Component(s) of Sendai Virus That Can Induce Interferon in Mouse Spleen Cells

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1 INFECTION AND IMMUNITY, Mar. 1983, P /83/ $02.00/0 Copyright C 1983, American Society for Microbiology Vol. 39, No. 3 Component(s) of Sendai Virus That Can Induce Interferon in Mouse Spleen Cells YASUHIKO ITOlt* AND YASUHIRO HOSAKA2 Germfree Life Research Institute, Nagoya University School of Medicine, Nagoya 466, Japan,1 and Research Institute for Microbial Diseases, Osaka University, Osaka 565, Japan2 Received 25 June 1982/Accepted 16 November 1982 To identify the active component of Sendai virus that induces interferon in mouse spleen cells, infectious and noninfectious viruses, envelope particles derived from them, and isolated hemagglutinin-neuraminidase (HN) glycoproteins were examined for interferon induction. The interaction between membranous structures containing Sendai virus HN glycoprotein and the receptors on the cell surface was shown to be sufficient for interferon induction in mouse spleen cells, suggesting that the actual inducer of interferon in mouse spleen cells is the HN glycoprotein of Sendai virus. When mouse spleen cells were stimulated in vitro with Sendai virus grown in eggs or LLC-MK2 cells or with membranous structures containing glycoproteins obtained from these viruses, interferon could be detected in the culture fluid. Furthermore, isolated HN glycoprotein per se could induce interferon in the cells. A linear correlation was found between the titer of interferon induced and the hemagglutinating activity of the membranous structure containing the HN glycoprotein. It was concluded from these findings that HN glycoprotein was the active component of Sendai virus responsible for interferon induction in mouse spleen cells and that viral RNA and F glycoprotein were not required. The results also showed that the interaction between HN glycoprotein and receptors on the cell surface triggered production of type I interferon (IFN-a and IFN-P). Although when Sendai virus was incubated at 56 C for 5 min it lost its hemolytic and hemagglutinating activities, it induced a considerable amount of interferon in the culture fluid of mouse spleen cells. The interferon-inducing ability of heat-inactivated virus could be absorbed with mouse spleen cells but not with sheep erythrocytes or mouse erythrocytes, indicating that the inactivated virus retained ability to bind to mouse lymphoid cells. Previously we found that Sendai virus that had been subjected to UV irradiation for 2 h could induce interferon in mouse spleen cell cultures as efficiently as untreated virus (6). Furthermore, Sendai virus grown in HeLa cells, which is unable to penetrate into tissue culture cells, was found to stimulate interferon production in mouse spleen cells but not in mouse fibroblast cells (L cells) (6). We recently reported that proteolytic cleavage of Fo glycoprotein of Newcastle disease virus was essential for interferon induction in L cells but not for interferon induction in mouse spleen cells (Y. Ito, Y. Nagai, and K. Maeno, J. Gen. Virol., in press). Membranous particles containing Sendai virus glycoproteins also induced interferon in mouse spleen cells but not in L cells (6). These results show that the actual inducer of interferon in mouse spleen cells is not viral nucleic acid, but t Present address: Department of Measles Virus, National Institute of Health, Tokyo , Japan. some other viral component(s), probably viral glycoprotein(s) of Sendai virus. In the present study, we analyzed the viral component(s) of Sendai virus that is essential for interferon induction in mouse spleen cells. The results showed that hemagglutinin-neuraminidase (HN) glycoprotein alone was sufficient for interferon induction in mouse spleen cells. Furthermore, induction of interferon with Sendai virus, which lost hemagglutination (HA) activity by heating at 56 C, was analyzed. MATERIALS AND METHODS Mice. Male C57BL/6 mice weighing 25 to 30 g were used in the present study. These mice were regarded as having no history of previous infection with Sendai virus, because the anti-sendai virus HA inhibition titers in their sera were less than 4. Virus. Sendai virus, Nagoya and Z strains, was propagated in the allantoic cavities of 10-day-old embryonated eggs by inoculation of a 102 or 10-' dilution of infected allantoic fluids. After the eggs were incubated at 35 C for 3 days, the allantoic fluids were 1019

2 1020 ITO AND HOSAKA TABLE 1. Interferon induction by Sendai virions or subviral components Interferon titera Glycoprotein Protein Interferon inducer Glcomprosteion content Mouse lnterferonin rcomposition (g/ml) L cells spleen cells Egg-grown virus HN, F LLC-SV HN, Fo 3 <4 4 REP from egg virus HN, F 125 <4 320 REP from LLC-SV HN, Fo 23.8 <4 4 Vesicles with HN glycoproteinb HN 48 <4 120 Heated egg virusc HN, F 410 <4 Heated HN glycoproteinc HN 48 <4 48 a Samples (0.1 ml each) of virions or subviral components (ca. 8,000 HAU) were added to L cell monolayers and mouse spleen cells (107 cells) and incubated at 35 C for 16 h. The culture fluid was then assayed for interferon. b The contamination of F protein was less than 1%. c Samples were heated at 50 C for 30 min. HA activity could not be detected in these samples. harvested and stored at - C. Tissue culture-grown noninfectious Sendai virus was propagated in LLC- MK2 cells, a continuous line of Rhesus monkey kidney cells, by infection of these cells with egg-grown virus, Z strain (LLC-SV) (2). The harvested virus was purified by sucrose density gradient centrifugation (3). Preparation of subviral components. Envelope particles were reassembled from Nonidet P-40-solubilized envelopes by dialysis through Spectrapore no. 2 tubes (Spectrapore Medical Industries Inc., Los Angeles, Calif.) against phosphate-buffered saline containing 1 mm MgCl2 (4) in the presence of SM2 beads (Bio-Rad Laboratories, Richmond, Calif.) (9). The dialysates were centrifuged at 100,000 x g for 60 min, and the pellets were used as reassembled envelope particles (REP). These particles contained two glycoproteins, HN and fusion (F or FO) proteins. Sendai virus glycoproteins were separated by using glutaraldehyde-treated erythrocytes (1). The HN proteins obtained were associated with membranous structures (1). Antisera. Antivirus serum was raised in rabbits by repeating intramuscular injections of the purified virions. Anti-HN and anti-f monospecific antisera were obtained from rabbits immunized, respectively, by HN and F glycoproteins separated by using glutaraldehyde-treated chicken erythrocytes (1). Cell cultures. LLC-MK2 cells were grown in Eagle minimum essential medium (MEM) supplemented with 10% fetal calf serum, and mouse L fibroblast cells were grown in the same medium further supplemented with 10%o tryptose-phosphate broth. The maintenance medium after virus infection was not supplemented with serum for L cells but with 2% serum for LLC- MK2 cells. Mouse spleen ceul cultures. The method used for preparation of mouse spleen cells was described previously (6). The spleen cells were cultured in Eagle minimum essential medium with 10%o fetal calf serum. Titration of HA and hemolytic activity. HA titration was done in microplastic trays by using 0.7% erythrocytes from various species. For hemolysis assay, the virus samples in 1 ml of phosphate-buffered saline were distributed in test tubes, and 2 ml of a 1% suspension of chicken erythrocytes in phosphate-buffered saline was added. The test tubes stood at 4 C for 1 h and were then incubated in a water bath at 37 C for 1 INFECT. IMMUN. h with occasional shaking. The released hemoglobin was measured spectrophotometrically at 540 nm against a blank of the erythrocytes without virus. Interferon titration. Interferon was assayed by the cytopathic effect inhibition microassay method with mouse L cells and vesicular stomatitis virus as the challenge virus (8). The reciprocal of the highest dilution of the sample causing 50%o protection was taken as the interferon titer. One unit of the interferon was found to be equivalent to two reference units of mouse interferon. Possible effects of infectious virus contaminated in the interferon samples were eliminated by treatment of the samples with anti-sendai virus serum or by dialysis of the sample at ph 2.0 before interferon assay. Chemical determination. Protein content was measured by the method of Lowry et al. (7). RESULTS Interferon induction by virions or subviral components of Sendai virus in mouse L cells and spleen cells. Volumes (0.1 ml each of virions or subviral components ca. 8,000 HA units [HAU]) (Table 1) were added to L cell monolayers (2 x 106 cells) or mouse spleen cells (107 cells) in 1 ml of Eagle minimum essential medium (Falcon 3001). After incubation of these cells at 35 C for 16 h, the culture fluids were collected and assayed for interferon production. Of the tested materials, only egg-grown Sendai virus induced interferon in L cells; the other materials did not (Table 1). On the other hand, all the virions and subviral components used induced interferon in mouse spleen cells (Table 1). No interferon activity was detected in control culture fluid of L cells or mouse spleen cells (data not shown). These results show that infectious virus was required for interferon induction in L cells, whereas membranous structures containing HN glycoproteins could induce interferon in mouse spleen cells. Dose responses of virions or subviral components of Sendai virus for interferon induction.

3 VOL. 39, B Izi 42 $ <20 SENDAI VIRUS AND INTERFERON PRODUCTION 1021 anti-hn serum but not by anti-f serum (Table Dilution of Virus Fluid 3). A similar inhibition by anti-hn but not anti-f FIG. 1. Dose-response curve of virions or subviral serum was also observed with the interferoncomponent' (s) for induction of interferon in mouse inducing ability of untreated Sendai virus (data spleen cell s. Serial twofold dilutions of viiions or not shown). These findings suggested that the subviral coi mponent(s) were added to the mouse spleen interferon-inducing ability of the heated as well cells, and after incubation at 35 C for 16 h, the culture as untreated virus involved HN protein but not fluids were collected and assayed for interferon. Sym- F protein. Furthermore, heated (50 C) HN gly- virus, initially 8,000 HAU; 0, coproteins retained considerable ability to in- bols: 0, elgg-grown LLC-SV, iinitially 8,000 HAU; A, REP from egg- duce interferon (Table 1). This heated protein grown viru increased linearly to the same level. This finding A A suggested that the membranous structure con- +~ ^ * A* o taining HN glycoprotein used in this experiment _* A had a lower interferon-inducing ability than virions or membranous structures containing both HN and F or Fo glycoproteins. Effect of heating at 56 C on interferon-inducing ability of Sendai virus. Hemolytic and HA activities were completely lost with heating at 56 C for 5 min (Table 2). The interferon-inducing activity of this heated virus in L cells was completely lost, whereas one-sixth of its activity in mouse spleen cells remained (Table 2). On further heating of the virus at 56 C, its interferon-inducing activity in mouse spleen cells did not decrease (Table 2). This finding showed that most of the interferon-inducing activity of Sendai virus was heat labile but that a minor portion (about one- * X I I I ^ sixth) was heat stable. The interferon-inducing ability of the heated virus was neutralized by is, initially 25,600 HAU; *, REP from did not exhibit HA activity, and therefore this LLC-SV, i] nitially 25,600 HAU; U vesicles with glycoprotei HN finding was principally consistent with the inns, initially 8,00 HAU. ducibility of the heated virus. Interferon induced by the heated virus was neutralized by anti-l-cell Newcastle disease vi- dilutions of infectious and nonin- rus antiserum (data not shown), which sug- Serial twoofold fectious viiruses, REP from the viruses, or HN gested that this interferon was also type I (IFN-a glycoprotesins were incubated with mouse spleen and IFN-P). Thus, the ability of induction of cells to dcetermine the dose responses of these type I interferon was found not only in the heat- interferon induction. As shown in labile components of Sendai virus but also in its materials ifor Fig. 1, siimilar high titers of interferon were heat-stable components lacking HA activity. induced o' ver the range of 250 to 25,600 HAU of Absorption of interferon inducibility of the either vinuses or REP. However, with similar heated virus with mouse spleen cells. Table 4 doses of IHN glycoprotein, interferon induction shows that sheep erythrocytes and mouse eryth- TABLE 2. Effect of heating at 56 C on Sendai virus interferon-inducing activity Interferon titer' Heating Hemolytic time HAU activity Mouse (min) (OD540)a L cells spleen cells , < <0 10 < <0 30 < <0 60 < <2 120 a Heated samples were diluted three times with phosphate-buffered saline and tested. OD540, Optical density at 540 nm. b The assay method was similar to that described in footnote a of Table 1.

4 1022 ITO AND HOSAKA TABLE 3. Inhibition with anti-hn monospecific antiserum of interferon induction by heat-inactivated Sendai virusa Antiserumb Interferon titee Control (minimum essential medium) Anti-HN monospecific serum... 4 Anti-F monospecific serum a Egg-grown Sendai virus (200 HAU) was heated at 56Cfor 1 h. b A 200-i&l amount of the heated virus was incubated with 10,ul of minimum essential medium or antiserum at 37 C for 30 min, and 100-pl samples of the reaction mixtures were then added to mouse spleen cells (10' cells). c The assay method was similar to that described in footnote a of Table 1. rocytes did not absorb the interferon-inducing activity of the heated virus, in contrast to the case of untreated virus, in which all the interferon inducibility of the virus was absorbed by repeated mixing with erythrocytes (Fig. 2). However, the interferon inducibility of the heated virus was absorbed with mouse spleen cells. This result indicated that the heated virus still had an adsorbing capacity for mouse spleen cells. DISCUSSION The previous hypothesis (5, 6) that the penetration of Sendai virus and functional viral RNA are needed for interferon induction in mouse fibroblast cells (L cells), whereas contact of the viral glycoproteins with the cell surface is sufficient for interferon induction in mouse spleen cells was verified by the present results. Furthermore, it was shown that the interaction between HN glycoproteins and cell surfaces was sufficient for interferon induction in mouse spleen cells, suggesting that the actual inducer of interferon in mouse spleen cells is the HN glycoprotein. Of infectious and noninfectious virions and subviral components of Sendai virus tested, only egg-grown infectious virions induced interferon in L cells. In contrast, infectious and noninfectious Sendai viruses and REP from egg-grown virus and LLC-SV induced interferon in mouse spleen cells. Even isolated HN glycoproteins could induce interferon in the spleen cells. Thus, we concluded that the HN glycoprotein was the active component for interferon induction in the spleen cells and that neither viral RNA nor F glycoprotein was needed for interferon induction in these cells. The results imply that the interaction between the HN glycoproteins and the cell surface is not only a simple contact but INFECT. IMMUN. also induces more complex processes, such as triggering of interferon production. It is interesting that the interaction between mitogenic lectin and lymphocytes triggers IFN--y production, whereas the interaction of the HN glycoprotein and lymphocytes triggers IFN-a and IFN-P production. Further studies are in progress on the mechanisms responsible for this difference. The membranous structures containing HN glycoproteins had less ability to induce interferon than virions or membranous structures containing HN and F or Fo glycoproteins. There are two possible explanations: F or Fo glycoprotein may contribute to the fortification of attachment of the HN glycoprotein-containing particles, or the HN vesicles used were very artificial in their conformation and their attachment ability was weaker than that of REP. There were some examples in which the efficiency of interferon induction was influenced by the nature of viral membranous structures; previously (6) we found that when Sendai virus was treated with 0.1 M K104, its infectivity and hemolytic and neuraminidase activities were destroyed, but considerable HA activity remained. Although the treated virus had no interferon-inducing ability itself, its binding to erythrocytes resulted in the recovery of the ability, indicating the importance of conformation of the surface structures of the inducer. Aggregation of the viral glycoproteins formed by a suitable quantity of antienvelope antiserum enhanced their interferon induction in mouse spleen cells (6). TABLE 4. Absorption of the activity to induce interferon in mouse spleen cells of heated Sendai virus with various cellsa Cells used for absorption No. of absorptions Interferon titer None 24 Sheep erythrocytes Mouse erythrocytes Mouse spleen cells <2 a A 0.2-ml amount of the heated Sendai virus (56 C for 60 min; initially 240 HAU) was mixed with 109 sheep or mouse erythrocytes or 108 mouse spleen cells at 40C for 30 min. The mixtures were centrifuged at 3,000 rpm for 5 min, and the supernatants were tested for ability to induce interferon in mouse spleen cells, as described in footnote a of Table 1.

5 VOL. 39, 1983 SENDAI VIRUS AND INTERFERON PRODUCTION 1023 A 12 R $4.0, 49 m 4p4 320 _ _ 5 '5 -IwQ a a A c 1 \ I - m $4 444 H 320 _- 160 _ r 10 5 'C5I o1 io-2 HAU of Serx]ai Virus Tixis of RBC Absorption FIG. 2. (A) Adsorption of Sendai virus by sheep erythrocytes and interferon induction in mouse spleen cells. A 1-ml amount of a 10%o sheep erythrocyte suspension was centrifuged at 3,000 rpm for 5 min, and the supernatant was decanted. Subsequently, 1 ml of Sendai virus preparation (1,000 HAU) was mixed with the pellet at 4 C for 30 min, and the mixture was centrifuged at 3,000 rpm for 5 min in a cold room. Absorption was carried out four times. The HA activity and ability to induce interferon in mouse spleen cells of the supernatants were assayed. Symbols: 0, HAU; 0, titer of interferon induced in mouse spleen cells. (B) Dose-response curve for induction of interferon by Sendai virus in mouse spleen cells. Serial 10-fold dilutions of preparations containing egg-grown Sendai virus were added to the culture fluids of mouse spleen cells. After incubation of these cells at 35 C for 16 h, the culture fluids were collected and assayed for interferon. Although the Sendai virus heated at 56 C for 5 min had no hemolytic or HA activity, it induced a considerable amount of interferon in the mouse spleen cell cultures. Moreover, prolonged heating of the virus caused no further decrease in its interferon-inducing activity in mouse spleen cells. Interferon induced by the heated virus was stable at ph 2 and was neutralized by anti-l cell Newcastle disease virus interferon (IFN-a and IFN-P) antiserum, suggesting that it is also type I interferon. The heated virus did not induce interferon in L cells. Since the interferon-inducing ability of the heated virus was neutralized by anti-hn antiserum but not by anti-f antiserum, the active component of heated virus that induced interferon seemed to be HN glycoprotein or dependent on the protein. The interferon-inducing ability of heated virus could be absorbed with mouse spleen cells but not with sheep or mouse erythrocytes, indicating that the heated virus retained ability to bind to mouse lymphoid cells. ACKNOWLEDGMENT This study was supported in part by grant (19 and 1981) from the Ministry of Education, Science, and Culture of Japan. LITERATURE CITED 1. Hosaka, Y. 19. Separation of Sendai virus glycoproteins by using glutaraldehyde-treated erythrocytes and preparation of monospecific antisera against the glycoproteins. Infect. Immun. 30: Hosaka, Y., Y. Fukami, Y. Yasuda, and J. A. Bonglla. 19. Complement-dependent antiviral monospecific antibodymediated lysis of murine cells coated with Sendai virus or its envelope component. Infect. Immun. 27: Hosaka, Y., and Y. Hosokawa Purification of Sendai virions with glutaraldehyde-treated red blood cells. Intervirology 8: Hosaka, Y., and Y. K. Shimlzu Artificial assembly of envelope particles of HVJ (Sendai virus). I. Assembly of hemolytic and fusion factors from envelopes solubilized by Nonidet P40. Virology 49: Ito, Y., Y. Kimura, I. Napta, and A. Kunii Production of interferon-like substance by mouse spleen cells through contact with BHK cells persistently infected with HVJ. Virology 60: Ito, Y., Y. Nshiyama, K. Shimokata, I. Nagata, H. Takeyama, and A. Kunil The mechanism of interferon induction in mouse spleen cells stimulated with HVJ. Virology 88: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Shimokata, K Studies on the pathogenicity of human-origin parainfluenza virus in the brain of mice. Microbiol. Immunol. 22: Volsky, D. J., and A. Loyter An efficient method for reassembly of fusogenic Sendai virus envelopes after solubilization of intact virions with Triton X-100. FEBS Lett. 92: