Retrovirus entry into cells depends on the presence of specific proteins that bind the viral Env protein and help trigger conformational changes in Env that lead to fusion of the virus and cell membranes and entry of the virus core into the cell. A wide variety of proteins have been found to serve as receptors for different retroviruses, based primarily on their ability to promote virus entry after expression in cells that are not naturally permissive for virus entry (). In most cases, a single protein suffices to render otherwise nonpermissive cells susceptible to virus entry. Typically, these proteins promote virus binding, and may also promote virus fusion with the cell membrane. For other viruses (for example, HIV) there are distinct binding and fusion receptors that are required for virus entry. Retrovirus receptors are key determinants of the species and cell types that a retrovirus can infect, and thus are primary determinants of the host range and the type of disease induced by the virus.
To identify the cell-entry receptor for JSRV we used a retroviral vector that encodes human placental alkaline phosphatase (AP) and that was packaged into virions bearing the JSRV Env protein on the virion surface [11
]. In early experiments we found that this vector could transfer and express (transduce) the AP marker protein gene to sheep and human cells, but not to rodent cells including those from mice, rats, and hamsters. This allowed us to develop a genetic screen to identify the human gene that when expressed in rodent cells would allow vector transduction. As target cells we used a set of 80 hamster cell lines carrying different fragments of DNA that had been produced by fusing hamster cells with irradiated human cells. This allowed us to identify the chromosomal location of the receptor within a few hundred kilobase pairs of DNA [11
]. We were lucky to find that this region had been cloned as a set of overlapping cosmid clones, and it was relatively straightforward to identify the gene encoding the receptor by testing hamster cells for JSRV vector susceptibility following transfection of individual cosmids into the cells [8
]. This genetic analysis indicated that only one gene served as a receptor for JSRV, but to reinforce this conclusion, we tested all of the human hyaluronidase family members for receptor activity, and found no activity associated with human Hyal1, Hyal3, Hyal4, or Spam1 [8
]. These data indicate that Hyal2 is the only protein in the human genome that functions as a JSRV receptor.
We also tested hyaluronidase family members from other species for receptor function to determine if receptor function correlates with the ability of the JSRV vector to transduce cells from different species (). Indeed, mouse Hyal2 functioned poorly as a receptor for JSRV vector cell-entry when expressed in mouse or hamster cells, consistent with the inability of the JSRV vector to transduce mouse cells [8
]. In contrast, sheep Hyal2 functioned well as a receptor for JSRV vector cell-entry when expressed in mouse or hamster cells, consistent with the high susceptibility of sheep cells to JSRV vector transduction [10
]. An intermediate result was obtained for rat Hyal2, where overexpression of rat Hyal2 in mouse, hamster, or rat cells rendered the cells susceptible to JSRV vector transduction, but rat cells are normally resistant to vector transduction [12
]. Additional experiments showed that JSRV Env binds rat Hyal2 less well than it does human Hyal2, supporting the interpretation that higher levels of rat Hyal2 are required to promote efficient JSRV vector transduction than are normally expressed on rat cells [12
]. In conclusion, these experiments show that expression of a functional Hyal2 protein is the primary determinant of JSRV Env-mediated virion entry into cells.
Receptor activity of Hyal2 orthologs (bold) and paralogs
Using a retroviral vector encoding AP packaged into virions bearing either the ENTV or JSRV Env proteins on the virion surface, we found that ENTV Env promotes infection of a more restricted range of cell types than does the JSRV Env [10
]. Given the similarity in ENTV and JSRV Env amino-acid sequences, we first tested whether ENTV Env might use Hyal2 for cell entry as does JSRV Env. Indeed, both sheep and human Hyal2 can serve as cell-entry receptors for virus bearing the ENTV Env. Interestingly, Hyal2 expression was not sufficient to promote entry of the ENTV vector into all cells, indicating that other factors are important for ENTV Env-mediated entry into cells [13