a similar pattern in a persistently infected culture of the same cell line, although the crises were less severe and after 40 to 45 passages, infectious viruses ceased to be produced (11). In contrast, HSV-1 persistence in human B and T cell lines is
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not characterized by such periods of enhanced viral replication and cytopathic effect (25). The cyclic nature of virus production with concomitant macrophage death may be explained by two coexisting populations of cells in the culture, one persistently infected, and the other non-infected but susceptible of being infected and lysed with a subsequent production of highly infectious virus titer. Surviving cells were mostly persistently infected, therefore resistant to reinfection (20), and few were noninfected. Consequently, some passages were required to reach the ratio between non-infected/persistently infected cells to originate a crisis. Virus isolated from P388D1per passages 47 and 60 showed phenotype changes with respect to HSV-1. In Vero monolayers (permissive host), they exhibited restricted growth (smaller plaque size, Figure 3) and different cytopathic effect; while HSV-1 gave the characteristic syncytia of a syn phenotype; in contrast, the phenotypes of HSVB and HSVA were clearly syn1 or undefined (Figure 4) (26). Our results agree with the reported phenotypic changes in HSV-1 obtained from human T lymphoblastoid, Chinese hamster, MDBK, Raji, and BJAB cell lines persistently infected (20,22,23,27). The observed difference in P388D1 protein synthesis in infected cells HSV-1 vs. P388D1per implies that HSV-1 did not shut off host protein synthesis in the persistently infected culture. This finding is congruent with the report on neuronal cells, where no reduction in protein synthesis was observed after HSV-1 infection (28). This effect might favor establishment and maintenance of HSV-1 persistence. Although specific HSV-1 proteins were present in P388D1per and in HSV-1-infected P388D1, they cannot be identified by their electrophoretic mobility. Their migration is influenced by factors such as viral strain, virus propagation history (host cell and rounds of replication), and assay conditions. Moreover, 11 of the 37 HSV-1 proteins expressed in cell cultures are glycosidic; their apparent MW varies with the number of radicals which, in turn, differ from the MW of the host used to propagate the virus (29,30). Viral proteins can be identified with monoclonal antibodies, although it should be kept in mind that modifications in antigen epitopes alter the recognition by the monoclonal antibodies (31). Studies designed to identify ICP expressed in the P388D1per culture and their changes are in progress. A persistent HSV-1 infection in murine macrophages allows studies using cells that are important in disseminating the infection. Furthermore, a large number of cellular and humoral reagents available for the study of viral immunity in mice can be used.
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ORIGINAL ARTICLE
Characteristics of a Macrophage Culture Persistently Infected with Herpes Simplex Virus Type 1 Jaime Bustos*,** and Beatríz Gómez * *Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), México, D.F., Mexico **Departamento de Atención a la Salud, Universidad Autónoma Metropolitana (UAM)-Xochimilco, México, D.F., Mexico Received for publication October 19, 1998, accepted April 6, 1999 (98/133).
Background. Persistence of herpes simplex type 1 (HSV-1) has been reported in sensory neurons, corneal epithelium, and lymphocytes, although other cell types such as macrophages should also be considered as hosts for HSV-1 persistence. Here we report the establishment and characterization of HSV-1 persistence in an immortalized murine macrophage-like cell line (P388D1). Methods. The persistently HSV-1 infected culture (P388D1per) was obtained from surviving P388D1 macrophages infected with HSV-1 MP strain at multiplicity of 0.001. P388D1per was characterized by: extracellular production of viruses, cells expressing viral antigens, and cells releasing infectious viruses. Viral plaque size and cytophatic effect were determined in viruses (HSVA and HSVB) obtained from two different P388D1per passages. Host and viral proteins were detected in P388D1per and in P388D1 cells infected with HSV-1 by metabolic [35S]-methionine labeling assays. Results. P388D1per culture was characterised by cyclic production of infectious viruses from non-detectable to 106 TCID50/mL; from 1.0 to 15.0% cells expressing viral antigens and macrophages released infectious viruses from 0.008 to 12.5%. Differences in viral plaque size and cytopathic effect morphology between HSVA, HSVB and HSV-1 were observed. Similar patterns of viral proteins were observed in P388D1per and in P388D1 infected with HSV-1. Nonetheless, the characteristic interference effect of HSV-1 on host protein synthesis was not observed in P388D1per culture. Conclusions. An HSV-1 persistently infected immortalized macrophage culture was established and characterized. Virus produced during persistence showed phenotypic alterations with respect to the original virus. P388D1per cell protein synthesis was not affected by the presence of HSV-1. © 1999 IMSS. Published by Elsevier Science Inc. Key Words: P388D1, HSV-1, Persistence, Virus phenotypic alterations.
Introduction A remarkable feature of herpes simplex virus type 1 (HSV-1) is its ability to persist in the natural host (human) after primary infection in a latent or subdetectable form (1). Persistence of HSV-1 has been documented in patients with recurrent HSV-1 infections, herpes keratitis, and urogenital
Address reprint requests to: Dra. Beatríz Gómez, Departamento de Microbiología y Parasitología, Facultad de Medicina, UNAM. México, D.F. 04510, México. Tel.: (1525) 6-23-24-69; FAX: (1525) 6/23-23-86; E-mail: begomez @ servidor.unam.mx
tumors (2–4). Nonetheless, the sites where HSV-1 maintains itself in the human organism vary with the clinical manifestations. Until now, persistence of the virus has been reported in neurons, corneal epithelium, and lymphocytes (3–5), although other cell types such as macrophages should also be considered as hosts for HSV-1 persistence. Macrophages are widely distributed in the body, are important effector cells in host responses to virus infection, and participate in both innate and acquired immunity. Their presence in major organs, tissues, serous spaces and sites of inflammation suggests that they are the first immune cells encountered by the invading virus (6). In addition, microphages are among the cell types selected for the persistence of
0188-0128/99 $–see front matter. Copyright © 1999 IMSS. Published by Elsevier Science Inc. PII S0188-0128(99)00 0 2 1 - 4
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DNA and RNA (7); therefore, it is likely that HSV-1 can remain in these cells. Persistence of HSV-1 in human mononuclear cells has not been documented, although circumstantial evidence suggests that the virus can maintain itself in these cells. HSV-1 persistence has been reported ex vivo in a primary murine spleen macrophage culture obtained from mice resistant to infection (8) and in vitro in a promonocyte cell line (9,10), and in a macrophage-like cell line (11). In cells from patients with recurrent HSV-1 infections, higher concentration of HSV-1 DNA are present in the recrudescent phase than in the convalescent phase, although it is not clear if the increase in DNA results from replication of latent viruses or by exogenous infection (2). Moreover, mononuclear phagocytes (human and murine) are ex vivo–susceptible to HSV-1 infection with varied permissivity, which seems to be related to the particular differentiation state in which monocytes are restrictive (9,12,13). In this report, we readdress the issue of HSV-1 persistence in macrophages. Due to the intrinsic heterogeneity of macrophages from primary cultures as hosts for virus persistence, we selected a well-defined macrophage-like cell line, P388D1, which displays a number of macrophage characteristics and is considered as a mature macrophage (14). We describe here the establishment and characterization of a stable, productive, persistently infected macrophage culture that has been maintained for more than 2 years. Viral persistence was confirmed and monitored by measuring extracellular virus titer, presence of cell-viral antigen, and macrophage production of infectious viruses. In addition, the phenotype of viruses obtained from the persistently infected culture was determined together with the presence of viral and host proteins in the persistent culture and in HSV1-infected macrophages.
Materials and Methods Virus. Herpes simplex virus type 1 (HSV-1) macroplaque strain (MP), originally obtained from Dr. B. Roizman (15), was used because of its large plaque size. Viruses obtained from passages 47 and 60 of the persistently infected culture were named HSVA and HSVB, respectively. Viral propagation and titration were carried out by standard techniques in Vero cells (16). Cell cultures. P388D1 cells, a continuous DBA/2 mouse macrophage-like cell line (ATCC TIB 63) (17), courtesy of Dr. A. Tingle, University of British Columbia, Vancouver, Canada, were grown as a monolayer in RPMI 1640 medium (Gibco BRL, Inc., Gaithersburg, MD, USA) supplemented with 10% heat-inactivated (568C/30 min) fetal bovine serum (FBS) (Bioexport, México, D.F., México), antibiotics (100 ui/mL penicillin, 100 mg/mL streptomycin) (Sigma
Chemical Co., St. Louis, MO, USA), and 0.00001% b-mercaptoethanol (Sigma Chemical Co., complete medium). Vero cells originally from ATCC (CCL 81) were used and maintained in MEM medium (Gibco BRL) supplemented with antibiotics, 10 mg/mL amphotericin B (Sigma Chemical) and 5% heat-inactivated FBS. Establishment of persistence. Persistently infected cultures were obtained by the infection of macrophage monolayers using HSV-1 at multiplicity of infection (MOI) 0.001–0.1 after being washed with PBS. Non-adsorbed virus was removed after 1 h of adsorption at 378C in a 5%-CO2 humid atmosphere. Subsequently, RPMI 1640 medium was added, with antibiotics but without serum. The cultures were maintained for 7 days with a daily change of the medium. Thereafter, the cells were subcultured every 2–3 days at a split ratio of 1:2 with a daily change of complete medium. At each passage, the supernatant was collected for virus titration. Immunofluorescence assay. Cell-viral antigens were detected by direct immunofluorescence (IF). The Syva Microtrak HSV1/HSV2 Culture Identification/Typing Test Kit (Syva Co., Palo Alto, CA, USA) was used using the technique recommended by the manufacturer. The percentage of fluorescent cells was determined by observing three separate slides of the same passage. A minimum of 100 cells was observed and an average was calculated. Infectious center assay. Cells releasing infectious virus were detected by the infectious center assay. This was carried out on a monolayer of Vero cells grown on 24-well microtiter plates (Costar, Cambridge, MA, USA). Persistently infected cells were extensively washed (.20 times) with PBS to eliminate extracellular virus, and were later detached from the surface of the Petri dish with a pipette. Subsequently, a serial three-fold limiting dilution of the cell suspension was made with RPMI 1640 medium containing 1% FBS. One hundred microliters of each dilution were delivered per well. Thereafter, 200 mL of MEM medium with 1% FBS and 0.7% agar (Difco Laboratories, Detroit, MI, USA) were added and incubated for 5 days at 378C. The cultures were MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide: thiazolyl blue (Sigma Chemical) stained or fixed with 3% formaldehyde in PBS and stained with crystal violet. The plaques were counted and the percentage of cells releasing infectious virus was calculated from an average of three determinations of the same passage. Plaque size and cytopathic effect. Vero cell monolayers were infected with ten-fold serial dilutions of HSV-1, HSVA, or HSVB. Non-adsorbed virus was removed after 1 h of adsorption at 378C, and medium with 0.7% agar was added. Cultures were incubated for 5 days and fixed with 3% formaldehyde in PBS and stained with crystal violet. The cytopathic effect
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Figure 1. Extracellular virus titers of HSV-1-persistently infected macrophage-like culture (P388D1per).
(CPE) in Vero and P388D1 cells infected at a MOI of 1.0 was examined after 48 h of infection under the microscope. Preparation of cellular extracts. Persistent acute infected (MOI 1 at 48 h post-infection) or non-infected cells were extensively washed with PBS and then resuspended in 100 mL of lysis buffer (10 mM Tris-HCl pH 7.5; 140 mM NaCl; 5 mM EDTA; 1% Triton X-100 and 1% sodium deoxycholate) and centrifuged at 12,000 3 g for 5 min. The supernatants were kept at 2208C until used. Protein content was measured by the method of Lowry (18). Polyacrylamide gel electrophoresis of radioactively labeled cellular extracts. Radioactively labeled cellular extract analysis was performed essentially as described elsewhere (19). Control cells, acute infected cells (MOI of 1.0, 24 h postinfection), and persistent cell cultures were incubated for 2 h at 378C in a methionine-free MEM medium (Gibco BRL). Then, 100 mCi/mL of Tran35SLabel (ICN Pharmaceuticals, Inc., Costa Mesa, CA, USA) was added. After 4 h, the cellular extracts were prepared and separated by 10% SDS-PAGE electrophoresis (40 mg/well). The radioactive proteins were detected by autoradiography. For determination of apparent molecular weights (MW), protein markers (Bio-Rad Laboratories, Inc., Richmond, CA, USA) were included and autoradiograms were scanned in a Bio-Rad densitometer Model GS-670 (Bio-Rad Labora-
tories). Data were analyzed with the computer-aided Molecular Analyst program (Bio-Rad Laboratories).
Results Permissivity of P388D1 to HSV-1 infection. Permissivity of P388D1 was evaluated by infecting the cultures at MOI
Figure 2. Direct fluorescent antibody staining of P388D1 persistently infected cells.
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Table 1. Percentage of P388D1per cells capable of relasing infectious virus and expressing viral antigens
Passage 63 64 66 68 70 71 72 74 75
Extracellular virus titer (PFU/ml)
Cells releasinga infectious virus (%)
Cells expressingb viral antigens (%)
1.6 3 104 6.3 3 101 1.0 3 104 5.0 3 102 3.2 3 105 6.3 3 104 5.0 3 104 3.9 3 104 5.0 3 104
2.6 6 0.40 0.008 6 0.002 ND 0.05 6 0.03 11.0 6 0.50 5.6 6 0.45 9.0 6 0.30 7.9 6 0.72 12.5 6 0.64
ND ND 15.0 6 0.80 1.0 6 0.42 10.0 6 0.62 ND 7.8 6 0.38 4.5 6 0.30 ND
a
Determined by infectious center assay. b Determined by direct immuno fluorescence. ND: not determined.
from 0.001–1.0, determining infectious extracellular viruses and culture survival time. The highest titer value (105.6 TCID50/mL) and the shortest survival time (3 days) was obtained with MOI of 1; at a lower MOI, a decrease in virus titer with a concomitant increase of monolayer survival time was observed. Viral titers of 104.5, 104, and 103 TCID50/mL and survival times of 4, 8 and 12 days were obtained with MOI of 0.1, 0.01, and 0.001, respectively. However, at MOI of 1.0 viral, yields in macrophages were lower than in permissive cells (Vero). Titers of 105.6 vs. 109 TCID50/mL were obtained, respectively.
Establishment of persistently infected cultures. To obtain persistently infected cultures, P388D1 cells were infected at MOI from 0.001–0.1, surviving cells were additionally propagated, and viral persistence was confirmed. Cultures infected at MOI higher than 0.001 survived few passages; on the contrary, when the infection was at MOI of 0.001, a high percentage of the macrophages survived and were able to be further propagated. In these cultures, the presence of HSV-1 was confirmed; one (P388D1per) has been subcultured for more than 2 years (75 passages). Persistently infected cultures were highly reproducible by infecting at low MOI.
Figure 3. Plaque size on Vero cells of HSV-1 and virus isolated from persistently infected cells. Vero monolayers 5 days after inoculation with (A) HSV-1, (B) HSVA, and (C) HSVB.
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Confirmation of HSV-1 persistence in P388D1per. HSV-1 persistence was verified and monitored by determining extracellular virus production, presence of cell-viral antigen, and macrophage producing infectious viruses. The pattern of extracellular virus yield vs. passages given by P388D1per is shown in Figure 1. Throughout the passages of the culture, viruses with cyclic fluctuating titers from 106 TCID50/mL to no detectable infectious particles were produced. Macrophages in P338D1per propagated as effectively as in non-infected cells except for periods of crises, which were followed by the highest viral titers and characterized by extensive cytopathic effect, cell lysis, and drastic decrease in viable cells. When more than 200 cells survived, the culture could be propagated further. The first passages (1–20) were characterized by severe crises; thereafter, the crises became less severe. Nevertheless, another severe crisis occurred after passage 73. Percentage of cells with cell-viral antigen expression and infectious virus production. The presence of cell-viral antigens and the percentage of cells expressing in P388D1per passage were studied by IF (Figure 2), and the number of
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macrophages that produced infective viruses was analyzed by the infectious center assay. In nine passages that produced extracellular virus with titers fluctuating between 6.3 3 101 to 3.2 3 105 PFU/mL, the number of cells that expressed viral antigens and produced infectious viruses was determined (Table 1). Cells expressing viral antigens varied from 1.0% to 15.0% and macrophages releasing infective viruses, from 0.008% to 12.5%. Characteristics of viruses obtained from P388D1per. Viruses obtained from passages 47 (HSVA) and 60 (HSVB) were characterized by plaque size and cytophatic effect. The plaque sizes on Vero of HSVA and HSVB were smaller than those of the original virus. Their diameter varied from 1.0– 2.5 mm for HSVA, and from 0.5–1.0 mm for HSVB (Figures 3B and 3C). In contrast, the diameter of the plaques obtained with HSV-1 were from 2.0–5.0 mm (Figure 3A). The CPE of HSV-1, HSVA, and HSVB on Vero and P388D1 cells was examined. The CPE caused by the three viral strains on Vero cells differed, as shown in Figure 4. In Vero cells, original HSV-1 presented the characteristic syncytia of the MP strain in a permissive cell line (Figure 4A).
Figure 4. Cytopathic effect on Vero and P388D1 cells of HSV-1 and virus isolated from persistently infected cells. Vero monolayer infected with (A) original HSV-1, (B) HSVA, and (C) HSVB. Macrophage monolayer infected with (D) original HSV-1, (E) HSVA, and (F) HSVB.
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HSVA gave smaller syncytia with fewer fused and rounded cells (Figure 4B). In contrast, the CPE of HSVB was characterized by rounded cells and no visible or distinguishable fused cells (Figure 4C). No great differences in the cytophatic
effect presented by the different viruses were observed in P388D1 cells. HSV-1 produced some syncytia and rounded cells, while HSVA and HSVB presented only rounded cells without syncytia formation (Figures 4D to 4F). When P388D1 was infected with HSV-1, HSVA or HSVB, no significant differences in viral yield were observed. The resulting values were 4.5 3 106, 9 3 105, and 7.6 3 105 PFU/ mL. On the contrary, when the infection was carried out in Vero titers of 2 3 109, 1.4 3 107, and 1.9 3 107 PFU/mL were obtained respectively, implying that during persistence viral infectivity to the permissive host was reduced but remained unchanged to the cells where it persists. Pattern of infected cell proteins in P3881 per cultures and in cells infected with HSV-1. Protein synthesis of HSV-1 infected P388D1 cells and in P388D1per culture was determined by metabolic labeling with [35S]-methionine. Infected cell proteins (ICP) in a MW range from 25–151 kDa were observed, 12 in P388D1per culture (Figure 5, lane 2), and 14 in P388D1 infected with HSV-1 (Figure 5, lane 3). In addition, a higher reduction in the synthesis of cellular proteins was observed in HSV-1–infected cells than in noninfected cells and P388D1per (Figure 4, lanes 1–3). Discussion
Figure 5. Autoradiograms of the electrophoretic profiles of [35S]-methionine labeled cellular extracts of persistent infection and lytic infection with viruses isolated during persistence. Mock infection (lane 1); P388D11 HSV-1 (lane 2); P388D1per (lane 3). Molecular weight of protein standards is shown on the left.
Macrophages play an important role in the control of herpes virus infection (6) and might be the reservoir for HSV-1 persistence in humans. Therefore, the study of the HSV-1-macrophage interaction is important. Immortalized macrophage-like cell lines are a convenient system to study in vitro interactions between HSV-1 and macrophage, i.e., persistence. HSV-1 persistence in macrophage cell lines has been documented in vitro: in a promonocyte cell line, which produced infective viruses by differentiation (9), and in a macrophage-like cell line, a virus-productive culture which spontaneously shifted from productive to non-productive (11). The HSV-1 stable productive persistently infected culture described here was obtained in a murine (P388D1) cell line in which the viral yield was lower than in Vero monolayer. These P388D1 characteristics and the conditions used to establish the culture favored viral persistence, low MOI, and subculturing 7 days post-infection. HSV-1 persistence in cell lines can be increased by maintaining the infected culture without subculturing for longer periods (20). Therefore, it is likely that the virus, which persisted in the culture, emerged after various rounds of replication in macrophages. The pattern observed in our culture of a sharp rise in virus titer with a subsequent decrease in the number of viable cells (Figure 1) is frequently observed in HSV-1 persistently infected cell cultures (21–24). We observed a similar pattern in a persistently infected culture of the same cell line, although the crises were less severe and after 40 to 45 passages, infectious viruses ceased to be produced (11). In contrast, HSV-1 persistence in human B and T cell lines is
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not characterized by such periods of enhanced viral replication and cytopathic effect (25). The cyclic nature of virus production with concomitant macrophage death may be explained by two coexisting populations of cells in the culture, one persistently infected, and the other non-infected but susceptible of being infected and lysed with a subsequent production of highly infectious virus titer. Surviving cells were mostly persistently infected, therefore resistant to reinfection (20), and few were noninfected. Consequently, some passages were required to reach the ratio between non-infected/persistently infected cells to originate a crisis. Virus isolated from P388D1per passages 47 and 60 showed phenotype changes with respect to HSV-1. In Vero monolayers (permissive host), they exhibited restricted growth (smaller plaque size, Figure 3) and different cytopathic effect; while HSV-1 gave the characteristic syncytia of a syn phenotype; in contrast, the phenotypes of HSVB and HSVA were clearly syn1 or undefined (Figure 4) (26). Our results agree with the reported phenotypic changes in HSV-1 obtained from human T lymphoblastoid, Chinese hamster, MDBK, Raji, and BJAB cell lines persistently infected (20,22,23,27). The observed difference in P388D1 protein synthesis in infected cells HSV-1 vs. P388D1per implies that HSV-1 did not shut off host protein synthesis in the persistently infected culture. This finding is congruent with the report on neuronal cells, where no reduction in protein synthesis was observed after HSV-1 infection (28). This effect might favor establishment and maintenance of HSV-1 persistence. Although specific HSV-1 proteins were present in P388D1per and in HSV-1-infected P388D1, they cannot be identified by their electrophoretic mobility. Their migration is influenced by factors such as viral strain, virus propagation history (host cell and rounds of replication), and assay conditions. Moreover, 11 of the 37 HSV-1 proteins expressed in cell cultures are glycosidic; their apparent MW varies with the number of radicals which, in turn, differ from the MW of the host used to propagate the virus (29,30). Viral proteins can be identified with monoclonal antibodies, although it should be kept in mind that modifications in antigen epitopes alter the recognition by the monoclonal antibodies (31). Studies designed to identify ICP expressed in the P388D1per culture and their changes are in progress. A persistent HSV-1 infection in murine macrophages allows studies using cells that are important in disseminating the infection. Furthermore, a large number of cellular and humoral reagents available for the study of viral immunity in mice can be used.
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