Human Cytomegalovirus

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1975, p Copyright ) 1975 American Society for Microbiology Vol. 2, No. 4 Printed in U.S.A. Demonstration of Immunoglobulin G Receptors Induced by Human Cytomegalovirus TORU FURUKAWA,* ELIZABETH HORNBERGER, SADATOSHI SAKUMA, AND STANLEY A. PLOTKIN The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 1914 Received for publication 11 July 1975 Human cytomegalovirus induced a new immunoglobulin G receptor in human fibroblasts. The immunoglobulin G receptor was well localized in the perinuclear region at 48 h postinfection, and antiviral agents blocked its synthesis. The immunoglobulin G receptor bound immunoglobulin G of man and several other species. It may be a source of error in the performance of indirect fluorescence tests for human cytomegalovirus antibody. Human cytomegalovirus (HCMV) infection induces various morphological and biochemical changes in cells (2, 7). The most distinct morphological change in infected human fibroblasts is the formation of nuclear and cytoplasmic inclusion bodies. The unique nuclear inclusion bodies (NI) have been the most reliable marker of HCMV infection and have been extensively studied by electron microscopy (3, 4). However, little is known concerning the nature of the cytoplasmic inclusion bodies (CI). Many serological methods, including complement fixation (CF), indirect hemagglutination, precipitin, and neutralization tests have become available to measure antibody for HCMV. The indirect immunofluorescent staining technique has been one of the fastest and most sensitive tests for measuring antibody levels and for identifying HCMV. During time course experiments, in which cells were infected with HCMV and stained by immunofluoresence, it was noted that HCMV induced an immunoglobulin G (IgG) receptor on the infected cell. In this paper we describe the characteristics of the IgG receptor and its potential clinical significance. While this work was in progress Keller et al. described a similar phenomenon in a published abstract (R. Keller, R. Pettchel, J. Goldman, and M. Goldman, Abstr. Annu. Meet. Am. Soc. Microbiol., 1975, S264, p. 257). MATERIALS AND METHODS Virus. The Towne strain of HCMV was used in these experiments. The history of this strain has been described elsewhere (1). Cells. WI-38 human diploid cells obtained from L. Hayflick were used between the 2th and 3th passage levels. The cells were grown in Eagle minimal essential medium (MEM) supplemented for growth or maintenance with 1% or 2% fetal calf serum, respectively. Infectivity assay. The plaque assay, according to the method of Wentworth and French (1), was used. Inoculated cultures were incubated in a CO2 incubator under a final concentration of.3% agarose. The plaques were counted by microscopic observation 14 days postinfection (PI), after staining with methylene blue. Cytological study. To observe cytopathology, cells were grown on coverslips in petri dishes. After the cells were fixed with Bouin fixative, they were stained with 5% May-Greenwald-Giemsa or hematoxylin eosin. CF test. The CF test was performed using the microtiter technique with 2 U of antigen obtained from Flow Laboratories (Rockville, Md.) and 2 U of complement. Sera. Sera were collected for clinical diagnosis from various sources and stored at -2 C until use. All sera were heat inactivated for 3 min at 57 C. The sera used were from children, newborn to 5 years of age. Cord sera were obtained from the Department of Obstetrics, University of Pennsylvania. Immunofluorescent study. For the immunofluorescent study, cells grown on Lab Tek chamber slides or cover slips were fixed with cold acetone for 1 min and air dried. The following fluorescein isothiocyanate (FITC) conjugates were used: (i) rabbit anti-human gamma globulin prepared by Behring Diagnostics (Laurel Springs, N.J.); (ii) rabbit antihamster gamma globulin prepared by Cappel Laboratories (Downingtown, Pa.); (iii) rabbit anti-guinea pig gamma globulin prepared by Cappel Laboratories; (iv) goat anti-rabbit gamma globulin prepared by Cappel Laboratories; (v) rabbit anti-human IgM globulin prepared by Behring Diagnostics; and (vi) human IgG and human IgM obtained from Cappel Laboratories. Method of fluorescent antigen staining. Either freshly fixed cells or those stored for up to 3 weeks at -2 C were used for fluorescent antibody. Preparations were dipped in phosphate-buffered saline (PBS), drained, and flooded with primary antiserum (HCMV, CF positive; HCMV, CF negative) or PBS. These were incubated at 37 C for 3 min, washed three times with PBS, and flooded with a secondary 332

2 VOL. 2, 1975 IgG RECEPTORS IN HUMAN CYTOMEGALOVIRUS 333 antiserum which was FITC conjugated. After a second 3-min incubation, the preparations were again washed three times with PBS and flooded with.1% Evan blue in deionized water. After 7 min of incubation at room temperature, slides were washed twice with deionized water, air dried, and mounted in Elvanol. The preparations were examined with a Leitz microscope equipped with a HBC 2 mercury lamp and a dry dark field condensor. A primary BG3 filter and a secondary 44 filter were used. Hemadsorption (HA). The method used was essentially that described by Watkins (9) and Furukawa et al. (2a). Briefly, sheep erythrocytes were sensitized by adding.1 ml of rabbit anti-sheep erythrocyte serum to 1. ml of a 4% suspension of sheep erythrocytes in MEM. After incubation at 37 C for 1 h, the erythrocytes were washed three times in PBS and resuspended in MEM to a final concentration of 1%. At various intervals PI, cover slips were washed, and 1% sensitized sheep erythrocytes were added. After incubation for 2 h at 37 C, the cover slips were washed three times with PBS, stained with Giemsa, and examined microscopically. Chemicals. Cytosine arabinoside and cycloheximide were purchased from Nutritional Biochemicals Corp. (Cleveland, Ohio). RESULTS WI-38 cells were infected at a multiplicity of 1 plaque-forming units (PFU) of virus per cell. At various intervals the cultures were assayed for HA, cell-associated infectious virus, and the presence of inclusion bodies. The results of these tests are shown in Fig. 1. Viral replication was evident at about 48 h PI, and cellassociated virus reached a peak 5 days PI. NI were not demonstrated by ordinary staining until 3 days PI. Immunofluorescent staining with CF-positive sera revealed that faint nu- HOURS POST INFECTION FIG. 1. Time course of appearances of HA and immunofluorescent antigens in relation with growth cycle of HCMV. Symbols:, infectivity; A, HA; A, NI immunofluorescence; OI, CI immunofluorescence. clear staining appeared 9 to 24 h PI. At 48 h PI immunofluorescent antigens were seen in the nucleus in a honeycomb-like pattern. Fluorescence was also seen in the perinuclear region. At 72 h PI, antigen was present in pronounced NI and, additionally, in diffusely stained but well-localized perinuclear CI (Fig. 2A). These inclusions were also demonstrated by hematoxylin eosin staining. HA was observed in 6% of the cells at 48 h PI and reached a peak of 78% at 72 h PI. In contrast to these findings with CF-positive sera, only cytoplasmic immunofluorescent staining, coinciding with CI, was seen with CFnegative sera (Fig. 2B). Effect of antiviral agents on cytoplasmic inclusions. Confluent monolayers of WI-38 cells were infected with HCMV at a multiplicity of 1 PFU per cell. After 1 h of adsorption the medium was replaced with MEM containing 2% fetal calf serum and cycloheximide (2,g/ml) or cytosine arabinoside (1 A.tg/ml). At 3 days PI, cells were fixed with acetone and stained using the indirect immunofluorescence technique. In cells treated with either inhibitor the NI were not stained; in addition, CI were not demonstrated with sera either positive or negative for HCMV antibody. In cytosine arabinoside-treated cells, faint diffuse nuclear staining was seen corresponding to the early antigen reported by The et al. (8). Under these conditions (treatment with cytosine arabinoside or cycloheximide) more than 9% of infectious virus production was inhibited (5, 8). These results indicated that CI were induced by HCMV infection and could be inhibited by inhibitors of either DNA or protein synthesis. Antibody distribution among human sera. Selected sera from infants and children, newborn to 5 years of age, and umbilical cord sera were assayed for immunofluorescent staining of CI or NI and by CF tests. All CF-positive sera stained for CI and NI, and the titers varied from 1:8 to 1:32 for NI and 1:22 to 1:528 for CI İn contrast to these findings, CF-negative sera showed only staining of CI. The titers were comparable to those of CF-positive sera (Fig. 3). The immunofluorescent antibody titer for NI was negative when the CF titer was negative. CF-positive sera also had higher staining titers against CI than against NI, but no difference in staining titers for CI was observed between CF-positive and CF-negative sera. These data indicated that "antibody" for CI might be some type of normally existing Ig instead of specific antibody for HCMV. Therefore, the subsequent experiments were carried out to determine the specificity of CI staining.

3 334 FURUKAWA ET AL. J. CLIN. MICROBIOL. Downloaded from FIG. 2. Fluorescent micrograph of infected WI-38 human diploid cells by HCMV (X4). (A) Indirect staining by CF-positive human serum; (B) indirect staining by CF-negative human serum. Infected monolayers on cover slips were fixed with cold acetone at 3 days PI. Immunofluorescent staining was performed with standard CFnegative serum by the indirect method using labeled anti-human IgG and IgM. Direct staining was also performed with labeled human IgG and IgM using PBS instead of intermediate serum. The results are summarized in Table 1. Only anti-igg fluorescent sera stained CI by both the direct and indirect methods. These results suggested that the binding site(s) of CI was an IgG receptor for human sera. The same type ofexperiments were carried out using hamster, rabbit and guinea pig sera. All were positive, indicating that CI had a common receptor for the IgG of different species. The presence of an IgG receptor was also confirmed by a blocking test (Table 2). Fluores- on October 19, 218 by guest

4 VOL. 2, 1975 IgG RECEPTORS IN HUMAN CYTOMEGALOVIRUS 335 LL '32 e a > 32 U z -oj Z L (f, Z -jc O ) w LA- I- < CF TITER Z (I) < Z I-o 8 m, U z Z LLZ FIG. 3. Relationship between CF antibody and immunofluorescent titers ofni and CI. Symbols:, NI;, b TABLE 1. Indirect immunofluorescent staining of inclusion bodies Sera and methods employed Serum 1 Serum 2" I O I- w Cytoplasmic Nuclear Human CF positive Anti-human IgG FITC + + Human CF negative Anti-human IgG FITC + PBSb Human IgG FITC + PBSb Human IgM FITC Human CF negative Anti-human IgM FITC Rabbit serum Anti-rabbit IgG FITC + Guinea pig serum Anti-guinea pig IgG FITC + Hamster serum Anti-hamster IgG FITC + Dilution 1:2. PBS was used instead of serum. Indirect fluorescent staining TABLE 2. Blocking of IgG receptor by IgG globulin Dilution" 1:1 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:124 IgG FITC IgG plus Anti-IgG FITCb IgG plus IgG FITCb " +, Positive staining of CI. b cein-conjugated normal human IgG did stain CI by the direct method but failed to stain after pretreatment of infected cells with IgG-containing serum. This supported the hypothesis that CI had an IgG receptor. DISCUSSION HCMV induced an IgG receptor that appeared to correspond to the CI. The IgG receptor was well localized in the perinuclear region

5 336 FURUKAWA ET AL. and appeared 24 h PI, before infectious virus appeared. Virus inhibitors, such as cytosine arabinoside and cycloheximide, blocked synthesis of the receptor, which suggested that the CI is coded for by virus production. Recently Westmoreland and Watkins (11) reported that herpes simplex virus induced an IgG receptor on the surface of infected cells and that the receptor was specific for the F, fragment of the IgG molecule of several species. Their search for a receptor stemmed from evidence that plasma membranes of infected cells developed the ability to bind sheep erthrocytes sensitized with rabbit anti-sheep erthrocyte serum (11). Previously, we reported the same phenomena in HCMV-infected WI-38 cells (T. Furukawa, S. Tanaka, A. Fioretti, and S. A. Plotkin, 1973, Abstr. Annu. Meet. Am. Soc. Microbiol. 1973, V374, p. 256). The IgG receptor described in this paper was well localized in the perinuclear region and therefore differed from the surface IgG receptor found in herpes simples virus infection (11). These differences could be due either to technical factors or to a difference between the virus. Nevertheless, the findings provided two important pieces of information: (i) the lack of significance of staining of CI in serological diagnosis of HCMV infections, and (ii) the nature of the CI. From the clinical point of view, serological methods such as CF, indirect hemagglutination, precipitin, and neutralization are frequently used for HCMV antibody testing. The immunofluorescence test has been widely used for diagnosis, and a commercial kit is available. However, one might expect misleading results if CI fluorescence is taken as the indicator of the presence of antibody. We have actually observed this type of misreading in a clinical diagnostic laboratory. Previous studies on CI showed the presence of lipid, carbohydrate, deoxyribonucleic acid, and viral antigen. The presence of viral antigen was inferred from immunofluorescence of CI using human serum (5, 6). This inference may not have been sound, although morphological evidence does indeed verify that CI contains virus. These inclusions were comprised ofhomogeneous dense bodies, smooth endoplasmic reticulum, vesicles, and various numbers of viral structures. They were surrounded by a cuff of Golgi apparatus, which in turn was enclosed by lysosomes, rough endoplasmic reticulum, and free ribosomes. Maturation of the virus was J. CLIN. MICROBIOL. observed within these inclusions (3, 4). From our study we could not determine what type of CI corresponded to CI having IgG receptor, but judging from the location and the time of appearance, it could be the C-2 inclusion (3). Further study is in progress to clarify the nature of the IgG receptor protein and its clinical significance with respect to the autoimmune phenomena and to HCMV antibody determination. ACKNOWLEDGMENTS This research was supported by Public Health Service grants AI from the National Institute of Allergy and Infectious Diseases and RR 554 from the Division of Research Resources and by a grant from Smith, Kline & French. LITERATURE CITED 1. Furukawa, T., A. Fioretti, and S. A. Plotkin Growth characteristics of cytomegalovirus in human fibroblasts with demonstration of protein synthesis early in viral replication. J. Virol. 11: Furukawa, T., S. Tanaka, and S. A. Plotkin Stimulation of macromolecular synthesis in guinea pig cells by human CMV. Proc. Soc. Exp. Biol. Med. 148: a. Furukawa, T., S. Tanaka, and S. A. Plotkin Inhibition of human cytomegalovirus by rifampin. J. Gen. Virol. 28: Kanich, R. E., and J. E. Craighead Human cytomegalovirus infection of cultured fibroblasts. I. Cytopathologic effects induced by an adapted and a wild strain. Lab. Invest. 27: Kanich, R. E., and J. E. Craighead Human cytomegalovirus infection of culture fibroblasts. II. Viral replicative sequence of a wild and an adapted strain. Lab. Invest. 27: McAllister, R., J. E. Filbert, and C. R. Goodheart Human cytomegalovirus. Studies on the mechanism of viral cytopathology and inclusion body formation. Proc. Soc. Exp. Biol. Med. 124: McAllister, R. M., R. M. Straw, J. E. Filbert, and C. R. Goodheart Human cytomegalovirus, cytochemical observations of intracellular lesion development correlated with viral synthesis and release. Virology 19: Tanaka, S., T. Furukawa, and S. A. Plotkin Human cytomegalovirus stimulates host cell RNA synthesis. J. Virol. 15: The, T. H., G. Klein, and M. M. A. C. Langenhuysen Antibody reaction to virus-specific early antigens (EA) in patients with cytomegalovirus (CMV) infection. Clin. Exp. Immunol. 16: Watkins, J. F Adsorption of sensitized sheep erythrocyte to HeLa cells infected with herpes simplex virus. Nature (London) 22: Wentworth, B. B., and L. French Plaque assay of cytomegalovirus strains of human origin. Proc. Soc. Exp. Biol. Med. 135: Westmoreland, D., and J. F. Watkins The IgG receptor induced by herpes simplex virus: studies using radioiodinated IgG. J. Gen. Virol. 24: