The major and minor group receptor families contain all but one human rhinovirus serotype

VIROLOGY

180,

814-817

(1991)

The Major and Minor Group Receptor CAROL Department

R.

UNCAPHER,

of Virus & Cell Biology,

Sharp

September

Contain

All but One Human

DEWIT,

AND

M.

CORRILLE

Merck

Received

Families

RICHARD

& Dohme

Research

Laboratories,

20,

accepted

Ocfobei

1990;

Rhinovirus

Serotype

J. COLONNO’ West Point,

Pennsylvania

19486

22, 1990

Previous studies have assigned 88 human rhinovirus (HRV) serotypes to major and minor receptor groups. Extension of these studies to include the remaining 14 unassigned serotypes indicated that 13 serotypes belong to the major group since their infection of HeLa cells is completely blocked by a monoclonal antibody that recognizes the major group receptor. This result indicates that the major group now accounts for 91 of the 102 known serotypes, while the minor group contains 10 serotypes. One serotype, HRV-87, appears to utilize neither the major nor minor group receptor and may represent a third receptor group. HRV-87 attachment cannot be blocked by other serotypes and displays a binding tropism similar to but distinct from minor group viruses. Unlike major and minor group serotypes, HRV-87 attachment and infection requires the presence of sialic acid on cellular receptors. o 19% Academic PESS, I~C.

Attachment of human rhinoviruses (HRVs) to specific cellular surface receptors is a prerequisite to successful infection. Utilizing competition binding and cell protection assays with a monoclonal antibody (MAb lA6) that recognizes the receptor for the vast majority of HRVs, we have previously demonstrated that 88 of the 102 known HRV serotypes can be divided into two receptor families ( 1, 2). The first family, referred to as the major group, contains 78 HRV serotypes and utilizes a single receptor identified as the intercellular adhesion molecule 1 (ICAM- ) (3-5). ICAM- is a cell surface ligand for the lymphocyte function-associated antigen 1 adhesion receptor (LFA-1 ), and its interaction with LFA-1 plays an important role in leukocyte adhesion and in the execution of immunological and inflammatory functions mediated by leukocyte adhesion (6, 7). The remaining 10 serotypes (minor group) compete for a second receptor, which has been tentatively identified as a 120-kDa cell surface protein of unknown function (8) Since the current number of recognrzed serotypes has been extended to 102 (9), we have proceeded to assign the remaining 14 HRV serotypes to receptor families. HRV serotypes and antisera were obtained from the ATCC and viruses propagated as previously described ( 7). Preparations of HRV-53, HRV-82, HRV87, and new serotypes HRV-90 to HRV-100 were titered by plaque assay and their serotypes confirmed by homologous antiserum neutralization (data not shown). ’ To whom addressed.

0042.6822/91

correspondence

and requests

$3.00

Copyright 0 1991 by Academic Press. lnc All rights of reproductvx in any form reserved.

for reprints

should

Earlier studies on the HRV major group have clearly demonstrated that attachment to the ICAM- receptor is absolutely required to achieve viral infection of HeLa cell monolayers since blockage by MAb lA6 completely abrogated infection (2). Taking advantage of this observation, we screened the 14 unassigned serotypes in a cell protection assay to determine if MAb 1A6 was capable of blocking viral infection. Confluent HeLa cell monolayers, in 48-well cluster plates, were either untreated (control) or pretreated with 1 pg of MAb 1A6 in a total volume of 0.1 ml of McCoy’s media for 30 min at 34” (2). After incubation, cell monolayers were challenged by the addition of 50 yl of the HRV serotype (m.o.i. = 1) to be tested and the cells incubated at 34” for 18-24 hr. Monolayers were then microscopically examined for cytopathic effect resulting from virus infection and control wells were compared to MAb lA6-treated cells. Results showed that only HRV-87 was able to infect cells and cause a visible cytopathic effect in the presence of MAb lA6, while monolayers infected with each of the other 13 HRV serotypes tested only showed evidence of viral infection in the absence of MAb lA6 (data not shown). Extended incubation for up to 48 hr failed to circumvent the MAb blockade. These results are consistent with earlier studies ( 1, 2) and further confirm that the vast majority (89%) of HRV serotypes utilize ICAM- as a cellular receptor. To determine if HRV-87 belonged to the minor HRV group, competition binding assays on HeLa cells were conducted with [35S] methionine-labeled HRV2 (minor group), HRV-36 (major group), and HRV-87 as previously described ( 7). Results (Table 1) showed

be

814

SHORT TABLE

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1

HRV BINDING TO CELL MONOLAYERS

Blocking Cell type

Virus

None

HRV-2

HeLa

HRV-2 HRV-36 HRV-87 HRV-2 HRV-36 HRV-87 HRV-2 HRV-36 HRV-87 H RV-2 HRV-36 HRV-87 HRV-2 HRV-36 HRV-87 HRV-2 HRV-36 HRV-87

28 18 22 29 1 17 6 2 28 14 1 24 19 2 5 8 1 6

5 14 20 6 1 16 3 ND ND 6 ND 23 7 ND ND 2 ND 5

BHK

CEF

L

SF9

Vero

Y/o Binding virus/antiserum

HRV-36 23 4 22 ND 1 ND ND ND ND ND ND ND ND 2 ND ND ND ND

HRV-87 23 18 30 24 1 17 ND ND 18 10 ND 20 ND ND 4 8 ND 2

HRV-87

Ab

ND ND 4 ND ND ND ND ND ND 2 ND ND 2 ND ND 5

Note. Cell binding studies were performed on confluent monolayers (1 O5 cells) in a 48-well cluster plate for 1 hr at 34” in a final volume of 0.15 ml as previously described (1). Blocking virus concentrations per assay were 5 X 1 O7 PFU (HRV-2), 3 X 1 O7 PFU (HRV36), and 8 X lo6 PFU (HRV-87). Values presented in the table are representative of data obtained from several assays. HRV-87 specific antiserum (l/40 dilution) was obtained from the ATCC and preincubated with HRV-87 for 15 min at 25”. ND, not done.

that attachment of both HRV-2 and HRV-36 were effectively blocked by the addition of excess unlabeled homologous virus but were not affected by the inclusion of heterologous serotypes. In contrast, the binding of HRV-87 was unaffected by the addition of unlabeled virus, including HRV-87. The reason for the lack of competition is unclear but may be due to the lower titers of the HRV-87 preparations used (4 to 6-fold lower than HRV-36 and HRV-2, respectively), a very high number of cellular receptors, or nonspecific binding. To control for nonspecific binding, neutralizing HRV-87 antiserum (ATCC) was included in the binding assay since previous studies have indicated that neutralization of HRV-14 interferes with attachment ( 10). Results (Table I) showed that HRV-87 binding was effectively blocked by neutralizing antiserum and indicates that the observed binding is the result of virus attachment. It should be pointed out that the absence of competitive binding has been observed previously with major group serotypes, HRV-1 1 and HRV-17, in that they also failed to block themselves ( I). However, unlabeled preparations of these two serotypes were able to block other major group serotypes and MAb lA6 blocked infection of both serotypes ( 1, 2).

815

To further pursue the question of whether HRV-87 is a minor group serotype, we took advantage of the different cellular tropisms displayed by the major and minor groups. The major group receptor (ICAM- ) is found only on cells of human and higher primate origin while the minor group receptor appears to be present on cells derived from a diversity of species (2). A series of binding studies were conducted on a variety of cell types in an attempt to assign HRV-87 to the minor group. The results of these studies are summarized in Table 1 and show that both the minor group serotype, HRV-2, and HRV-87 can bind to varying degrees to BHK (hamster), CEF (chicken), L (mouse), SF9 (insect), and Vero (monkey) cells, while the major group serotype, HRV-36, was unable to bind to any of these cell lines as predicted from previous studies (2). The poor binding of HRV-87 on SF9 and Vero cells again indicates that HRV-87 binding is selective since differential binding is observed on equivalent numbers of cells. The results in Table 1 support the MAb lA6 experiments above and indicate that HRV-87 is clearly not a major group virus. Instead, these binding experiments indicate that the receptor(s) for HRV-87 can be found on a diversity of cells and displays a tropism similar to the minor group serotype HRV-2. While HRV2 and HRV-87 bind comparably to four of the six cell lines tested, the weak binding of HRV-2 to CEF cells and strong binding to SF9 cells contrast with the HRV87 binding results and further suggest that HRV-87 may not bind to the minor group receptor. In an attempt to further characterize the binding site of HRV-87, the role of sialic acid was investigated since previous studies have demonstrated that sialic acid is not required for binding of major or minor group serotypes ( 11, 12). HeLa cell monolayers were treated with neuraminidase to remove sialic acid and used in virus binding studies. Surprisingly, results showed that HRV-87 attachment was dependent on the presence of sialic acid since neuraminidase treatment inhibited binding by nearly 90% (Table 2). The effect is clearly due to the addition of neuraminidase since control cells treated with low pH media alone support viral attachment (Table 2). Under identical conditions, the binding of HRV-2 and HRV-36 was unaffected. The sensitivity of HRV-87 binding to neuraminidase treatment was similar to influenza virus, which is dependent on sialic acid for binding ( 13) and was included as a positive control (Table 2). A similar inhibition of encephalomyocarditis virus binding has also been reported following treatment of human erythrocytes with neuraminidase ( 14). To demonstrate that removal of sialic acid had indeed removed an important receptor component required for HRV-87 infection, neuraminidase-treated

816

SHORT TABLE EFFECT

Virus HRV-2

HRV-36

H RV-87

FLU (WSN)

2

OF NEURAMINIDASE TREATMENT ATTACHMENT AND INFECTION

Treatment None Buffer Buffer None Buffer Buffer None Buffer Buffer None Buffer Buffer

+ NA

+ NA

+ NA

+ NA

COMMUNICATIONS

% Binding 20 20 16 21 18 21 27 30 3 40 33 11

ON VIRUS

PFU Ratio 1 .oo 0.93 1 .oo 1 .oo 0.92 1 .oo 1 .oo 2.57 0.002 ND ND ND

Nore. Confluent HeLa cell monolayers in 48.well cluster plates were incubated with 0.1 ml McCoy’s media adjusted to pH 6.0 (Buffer) or in the same media containing 2.5 U/ml of Arfbrobacter ureafaciens neuraminidase (Sigma) (Buffer + NA) for 2 hr at 37”. Following incubation, the media were removed and monolayers gently washed twice before being refed with 0.15 ml of fresh McCoy’s (pH 7) media. Cells were either used for binding assays as described in Table 1 and included influenza virus (FLU) prepared as described elsewhere (18) or challenged with 10 ~1 of HRVs (m.o.i. = 1) for 90 min at 34”. For cell protection assays, virus inoculum was removed after 90 min, washed twice with 0.3 ml media, and refed with 0.15 ml fresh McCoy’s media, and cells were incubated 18 hr at 34”. Plates were freeze/thawed to disrupt cell monolayers, cell debris was removed by centrifugation, and the virus present was titrated by plaque titration (1). PFU ratio, virus yield from treated monolayers/yield from control infections. ND, not done.

cells were challenged with each of the three representative viruses and the resulting progeny virus titered by plaque assay (Table 2). Only HRV-87 was unable to infect HeLa cells that had been pretreated with neuraminidase, showing a 500-fold drop in virus yield. This decrease in virus yield represents a near complete blockade since virus yields from all three viruses were also reduced to an equivalent extent when inoculum virus was treated with serotype-specific antiserum or when HRV-36-infected cells were pretreated with MAb lA6 (Ref. ( 15), unpublished data). The above results strongly support the conclusion that HRV-87 binds to cells at a receptor site different from that of the major and minor group HRVs. However, the possibility cannot yet be ruled out that HRV87 binds to the minor group receptor at a unique and different location than other minor group serotypes. It is unlikely that HRV-87 can bind to several different receptors containing sialic acid since attempts to demonstrate binding to purified ICAMreceptor containing sialic acid have been unsuccessful (unpublished data).

In light of these unexpected results, we have questioned whether HRV-87 is indeed a HRV. HRV-87 was the assigned designation given to prototype strain F02-3607-Corn in 1971 ( 16), and neutralizing antiserum (V. V. Hamparian, Columbus, OH) generated against the original human clinical isolate effectively neutralized our current preparations of HRV-87. To prove that HRV-87 is a HRV, a series of experiments were undertaken to further characterize radiolobeled HRV-87 particles. Size determination on rate-zonal sucrose gradients ( I) and particle banding on isopycnic metrizamide gradients (I) indicated that HRV-87 particles had the identical size and density of HRV-2 and HRV-36 (unpublished results). Analysis of [35S]methionine-labeled HRV-87 by SDS-PAGE (1) yielded three of the four expected structural proteins with molecularweights of 23, 24, and 29 kDa (data not shown). Similar to other picornaviruses, the fourth capsid protein, 1A (VP4), is probably not labeled due to the absence of methionine residues ( 1). Electron micrographs of HRV-87 particles also confirmed that they are picornaviruses (data not shown). Since a distinct characteristic of HRVs is their sensitivity to low pH, we incubated HRV-87 in pH 5.0 buffer for 1 hr at 25” and then measured viability of the resulting virus preparation by plaque titration. Results showed greater than a 6 log drop in virus titer following pH 5.0 treatment (data not shown) and taken with the characterization above, strongly suggest that HRV-87 is indeed a HRV. In summary, characterization of HRV serotypes has enabled the assignment of all but one of the 102 recognized HRV serotypes to either the major or minor receptor groups (Fig. 1). The major group contains 91

F 32

33 34 35 36

37 38 39 40

0

Major

Minor

Other

FIG. 1. Assignment of HRVs to receptor families. Each of the known HRV serotypes are depicted above and coded to reflect receptor families (white, major group; black, minor group; and striped, nonmajor, nonminor group virus).

SHORT

COMMUNICATIONS

serotypes and includes all of the newly numbered serotypes, HRV-90 to HRV-100, and 1 unnumbered clinical serotype, designated Hanks ( 17)) which appears to be a unique serotype. The minor group contains 10 serotypes which compete for binding to a yet unidentified receptor found on cells derived from a diversity of species. HRV-87, which is clearly not a major group virus, remains an anomaly since it shows cell binding tropisms similar to minor group viruses yet is unlike minor group viruses in requiring sialic acid for attachment. Further characterization will be required to determine whether the HRV-87 receptor is indeed unique, and subsequent sequencing of the HRV-87 RNA genome may yield important insights into the region(s) of the viral capsid involved in HRV attachment.

6.

10. 11. 12.

ACKNOWLEDGMENTS We wish to thank V. V. Hamparian for supplying HRV-81 ing antiserum and B. Wolanski for electronmicroscopy.

13. neutraliz-

14. 15.

REFERENCES 1. ABRAHAM, G., and COLONNO, R. J., /. viral. 51, 340-345 (1984). 2. COLONNO, R. J., CALLAHAN, P. L., and LONG, W. J., /. L&o/. 57, 7-12 (1986). 3. GREVE, J. M., DAVIS, G., MEYER, A. M., FORTE, C. P., YOST, S. C., MARLOR, C. W., KAMARCK, M. E., and MCCLELLAND, A., CeI56, 839-847 (1989). 4. STAUNTON, D. E., MERLU~I, V. J., ROTHLEIN, R., BARTON, R., MARLIN, S. D., and SPRINGER, T. A., Ce// 56, 849-853 (1989). 5. TOMASSINI, J. E., GRAHAM, D., DEWITT, C. M., LINEBERGER, D. W.,

16.

17. 18.

817 RODKEY, J. A., and COLONNO, R. J., froc. Nat/. Acad. SC;. USA 86, 4907-4911 (1989). MAKGOBA, M. W., SANDERS, M. E., GINTHER LUCE, G. E., GUGEL, E. A., DUSTIN, M. L., SPRINGER, T. A., and SHAW, S., Eur. J. Immunol. 18, 637-640 (1988). DUSTIN, M. L., STAUNTON, D. E., and SPRINGER, T. A., lmmunol. Today9, 213-215 (1988). MISCHAK, H., NEUBAUER, C., BERTHOLD, B., KUECHLER. E., and BLASS, D.. J. Gen. viral. 69, 2653-2656 (1988). HAMPARIAN, V. V., COLONNO, R. J.. COONEY, M. K., DICK, E. C., GWALTNEY, J. M., JR., HUGHES, J. H., JORDAN, W. S., JR., KAPIKIAN, A. Z., MOGABGAB, W. J.. MONTO, A., PHILLIPS, C. A., RUECKERT, R. R., SCHIEBLE, J. H., Stott, E. J.. and TYRRELL, D. A. J., \/irology 159, 191-192 (1987). COLONNO, R. J., CALLAHAN, P. L., LEIPPE, D. M., RUECKERT, R. R., and TOMASSINI, J. E., 1. Viral. 63, 36-42 (1989). TOMASSINI, J. E.. MAXSON, T. R., and COLONNO, R. J., J. Bioi. Chem. 264, 1656-1662 (1989). MISCHAK, H., NEUBAUER, C., KUECHLER, E., and BLASS, D., I/ire/ogy 163, 19-25 (1988). ROGERS, G. N., and PAULSON, J. C.. I/irology 127, 361-373 (1983). ALLAWAY, G. P., and BURNESS, A. T. H., /. viral. 59, 768-770 (1986). COLONNO. R. J., LAFEMINA, R. L., DEWITT, C. M., and TOMASSINI. J. E., “New Aspects of Positive-Strand Viruses ” (M. A. Brinton and F. X. Heinz, Eds.), ASM. Washington, D. C., pp. 257261, 1990. KAPIKIAN, A. Z., CONANT, R. M., HAMPARIAN, V. V., CHANOCK, R. M., DICK, E. C., GWALTNEY, J. M., JR., HAMRE, D., JORDAN, W. S., JR., KENNY, G. E., LENNETTE, E. H., MELNICK, J. L., MoGABGAB, W. J., PHILLIPS, C. A., SCHIEBLE, J. H., STOTI-, E. J., and TYRRELL, D. A. J., L%ology 43, 524-526 (1971). GWALTNEY, J. M., JR., MOSKALSKI, P. B., and HENDLEY, J. O., Ann. lnt. Med. 88, 463-467 (1978). KRUG, R. M., Viroiogy44, 125-136 (1971).