of B2 expression), even after infection at multiplicities high enough to ensure that ... role in the promotion of cell fusion and that glycoprotein C2 can act to suppress ... envelope with the plasma membrane of the host cell (3-5). Membrane fusion ...
Proc. Nati. Acad. Spi. USA Vol. 74, No. 9, pp. 3913-3917, September 1977
Cell Biology
Cell fusion induced by herpes simplex virus is promoted and suppressed by different viral glycoproteins* (membranes)
ROBERTO MANSERVIGItt, PATRICIA G. SPEARt§, AND ALEXANDER BUCHANS t Department of Microbiology, University of Chicago, Chicago, Illinois 60637; and 1 Department of Virology, University of Birmingham, Birmiingham B15 2TJ, England
Communicated by Hewson Swift, July 1, 1977
ABSTRACT Some of the factors that regulate membrane fusion resulting in polykaryocyte formation have been investigated, using the model system of human cells infected with mutants of herpes simplex virus (HSV) One of the mutant viruses used in this study (MP) failed to produce the viral glycorotein designated C2-a nonlethal defect that has previously been correlated with the polykaryocyte-inducing phenotype of this and other mutant strains (wild-type strains of HSV usually induce the aggregation of infected cells rather than their fusion). The other mutant virus (tsB5), a temperature-sensitive conditional-lethal mutant, failed to produce giYcoprotein B2 at nonpermissive temperature, whereas the synthesis of all other viral products appeared to be normal. We produced and isolated seven recombinants of MP and tsB5 that expressed both of the parental alterations iv glycoprotein synthesis. All of the recombinant viruses induced the fusion of infected cells at 340 (correlated with the absence of C2 expression) but were unable to cause cell fusion at 390 (correlated with the absence of C2 and of B2 expression), even after infection at multiplicities high enough to ensure that all cells in the cultures synthesized viral macromolecules. These results and studies on the dominance or recessiveness of the fusion-inducing phenotype in mixed infections provide evidence that glycoprotein B2 plays a critical role in the promotion of cell fusion and that glycoprotein C2 can act to suppress fusion.
The fusion of cellular membranes is an important component of several essential biological processes, including both endocytosis and exocytosis and also syncytium formation such as occurs during muscle development. Membrane glycoproteins may play a critical role in promoting membrane fusion, as indicated by studies done with certain enveloped viruses that are capable of inducing cell fusion (reviewed in refs. 1 and 2). Sendai virus, for example, specifies the synthesis of a membrane glycoprotein that is responsible for the ability of this virus to induce cell fusion and is also required for viral infectivity (3, 4), which probably depends in part upon fusion of the virion envelope with the plasma membrane of the host cell (3-5). Membrane fusion, whether virus-induced or directed by cellular products, must be regulated in some way so that the fusion occurs only between specialized regions of particular membranes in a single cell or between the surface membranes of specific differentiated cell types. An understanding of the process of membrane fusion, therefore, requires identification of membrane constituents or other products that can prevent or limit the fusion of adjacent membranes, in addition to characterization of the membrane constituents that promote fusion. This paper presents evidence that, in human cells infected with herpes simplex virus (HSV), the promotion and suppression of cell fusion are two separate viral functions associated with two different viral glycoproteins. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3913
Cells infected with HSV exhibit modifications in their social behavior, the nature of which depends upon properties of the particular virus strain used for infection (6-8). Most fresh isolates of HSV cause cell aggregation in which varying degrees of adhesiveness can be evident. Some strains of HSV induce cell fusion, however, and in certain instances it has been shown that the polykaryocyte-inducing phenotype results from a nonlethal mutation of cyto-aggregating virus (6, 9-13). It should be noted that expression of the polykaryocyte-inducing phenotype may depend in part on properties of the host cell because some HSV mutants (and laboratory strains) promote the fusion of certain cell lines or strains but not of others (6,11). Previous studies suggested that the kind of altered cellular interactions observed after HSV infection was determined at least in part by the spectrum and properties of viral glycoproteins present in plasma membranes of infected cells (14-16) and, in fact, the polykaryocyte-inducing phenotype of some HSV type 1 (HSV-1) mutants can be correlated with a specific defect in glycoprotein synthesis. Comparisons of the glycoproteins made by different HSV-1 strains revealed that cytoaggregating isolates specify the synthesis of at least four membrane glycoproteins (13, 15, 17), whereas some fusioninducing mutants are clearly defective in the synthesis or processing of the glycoprotein designated C2 (13, 15, 18). It has been observed that the wild-type phenotype (cell aggregation and normal production of all glycoproteins, including C2) is usually dominant over the fusion-inducing phenotype in mixed infections (6, 19, 20). These findings suggested to us that the glycoprotein whose synthesis is defective in infections with the polykaryocyte-forming mutants may normally act to suppress the fusion-inducing activity of another HSV-1 product. Previous studies have indicated that this fusion-promoting viral product may also be a membrane glycoprotein (21-24). In this paper we present evidence that HSV-1-induced cell fusion is governed by the activities of at least two viral glycoproteins, one of which (glycoprotein B2) promotes fusion whereas the other (glycoprotein C2) suppresses it. This conclusion is based on experiments done with HSV-1 mutants, including a polykaryocyte-forming mutant that fails to synthesize glycoprotein C2, a temperature-sensitive (ts) mutant that is unable to produce glycoprotein B2 at nonpermissive temperaAbbreviations: HSV, herpes simplex virus; HSV-1, HSV type 1; ts, temperature-sensitive; HEp-2, human epidermoid carcinoma no. 2; 199-V, medium 199 supplemented with 1% fetal calf serum; MOI, multiplicity of infection; PFU, plaque-forming units. * This paper is second in a series entitled "Membrane Proteins Specified by Herpes Simplex Viruses." The first paper is ref. 17. t Present address: Istituto di Microbiologia, Universita delgi Studi, 44100 Ferrara, Italy. § To whom reprint requests and correspondence should be addressed. R. Manservigi and P. G. Spear, unpublished data.
3914
Cell Biology: Manservigi et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 1. Numbers and morphologies of plaques formed by the parental mutant viruses and the double-mutant recombinant viruses at permissive and nonpermissive temperatures
Morphology of Number of plaques, PFU/ml Virus MP tsB5
340
390
Ratio 340/390
Parental viruses 0.5 6.0 X 108 1.1 X 109 6.8 X 104 1.7 X 109 2.5 X 104
plaques on HEp-2 cells* 340
390
pk. cytoagg.t
pk.
Double-mutant recombinants 5.2 X 104 pk. tsB5MP-1 3.4 X 108 6.5 X 103 1.0 X 105 pk. tsB5MP-2 3.0 X 108 3.0 X 103 3.2 X 104 pk. tsB5MP-3 2.2 X 109 7.0 X 104 4.2 X 104 pk. tsB5MP-4 2.1 X 108 5.0 X 103 >5.0 X 104 pk. tsB5MP-5 5.0 X 108
