Hemagglutination variant of measles virus

VIROLOGY 80, 441-444 (1977)

Hemagglutination Variant of Measles Virus ALAN M. BRESCHKIN, BARBARA WALMER, AND FRED RAPP Department of Microbiology, The Milton S. Hershey Medicat-~.enter, The Pennsylvania State University, College of Medicine, Hershey, P e n n ~ a n i a 17033 ? Accepted April 8, 1977 A hemagglutination variant (HAN-12) of measles virus was isolated following mutagenesis of the parental strain (CC). Cells infected with HAN-12 formed syncytia but did not adsorb monkey erythrocytes. Although HAN-12 produced normal yields of infectious virus, no hemagglutination or hemolysis activity was detected when assayed in isotonic phosphate-buffered saline. However, in hypertonic buffer the hemagglutination and hemolysis titers of HAN-12 were equivalent to that of CC. In isotonic medium, HAN-12 adsorbed to host cells as efficiently as the parental strain. Comparison of HAN-12 and CC virion-associated polypeptides by SDS-polyacrylamide gel electrophoresis did not reveal any differences in migration rates of the proteins. Our results are discussed in regard to previous reports of hemagglutination variants of measles virus.

Measles virus causes agglutination of baboon (1) and monkey (2, 3) erythrocytes. Virus adsorption to erythrocyte receptors has been presumed to be similar to adsorption to host cell receptors. However, unlike other paramyxoviruses, the measles subgroup lacks neuraminidase activity and does not adsorb to neuraminic acidcontaining receptors (4, 5). The chemical nature of the measles virus receptor is not presently understood. Hemagglutination (HA) reflects the activity of a virus glycoprotein on the envelope of the virion (6). In addition to HA, measles virus also causes lysis of monkey erythrocytes (1, 7, 8). The hemolytic (HL) activity has been separated from HA activity (6, 9, 10). HL and cell fusion activities of measles virus are closely related and apparently depend on the presence of a second virus glycoprotein on the envelope (6). Previous studies indicate that measles virus HA was affected by the tissue in which the virus was propagated (3) and the virus strain (11-14). Schluederberg and Nakamura (15) reported a salt-dependent HA activity from measles virusinfected cells, and recently Shirodaria et al. (16) found that several strains of measles virus hemagglutinated in hypertonic phosphate-buffered saline (PBS) but not in

isotonic buffer. However, the passage histories of these strains were not known, and so the process which led to their selection could not be determined. We have isolated a hypertonic HA variant following mutagenesis of a conventional HA strain. The parental virus, CC (17), was treated with 25 t~g/ml nitrosoguanidine (K and K Laboratories, Plainview, New York) for 15 min at 25°. The mutagenized virus was plated on BSC-1 cells and plaque progeny were picked (17). Each isolate was plated in duplicate on BSC-1 cells and tested for hemadsorption (17) using 0.5% African green monkey erythrocytes (Flow Laboratories, Rockville, Maryland) when typical measles virus cytopathic effect (CPE) was present. Out of approximately 900 isolates tested, one was very deficient in hemadsorption. Following plaque purification, this isolate continued to be hemadsorption negative and was designated HAN-12. Additional cell types such as Vero, primary green monkey kidney and human embryonic kidney (Flow Laboratories, Rockville, Maryland) infected with HAN-12, were also hemadsorption negative. At all incubation temperatures tested (33, 37, and 39°), HAN-12 replicated equally well, and the infected cells did not hemadsorb. 441

Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0042-6822

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The replication of HAN-12 was slightly delayed compared to CC, but the maxim u m titers were nearly equal (Fig. 1). For CC, the plaque-forming units (PFU) and HA titers increased concomitantly; the ratio of P F U to HA titers was approximately 1 × 108 (Table 1). In contrast, virus progeny from HAN-12 infected cells did not hemagglutinate to any detectable titer when assayed in isotonic PBS (see below). Unlike CC, HAN-12 was also deficient in causing hemolysis (Table 1). The HL and HA titers of CC were comparable, but HAN-12 did not exhibit either activity. However, the cell-fusion factor of HAN-12 was quite active as indicated by the formation of large syncytia. Other investigators have previously observed HA in the absence of ilL (6, 9,17) but not vice versa (6, 18). Since HAN-12 had an active cell-fusion factor (an HL-related function), we conclude that the HL deficiency of HAN-12 is a secondary effect of its HA deficiency. We suggest that HL can only occur after the virus has adsorbed to the erythrocyte. 108

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FIO. 1. Growth characteristics of C C and H A N 12. BSC-I cell monolayers were inoculated with 0.1 PFU/cell. Cultures were incubated at 37°. At the times indicated, virus was harvested from infected cells by sonication (30 sec). Samples were assayed for infectious virus (17) and isotonic HA (see Table 1).

TABLE 1 HEMAGGLUTINATION AND HEMOLYSIS TITERS OF CC AND H A N - 1 2

Virus

CC HAN-12

Infectivity ~

8 × 10~ 4 x 10T

Isotonic

Hypertonic

HA b HL c

HA ~ HL c

64 <1

32 <2

64 32

32 16

PFU/ml. b Reciprocal of highest dilution showing HA assayed in PBS (isotonic assay) or PBS containing 0.8 M (NH4)2SO4 (hypertonic assay) in 0.2 ml volume. For the isotonic assay, an equal volume of 0.5% African green monkey erythrocytes was added. For the hypertonic assay, a 2% suspension (16) was used. Results were read after 1-2 hr of incubation at 37°. Reciprocal of highest dilution showing HL assayed with 5% monkey erythrocytes in PBS (isotonic) or PBS containing 0.8 M (NH4)2SO4 (hypertonic). After 4 hr of incubation at 37°, blood cells were removed by low-speed centrifugation. Hemolysis was determined by absorbance at 410 nm on the supernatants. Erythrocytes in PBS were the control for spontaneous hemolysis.

In the presence of 0.8 M (NH4)2SO4, HAN-12 did hemagglutinate (Table 1). For CC, the isotonic and hypertonic HA titers were 64. This represents a fourfold enhancement of HA in the high salt because four times more erythrocytes were used in the hypertonic assay. The ratio of P F U to hypertonic HA titer was I × 10Gfor CC and HAN-12. Thus, the hypertonic HA titer of HAN-12 was equivalent to CC. In hypertonic PBS, HAN-12 also exhibited HL activity, and the hypertonic HL titers of CC and HAN-12 were equivalent. Measles virus HA has been thought to be functionally related to adsorption to host cells. To test HAN-12's adsorption to host cell receptors, we compared adsorption kinetics of CC and HAN-12. The strains were inoculated onto BSC-1 cell monolayers at 25° in isotonic PBS, i.e., conditions in which HAN-12 did not hemagglutinate. Beginning at 10 min postinoculation the inocula were removed, and the cultures were washed, and then overlaid with agar for a P F U assay (17). The results indicated that HAN-12 and CC were adsorbed equally efficiently; for both strains maximum adsorption occurred by 50 min postinoculation. This suggests that

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the receptor sites involved in HA and host cell adsorption are not identical, because the two processes can be independently altered. Thus, the nature of the measles virus receptor site remains unclear. To compare the virion-associated proteins of CC and HAN-12, we analyzed [3~S]methionine-labeled virus by SDSpolyacrylamide gel electrophoresis using the discontinuous Tris-glycine buffer system (19)~ The migration of the proteins (Fig. 2) is similar to previous results for measles virus (20, 21); six prominent bands were consistently resolved. Electrophoretic analysis of [3H]glucosamine-labeled (1 t~Ci/ml; 20 Ci/mmol; Amersham/ Searle) virions indicated that VP1 (molecular weight 77,000-78,000) was a glycoprotein (data not shown). This agrees with the results of Bussell et al. (21). In contrast, Hall and Martin (20) reported two glycosylated polypeptides (molecular weights 69,000 and 53,000) from measles virions. The representative slab gel shown in Fig. 2 clearly shows that the electrophoretic profiles of CC and HAN-12 are identical. [3H]Glucosamine labeling demonstrated that VP1 of HAN-12 was glycosylated. We compared the neurovirulence of CC and HAN-12 following intracranial inoculation (22) of newborn random-bred golden Syrian hamsters (Lakeview Hamster Colony, Newfield, New Jersey) with 104 PFU. Both strains caused an acute encephalitis which killed all of the animals by 10 days. Virus recovered by cocultivation on BSC-1 cells from the brains of HAN-12-inoculated animals (22) was still HA negative in isotonic PBS. Shirodaria et al. (16) reported that two measles strains which hemagglutinated only in hypertonic PBS were of recent human history. They suggested that salt-dependent HA is a characteristic of wild-type isolates. According to their hypothesis, conventional HA strains arise as the result of a mutation and subsequent selection during laboratory passage. Unlike the HA variants reported previously (16), the derivation of HAN-12 is completely defined. Our method of isolation suggests that HAN-12 arose as the result of a mutation

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FIG. 2. Photograph of autoradiogram developed from a 7.5% polyacrylamide slab gel of [3~S]methionine-labeled purified virus. Virus was purified from the media of BSC-1 cell monolayers on 150-mm plastic Petri dishes infected a t a multiplicity of infection of 0.1 PFU/cell after 48 h r of incubation at 37 °. After 24 hr, 5 ~Ci/ml of [3~S]methionine (400 Ci/mmol; Amersham/Searle) was added in Earle's salt solution supplemented with 1 × nonessential amino acids and 1 × vitamin solution. Virus was purified as described previously (20). Purified virus was dissolved in 0.0625 M Tris, pH 6.8, 2% SDS, and 5% mercaptoethanol, and boiled for 2 rain. Solubilized virus proteins were analyzed on 7.5% polyacrylamide slab gels using the discontinuous Tris-glycine buffer system (19). After electrophoresis the gels were fixed, dried, and exposed to Kodak no-screen X-ray film. Molecular weight markers were phosphorylase a (92,000), bovine serum albumin (69,000), and ovalbumin (45,000).

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induced by nitrosoguanidine. Alternatively, HAN-12 could represent a low frequency genetic variant present in the parental strain. In either case, the biological properties of HAN-12 indicate that its HA protein has a minor alteration at the erythrocyte receptor site. Apparently in high salt this defect is overcome due to electrostatic or conformational changes at the receptor site. To date we have not found any cell types in which the growth of HAN-12 is selected against, and the HA deficiency has remained stable through three virus passages in BSC-1 cells. ACKNOWLEDGMENTS We thank E. Morgan, J. Iltis, and P. Knight for helpful discussions. We especially thank P. Gupta for his help with the polyacrylamide gel electrophoresiN. This investigation was supported in part by Public Health Service research grant AI 12203 from the National Institute of Allergy and Infectious Diseases. A.M.B. is a recipient of Public Health Service Postdoctoral Research Fellowship Award 1 F32 AI05237 from the National Institute of Allergy and Infectious Diseases. REFERENCES 1. PERIES, J. R., and CHANY, C., C. R. Acad. Sci. 251, 820-821 (1960). 2. ROSEN,L., Virology 13, 139-141 (1961). 3. ROSANOFF,E. I. Proc. Soc. Exp. Biol. Med. 106, 563-567 (1961). 4. NORRBY,E., Arch. Ges. Virusforsch. 12, 164-172 (1962).

5. WATERSON,A. P. Arch. Ges. Virusforsch. 16, 5780 (1965). 6. HALL, W. W., and MARTIN,S. J., J. Gen. Virol. 22,363-374 (1974). 7. DEMEIo, J. L., Virology 16, 342-344 (1962). 8. NORRBY,E. C. J., and FALKSVEDEN,L. G.,Arch. Ges. Virusforsch. 14, 474-486 (1964). 9. NORRBY,E., Arch. Ges. Virusforsch. 14,306-318 (1964). 10. NORRBY,E., and HAMMARSKJOLD,S., Microbios 5, 17-29 (1972). 11. RUCKEL-ENDERS,G., Amer. J. Dis. Child. 103, 297-306 (1962). 12. RUCKEL-ENDERS,G., Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. 191, 217-236 (1963). 13. ODDO, F. G., CHIARINI, A., and SINATRA, A., Arch. Ges. Virusforsch. 22, 35-42 (1967). 14. TISCHER,I., Zentralbl. Bakteriol. Parasiten. , Infectionskr. Hyg. 18,446-476 (1967). /5. SCHLUEDERBERG,A., and NAKAMURA,M., Virology 33, 297-306 (1967). 16. SHIRODARIA,P. V., DERMOTT,E., and GOULD,E. A., J. Gen. Virol. 33, 107-115 (1976). 17. HASPEL,M. V., DUFF,R., and RAPP, F., J. Virol. 16, 1000-1009 (1975). 18. NORRBY,E., Virology 44, 599-608 (1971). 19. MAIZEL,J. V., JR., In "Methods in Virology" (K. Maramorosch and H. Koprowski, eds.), Vol. 5, pp 177-246. Academic Press, New York, 1971. 20. HALL,W. W., and MARTIN,S. J., J. Gen. Virol. 19, 175-188 (1973). 21. BUSSELL,R. H., WATERS,D. J., SEALS, M. K., and ROBINSON,W. S., Med. Microbiol. I m m u nol. 160, 105-124 (1974). 22. HASPEL, M. V., DUFF, R., and RAPP, F., Infect. Irnrnun. 12, 785-790 (1975).