Proc. Natl. Acad. Sci. USA Vol. 93, pp. 11407-11413, October 1996 Colloquium Paper This paper was presented at a colloquium entitled "Genetic Engineering of Viruses and of Virus Vectors," organized by Bernard Roizman and Peter Palese (Co-chairs), held June 9-11, 1996, at the National Academy ofSciences in Irvine, CA Cell-surface receptors for retroviruses and implications for gene transfer A. DUSTY MILLER Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Room C2-023, Seattle, WA 98109 ABSTRACT Retroviruses can utilize a variety of cell- occurring retroviruses can use a variety of different proteins surface proteins for binding and entry into cells, and the for cell entry, although in general individual retroviruses cloning of several of these viral receptors has allowed refine- appear to recognize- a single receptor. Utilization of addi- ment of models to explain retrovirus tropism. A single recep- tional cell-surface proteins for vector entry has been tor appears to be necessary and sufficient for entry of many achieved by incorporation of polypeptides into the Env retroviruses, but exceptions to this simple model are accu- protein to alter its receptor binding properties or by replace- mulating. For example, HIV requires two proteins for cell ment of the retroviral Env protein with surface proteins from entry, neither of which alone is sufficient; lOAl murine other viruses. These alterations can allow targeting of par- leukemia virus can enter cells by using either of two distinct ticular cells that express specific proteins or an expansion of receptors; two retroviruses can use different receptors in some the range of cells that can be transduced by targeting broadly cells but use the same receptor for entry into other cells; and expressed proteins. In this paper I will review the factors that posttranslational protein modifications and secreted factors govern retrovirus binding and entry into cells and implica- can dramatically influence virus entry. These findings greatly tions for the design of retroviral vectors. complicate the rules governing retrovirus tropism. The mech- anism underlying retrovirus evolution to use many receptors Virus Interference for cell entry is not clear, although some evidence supports a mutational model for the evolution of new receptor specific- Early evidence that retroviruses use multiple receptors for cell ities. Further study offactors that govern retrovirus entry into entry came from studies of virus interference. Infection of a cells are important for achieving high-efficiency gene trans- cell by a replication-competent retrovirus results in synthesis duction to specific cells and for the design of retroviral vectors of a retroviral Env protein that binds to the receptor used for to target additional receptors for cell entry. virus entry. This effectively blocks entry of the original virus and other retroviruses that target the same receptor, whereas Many features make retrovirus vectors a good choice for gene entry of retroviruses that use different receptors is unaffected. transfer into animal cells. Most importantly, these vectors Interference between retroviruses has been shown to occur at integrate efficiently into the target cell genome to promote the level of virus entry into cells and not at some other step in stable gene transfer, and integration is precise with respect to the virus life cycle. By interference analysis, retroviruses that the virus genome, resulting in unrearranged transfer of the infect human cells have been assigned to eight groups that use desired genes. The only other integrating vector is derived different receptors on human cells (Table 1). The genes from adeno-associated virus, but integration is inefficient (1) encoding these receptors are scattered on different chromo- and appears not to be precise with respect to the viral genome somes (Table 1), indicating that the receptors are different (2). In addition, retroviral vectors can transduce both dividing proteins. and non-dividing cells, although this is true of vectors derived from HIV (3) and not the commonly used vectors derived from Cloned Retrovirus Receptors murine leukemia viruses, which require cell division (4). Furthermore, retrovirus vectors can be designed to eliminate In 1984 CD4 (previously called T4) was identified as a all viral protein coding regions without affecting gene transfer receptor for HIV-1, and became the first known retrovirus rates, and can be made in the absence of replication-competent receptor (12, 13). Since then, six additional retrovirus re- virus by using retrovirus packaging cell lines, which supply all ceptors have been identified and their cDNAs cloned (Table of the viral proteins required for vector transmission. Gene 2). All except CD4 appear to be sufficient for entry of the transfer and expression mediated by such replication- corresponding retroviruses by the criteria that expression of incompetent vectors is called transduction to differentiate this these receptors in nonpermissive cells renders the cells process from virus infection followed by further virus replica- susceptible to infection. In contrast, CD4 transfer into tion. nonpermissive mouse cells does not allow infection by HIV. A key consideration in retroviral vector design is the HIV binds to all cells that express CD4, but another factor source of the viral envelope (Env) protein present on vector is required for HIV entry. Recently, a coreceptor for T-cell virions, because this protein binds to specific cell-surface tropic HIV-1 strains has been found and was named fusin to proteins and is the primary determinant of the range of cells indicate its presumed role in virus entry following HIV-1 that can be transduced by the vector. The name of the virus binding to CD4 (14). Expression of the human CD4 and fusin or the virus group from which the Env protein was derived proteins in mouse cells renders the cells susceptible to HIV-1 will be referred to as the pseudotype of the vector. Naturally infection, whereas either protein alone is insufficient. Even The publication costs of this article were defrayed in part by page charge Abbreviations: MLV, murine leukemia virus; AM-MLV, amphotropic payment. This article must therefore be hereby marked "advertisement" in MLV; MoMLV, Moloney MLV; CHO, Chinese hamster ovary; accordance with 18 U.S.C. §1734 solely to indicate this fact. GALV, gibbon ape leukemia virus; FeLV, feline leukemia virus. 11407 Downloaded by guest on October 6, 2021 11408 Colloquium Paper: Miller Proc. Natl. Acad. Sci. USA 93 (1996) Table 1. Retrovirus interference groups in human cells Interference Human chromosome group Virus Description that encodes receptor 1 RD114 Cat endogenous virus 19 SNV Avian spleen necrosis virus BaEV Baboon endogenous virus SRV-1 Simian retrovirus SRV-2 Simian retrovirus SRV-3 (MPMV) Simian retrovirus SRV-4 Simian retrovirus SRV-5 Simian retrovirus PO-1-Lou Spectacled langur retrovirus SMRV Squirrel monkey retrovirus 2 MLV-A Amphotropic murine leukemia virus 8 3 MLV-X Xenotropic murine leukemia virus 4 FeLV-C Feline leukemia virus 5 FeLV-B Feline leukemia virus -2 SSAV Simian sarcoma-associated virus GALV Gibbon ape leukemia virus 6 BLV Bovine leukemia virus 7 HTLV-1 Human T-cell leukemia virus 17 HTLV-2 Human T-cell leukemia virus ChTLV Chimpanzee T-cell leukemia virus STLV Simian T-cell leukemia virus 8 HIV-1 Human immunodeficiency virus 12 HIV-2 Human immunodeficiency virus SIV Simian immunodeficiency virus Interference data are from Sommerfelt and Weiss (5), and for SNV, from Kewalramani et al. (6). Chromosome localization data are from the following references: group 1 (7), group 2 (8), group 5 (9), group 7 (10), and group 8 (11). more recently, a second protein related to fusin and previ- addition, these proteins serve as receptors for distinct groups ously named CC-CKR-5 has been found to be a coreceptor of viruses in human cells (Table 1). for macrophage-tropic HIV-1 strains (15, 16). These results, showing that two proteins are required for The lOAl Retrovirus Can Use Either of HIV-1 entry, raise the possibility that coreceptors are re- Two Receptors for Cell Entry quired for entry of other retroviruses. However, their de- tection will require the identification of nonpermissive cells Studies of cloned retrovirus receptors and most virus inter- for which transfer of the known receptors does not render the ference data suggested that individual retroviruses bind to a single protein for entry into cells. When different viruses bind cells susceptible to infection. Some retroviruses have a very to the same receptor, they typically show reciprocal inter-fer- wide host range; thus, if other proteins are required for entry ence; that is, infection of cells by either virus blocks entry by of these viruses, functional homologs of these coreceptors the other virus. The finding of nonreciprocal interference must be distributed in cells from many species. widely between some retroviruses complicated this picture. In the Two of the cloned retrovirus receptors, Raml and Glvrl, are example shown (Table 3), transduction by a vector with an closely related at the protein sequence level (21, 22, 24), and amphotropic, a lOA1, or an ecotropic pseudotype was mea- both are sodium-dependent phosphate transporters (23). sured in NIH 3T3 mouse cells infected with amphotropic MLV These proteins are members of a large family of known and (AM-MLV), lOAl MLV, Moloney MLV, or no virus. A typical presumptive phosphate transporters from many organisms pattern of interference for viruses that use different receptors (Fig. 1). However, Raml and Glvrl are clearly distinct since for cell entry is shown by the amphotropic and ecotropic the genes encoding these proteins are located on different viruses, where ecotropic vector transduction is blocked by the chromosomes in humans and mice (8, 9, 30, 31) and they show presence of ecotropic MoMLV in the target cells, but is very different patterns of expression in animal tissues (23). In unaffected by the presence of amphotropic virus, and ampho- Table 2.