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
442
SHORT COMMUNICATIONS
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
,
i
I
36
A 107
U • CC
~'FU~
O • HAN-12
M
106
ZD
12 10 5
10 0
24 HOURS
48
72
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
442
SHORT COMMUNICATIONS
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
,
i
I
36
A 107
U • CC
~'FU~
O • HAN-12
M
106
ZD
12 10 5
10 0
24 HOURS
48
72
