Sindbis virus is an alphavirus with a very wide host range, being able to infect many birds and mammals as well as mosquitoes. We have isolated a monoclonal antibody that largely blocks virus binding to mammalian cells. This antibody was found to be directed against the C-terminal domain of the high-affinity laminin receptor, a 67-kDa protein present on the cell surface that binds with high affinity to basement membrane laminin and that is known to be important in development and in tumor invasion. This receptor is believed to be formed from a 295-amino-acid polypeptide that is modified in some unknown way after translation. The primary sequence of this 295-amino-acid protein is highly conserved among mammals. We found the hamster amino acid sequence to be identical to a mouse sequence and to differ at only two amino acids from a human sequence and at two amino acids from a bovine sequence. To verify the importance of the laminin receptor for infection by Sindbis virus, hamster cells were stably transfected with the gene encoding the 295-amino-acid protein under the control of a high-efficiency promoter. Such transfected hamster cells overexpressed the laminin receptor at the cell surface, bound severalfold more Sindbis virions than did the parental cells, and became infected by Sindbis virus with a higher efficiency. In contrast, cells transfected with the antisense gene expressed less laminin receptor on the surface and were less susceptible to the virus. Binding of the virus varied linearly with the amount of laminin receptor on the cell surface, whereas infectivity measured with a plaque assay varied with the 1.4 power of the receptor concentration, suggesting that interaction with more than one receptor aids virus penetration. By these criteria, the laminin receptor functions as the major receptor for Sindbis virus entry into mammalian cells. We also found that the anti-laminin receptor antibody partially blocked Sindbis virus binding to mosquito cells, suggesting that the laminin receptor is conserved in mosquitoes and functions as a Sindbis virus receptor in this host. The wide distribution of this highly conserved receptor may be in part responsible for the broad host range exhibited by the virus, which infects a wide range of mammals and birds as well as its mosquito vector and can infect many different tissues within these hosts.(ABSTRACT TRUNCATED AT 400 WORDS)