Cell-Surface Receptors for Retroviruses and Implications for Gene Transfer A

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 mechanism 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. Cloned retrovirus receptors Retrovirus Receptor Type* Function Refs. Human immunodeficiency virus CD4 TM1 Immune recognition 12, 13 Fusin, CC-CKR-5 - G protein-coupled (coreceptors) TM7 chemokine receptors 14-16 Simian immunodeficiency virus CD4 TM1 Immune recognition 17 Murine ecotropic retrovirus Recl TM14 Basic amino acid transport 18-20 Murine amphotropic retrovirus Raml TM10-13 Phosphate transport 21-23 Gibbon ape leukemia virus Glvrl TM10-13 Phosphate transport 23, 24 Bovine leukemia virus Blvr TM1 ND 25, 26 Avian leukosis virus type A Tva TM1 LDL receptor-like protein 27 Feline immunodeficiency virus CD9 TM4 Signaling protein? 28, 29 ND, not determined; LDL, low density lipoprotein. *TM followed by a number indicates the number of transmembrane domains in the protein. Downloaded by guest on October 6, 2021 Colloquium Paper: Miller Proc. Natl. Acad. Sci. USA 93 (1996) 11409

hRaml Table 3. Nonreciprocal interference between 1OAl and Homo sapiens amphotropic retroviruses rRaml Vector titer, FFU/ml Rattus norvegicus LAPSN 3T3 + 3T3 + 3T3 + cRaml Cricetulus griseus pseudotype 3T3 AM-MLV 1OAl MoMLV hGlvrl Amphotropic 5 x 106 40 3 4 x 106 Homo sapiens 1OAl 7 x 106 6 x 106 2 x 102 6 x 106 cGIvrl Ecotropic 3 x 106 2 x 106 2 x 106 40 Cricetulus griseus The LAPSN vector encodes alkaline phosphatase and neomycin mGlvrl phosphotransferase. LAPSN vector with an amphotropic, 1OA1, or Mus musculus ecotropic pseudotype was made by using PA317 retrovirus packaging cells, wild-type 1OAl virus, or PE501 packaging cells, respectively. B0222.2 Transduction was measured by staining cells for alkaline phosphatase C. elegans 2 days after exposure to the vectors. Data are from Miller and Chen B0222.3 (32). C. elegans YGO4 Some Receptors Can Promote Entry of Retroviruses That H. influenzae Normally Utilize Independent Receptors ORF .. -.. I In human cells, gibbon ape leukemia virus (GALV) exclusively Thermoautotrophicum uses Glvrl for entry and amphotropic retrovirus exclusively Pho4 uses Raml. These facts are reflected in the assignment of these Neurospora crassa viruses to separate interference groups for human cells (Table YB81 1). However, analysis of the hamster homolog of Raml shows S. cerevisiae that it can function as a receptor for GALV or amphotropic PitA retrovirus (34). In addition, certain chimeric receptors made E. cofi between Raml and Glvrl can also function as receptors for PitB both viruses (Fig. 3). In this example, the hybrid receptor RRG E. coli promotes transduction by GALV or amphotropic pseudotype vectors with efficiencies similar to those found for GALV PitH Streptomyces halstedii vector transduction of cells expressing the normal human Glvrl receptor (GGG) or vector transduction of Pit amphotropic Mycobacterium leprae cells expressing the normal rat Raml (RRR) receptor. Thus, the ability of retroviruses to utilize certain receptor homologs FIG. 1. Dendrogram of amino acid sequence similarities among for entry, and therefore the interference pattern of these phosphate transporters. Distances between sequences were computed by the Genetics Computer Group program PILEUP. Overall sequence identity for the branch point at the far left is about 21%. Sequences 1 were obtained from GenBank: hRaml, L20852; rRaml, L19931; cRaml, U13945; hGlvrl, L20859; cGlvrl, U13946; mGlvrl, M73696; B0222.2 and B0222.3, U50312; YGO4, P45268; M. thermoauto trophi- UNINFECTED A.N-. p9bFk cum ORF, S08522; Pho4, M31364; YB8I, P38361; PitA, P37308; PitB, CELLS P43676; PitH, P41132; Pit, U15187. tropic vector transduction is blocked by the presence of AM-MLV, but is unaffected by the presence of ecotropic MoMLV. Nonreciprocal interference is displayed by the amphotropic and lOAl viruses, where lOAl-pseudotype vector 1 1 transduction is unaffected by the presence of amphotropic virus in the target cells, but amphotropic vector transduction AM is blocked by the presence of lOAl virus. These data suggested INFECTED r- N., -% -." /0-*Or-N that lOAl virus can enter cells by using a different receptor CELLS than that used by amphotropic virus, and that lOAl virus can also bind to the amphotropic receptor and block amphotropic 1 1 virus entry (Fig. 2). Given that lOAl virus can bind to Raml, we tested the ability of Raml to mediate entry of the lOAl-pseudotype LAPSN vector. We also tested Glvrl due to its similarity to Raml and thus the possibility that Glvrl was the alternative 1 OAl receptor for entry of lOAl virus. We found that expression of INFECTED e4n.4.-' fin..., human Raml, rat Raml, human Glvrl, or mouse Glvrl CELLS rendered Chinese hamster ovary (CHO) cells susceptible to 'KNAM lOAl-pseudotype LAPSN vector transduction (Table 4). Thus, '0 En v () lOAl virus can bind and enter cells by using either of two ll1. Rann1 different retrovirus receptors. Amphotropic virus can enter EnvOAl Viruses CHO cells expressing human or rat Raml, but not those .-~- Glvrl /-\, Env expressing human or mouse Glvrl (data not shown). These results explain the nonreciprocal interference observed be- FIG. 2. Nonreciprocal interference between 1OAl and ampho- tween lOAl and amphotropic retroviruses. tropic retroviruses. Downloaded by guest on October 6, 2021 11410 Colloquium Paper: Miller Proc. Natl. Acad. Sci. USA 93 (1996) Table 4. A 1OAl-pseudotype retroviral vector can utilize Raml or homolog is expressed but is nonfunctional. For example, Glvrl for cell entry ecotropic retroviruses infect rodent cells, but do not infect Vector titer, human cells, even though human cells express a homolog of the Receptor Species FFU/ml murine ecotropic receptor that is 87% identical to the mouse protein. In this case, only two amino acid changes are required Ram-1 Human 1 x 106 to convert the human protein into a functional receptor, or to Rat 3 x 105 render the mouse protein nonfunctional as an ecotropic ret- Glvr-1 Human 6 x 105 rovirus receptor (35, 36). Mouse 7 x 105 Other examples of virus restriction in different species are None <500 provided by viruses that utilize Pit receptor family members for Retrovirus receptor cDNAs were expressed by using the retroviral entry. A simple example is the restriction of GALV entry into vector LXSN. CHO cells were seeded at 2 x 104 per well (d = 3.5 cm) mouse cells, which, like the restriction to ecotropic virus in multiwell dishes on day 1. On day 2, cells were cotransfected with infection of human cells, is not due to lack of receptor homolog 2.5 jig of 13-galactosidase expression plasmid and 2.5 ,ug of the receptor expression construct. On day 3, one set of dishes was stained for expression, but to minor changes in mouse Glvrl compared ,B-galactosidase to assess transfection efficiency, whereas the other set with human Glvrl. was infected with 2 ,u of the LAPSN vector pseudotyped with the 1OAl A more complicated example is provided by 1OAl receptor retrovirus in the presence of 50% medium conditioned by CHO cells. usage in different species. As noted above, 1OAl-pseudotype On day 4, cells were stained for alkaline phosphatase and foci of virus can use human Raml or human Glvrl to enter CHO cells stained cells were counted. Transfection efficiencies were similar for (Table 4) and to enter human cells (32). However, in rat cells all constructs, as measured by ,B-galactosidase staining. Data are from 1OAl virus infection does not block GALV-pseudotype vector Miller and Miller (33). transduction (Table 5), showing that 1OAl cannot bind to or retroviruses, will depend on the specific receptors expressed in enter cells by using the rat GALV receptor. Indeed, 1OAl has the target cells. the same interference properties as amphotropic retrovirus in rat cells, and thus uses the amphotropic virus receptor for Retrovirus Interactions with Homologous Receptor Proteins entry. Thus, 1OAl virus behaves like an amphotropic virus in from Other Species Are Complex rat cells, but like a combination of an amphotropic virus and GALV in human cells. Therefore, the interference and recep- Limitations to retrovirus entry into cells from different species tor utilization properties of 1OAl virus depends on the cell type are due to variable expression of the receptor or its homologs, used for the analysis. or to amino acid sequence differences or posttranslational modifications in the receptor homologs in different species Env Amino Acid Sequence Is Not Predictive that inhibit virus binding or entry. The former mechanism for of Receptor Utilization virus resistance is primarily applicable to different cells from the same organisms that express variable levels of the receptor. It is not clear how retroviruses have evolved to utilize such a An example of this is provided by HIV, which for entry diverse group of receptors for entry into cells. One possibility requires the CD4 receptor that is found primarily on T is that mutations in the Env protein promote weak binding and lymphocytes and not on cells from many other tissues. Many entry through interaction with new receptors, and if the new examples of the latter mechanism ofvirus resistance have been receptor specificity is beneficial for virus survival, selective found for cells from different species, in which a receptor pressure favors further mutations that promote more efficient GALV vector, Amphotropic vector, Construct NIH 3T3 cells CHO cells (Ram-1) RRR I I (Glvr-1) GGG GRR GGR RGG RRG GRG I GGrG 1I I 10-4 10-3 10-2 -10-1 10-3I ----r-10-2 10-1I looI Vector-transduced cell foci Vector-transduced cell foci ,B-galactosidase-positive cell foci p-galactosidase-positive cell foci

FIG. 3. GALV- and amphotropic-pseudotype vector transduction of cells expressing hybrid Ram-1/Glvr-1 receptors. CHO or NIH 3T3 cells were seeded in 3.5-cm dishes at 2 x 104 cells per dish. On day 2, hybrid constructs cloned in the retroviral vector LXSN were cotransfected (1:1) with a plasmid encoding ,B-galactosidase (2.5 ,ug each). Parallel dishes were stained for ,3-galactosidase, whereas the other set was infected with the retroviral vector LAPSN pseudotyped by amphotropic Env [LAPSN(PA317)] or a GALV Env [LAPSN(PG13)] at a multiplicity of infection of about 2. Cells were stained for alkaline phosphatase activity on day 5. Values are the number ofvector-transduced (alkaline phosphatase-positive) foci divided by the number of ,B-galactosidase positive foci. Results are averages of duplicate dishes. The experiment was performed three times with similar results. Data are from Miller and Miller (33). Downloaded by guest on October 6, 2021 Colloquium Paper: Miller Proc. Natl. Acad. Sci. USA 93 (1996) 11411 Table 5. 1OAl-pseudotype LAPSN vector can use the r FAIDS amphotropic receptor, but not the GALV receptor, for entrv into 208F rat cells FeLV-A FeLV-B LAPSN Vector titer, FFU/ml pseudotype 208F 208F + AM-MLV 208F + GALV 1 QAl 1OAl 1 x 107 500 1 X 107 AM-MLV Amphotropic 2 x 106 200 2 x 106 MoMCF GALV 2 x 105 2 x 105 100 LAPSN vector with a 1OA1, amphotropic, or GALV pseudotype was NZB made by using wild-type 1OAl virus, PA317 retrovirus packaging cells, FrMLV or PG13 packaging cells, respectively. Transduction was measured by staining cells for alkaline phosphatase 2 days after exposure to the MoMLV vectors. Data are from Miller and Chen (32). AKV utilization of the new receptor. Another model involves re- GALV placement of portions of the retroviral Env protein with of cellular proteins that recognize other cell-surface FIG. 4. Dendrogram of amino acid similarities between different portions retroviral Env proteins. Distances between sequences were computed proteins. For example, incorporation of a portion of the by the Genetics Computer Group program PILEUP. Overall sequence erythropoietin protein into an existing Env protein might allow identity for the branch point at the far left is about 42%. Sequences the erythropoietin receptor to function as a new target for were obtained from GenBank: FAIDS, feline AIDS virus, M18247; retrovirus binding and entry. This model for acquisition of new FeLV-A, feline leukemia virus subgroup A, M12500; FeLV-B, feline receptor specificities parallels the process of retrovirus acqui- leukemia virus subgroup B, X00188; 1OA1, 1OAl MLV, M33470; sition of cellular oncogenes. Just as oncogenes can improve AM-MLV, M33469; MoMCF, Moloney mink cell focus-forming virus, retrovirus replication and survival, the ability to utilize new J02254; NZB, NZB MLV, K02730; FrMLV, Friend MLV, Z11128; receptors for cell entry should improve the survival potential MoMLV, J02255; AKV, AKV MLV, J01998; and GALV, M26927. of a retrovirus. In addition, the existence of cellular proteins that bind cell-surface molecules with high affinity seems a IOAI virus, which targets Raml or Glvrl for entry (32, 38). more likely source for the development of radically altered However, because Raml and Glvrl are related proteins, this retrovirus receptor specificities compared with random Env change does not represent a dramatic switch in receptor mutations. specificity, and it will be interesting to see if minor amino acid Predictions of the model for alteration ofretrovirus receptor changes in Env proteins can result in more dramatic changes utilization by incorporation of cellular genes is that retrovi- in receptor utilization. ruses that utilize similar receptors should contain more closely related Env proteins than retroviruses that use other receptors, Endogenous Synthesis of Env Protein Can Block and Env proteins should contain regions of similarity with Retrovirus Entry cellular proteins. A comparison of Env proteins from several retroviruses shows that retroviruses that use different recep- Retrovirus receptors can be rendered nonfunctional due to tors can be more highly related than those that use the same blockade by Env protein synthesized by a replication- receptor (Fig. 4). For example, GALV, lOA1, and subgroup B competent retrovirus. This is the basis for the virus interfer- feline leukemia virus (FeLV-B) all can use Glvrl as a receptor, ence discussed above. Interference with receptor function can but FeLV-B is more closely related to FeLV-A and FAIDS, also result from synthesis of Env proteins from endogenous which use different receptors, than it is to GALV or the lOAl retroviruses or fragments of retroviruses that are inherited in virus. Likewise, the lOAl virus is more closely related to the animals. A well-documented example of this phenomenon polytropic (MoMCF; Moloney mink cell focus-forming virus), involves the Fv-4 locus in mice (39), the phenotype of which is xenotropic (NZB), and ecotropic (FrMLV, MoMLV, and due to a truncated endogenous ecotropic retrovirus that is AKV) retroviruses than to GALV or FeLV-B. The same missing the gag and part of the pol genes, but which contains overall dendrogram is obtained even if one compares only the an intact env gene. Synthesis of this endogenous env gene 200 amino acids at the amino termini of the processed Env product in mouse tissues blocks infection and leukemia caused proteins that are directly involved in receptor binding (not by ecotropic retroviruses by blocking the ecotropic retrovirus shown). Comparison of these amino terminal receptor-binding receptor. Other examples of this phenomenon have been found regions of the Env proteins from viruses in the FeLV, MLV, for avian leukosis viruses in chickens (40) and for MCF viruses and GALV groups reveals a similar amino acid framework in mice (41). surrounding two variable regions, with no common features in the variable regions that would predict the receptor utilization Hamster Cells Secrete a Factor That Blocks Retrovirus pattern (37). In addition, no similarities have been found Infection and a Similar Factor Is Found in Hamster Serum between regions of retroviral Env proteins and already se- quenced cellular proteins. Thus, the data to date favor a CHO cells are resistant to infection by many retroviruses. In mutational origin for new receptor specificities rather than a most cases, this resistance can be abrogated by prior treatment model involving acquisition of cellular proteins that can bind of the cells with the glycosylation inhibitor tunicamycin. The new cell-surface receptors. The mutational model can explain resistance to GALV and amphotropic retrovirus infection is the diversity in Env sequences among viruses that recognize due to a secreted protein factor that blocks infection (42). the same receptor as the result of convergent evolution of Thus, addition of CHO cell-conditioned medium to CHO cells different parental retroviruses. that have been made susceptible to infection by treatment with Another argument in favor of a mutational model for tunicamycin blocks infection by GALV and amphotropic acquisition of new receptor specificities is the finding that small viruses. In contrast, addition of the conditioned medium does changes in a virus Env protein can result in a new receptor not block infection of tunicamycin-treated CHO cells by an specificity. For example, no more than six amino acid changes ecotropic retrovirus, showing that the medium is not simply are required to convert an amphotropic Env, which targets toxic, and that the effect is specific for retroviruses with Raml, to one having the receptor utilization properties of particular Env proteins. Interestingly, the CHO cell- Downloaded by guest on October 6, 2021 11412 Colloquium Paper: Miller Proc. Natl. Acad. Sci. USA 93 (1996) conditioned medium does not block amphotropic vector trans- different pseudotypes. Approximate host ranges of packaging duction of human or mouse cells, nor does it block transduction cells derived using mammalian retroviruses are shown in Table of CHO cells made susceptible to amphotropic vector trans- 7. A listing of specific packaging cell lines can be found in ref. duction by prior introduction of genes expressing human or rat 46. The best vector pseudotype for a given application will be Raml (21, 22), indicating that the factor can bind to and block further influenced by the specific target tissue and the expres- the hamster receptor but not the human or rat receptors. sion of suitable levels of receptors with proper posttransla- Hamster serum contains a similar factor that can block tional modifications to allow efficient virus entry. For example, retrovirus infection of tunicamycin-treated CHO cells (Table Glvrl is overexpressed compared with Raml in hematopoietic 6). Addition of 5% serum from Chinese hamsters completely cells (23), and vectors with a GALV pseudotype have been blocked transduction by an amphotropic vector, and 12.5% found to transduce hematopoietic cells more efficiently than serum from Syrian hamsters also significantly inhibited trans- the same vectors with an amphotropic pseudotype (47, 48). duction. In contrast, addition of 25% fetal bovine serum had no effect on transduction of tunicamycin-treated CHO cells by Conclusions the amphotropic vector. Like the CHO cell-conditioned medium, the hamster sera had no effect on amphotropic vector Retroviruses utilize a diverse set of proteins for cell entry. infection of HeLa human cells (Table 6), indicating a species Single proteins are apparently required for binding and entry specificity for the factor. of most retroviruses, although two proteins are required for Thus, hamster serum contains a similar, potentially identi- HIV. Although virus entry is dependent on the level of cal, inhibitor of retrovirus infection to that secreted from CHO receptor expression in particular cells, there are many other cells. Based on the principle of virus interference, the factor factors that govern utilization of a receptor or its homologs in could be a fragment of an Env protein that is secreted from different species. Subtle alterations in the amino acid sequence cells and blocks infection by binding to the virus receptor. of receptor homologs in different species can dramatically Alternatively, it could be a normal cellular protein that nat- affect virus entry, either as a direct result of changes in the urally interacts with the phosphate transporter that serves as primary amino acid sequence or as an indirect result of altered a receptor for GALV and amphotropic viruses resulting in a protein modifications such as glycosylation. Indeed, restricted block to infection. virus host range is not generally due to a lack of expression of homologous receptor proteins, but is more often related to Receptor Glycosylation Can Affect Retrovirus Entry minor alterations in these proteins. In addition, soluble proteins secreted by some cells and present in some animals, and Retroviral interference can by reversed by inhibitors of gly- retroviral Env proteins synthesized from replication- cosylation that affect Env processing and subsequent binding competent viruses or from endogenous virus sequences, can to virus receptors (44). In addition, inhibitors of glycosylation block receptor utilization. These are all important consider- can have direct effects on a retrovirus receptor to modulate ations in the design of retroviral vectors for gene transfer in infection. For example, the ecotropic retrovirus receptor ho- cultured cells and in animals. molog on Mus dunni cells functions as a receptor for most Recently it has become clear that certain retroviruses can ecotropic retroviruses with the exception of MoMLV. Tuni- use more than one receptor for entry into some cell types, and camycin treatment renders the cells susceptible to infection by some receptors can promote entry of retroviruses that nor- MoMLV, and alteration of a single amino acid in the Mus mally utilize different receptors in other cells. These results dunni receptor to prevent glycosylation at that site results in a seriously complicate attempts to classify retroviruses into receptor that promotes MoMLV infection (45). Thus, subtle groups based on receptor utilization, as determined by inter- changes in receptor glycosylation can have a major effect on ference analysis, because these groupings depend on the the ability of a retrovirus to utilize the receptor for cell entry. particular receptors expressed on the cell type used for the analysis. In fact, this problem was appreciated long before the Vector Pseudotypes Available for Gene molecular basis for this phenomenon was determined (49). Transfer Applications Further development of retroviral vectors for gene transfer applications has involved the incorporation of Env proteins Given the complicated factors that govern retrovirus entry into cells from different tissues and different species, it is helpful Table 7. Host range of selected retrovirus packaging cells that there are a wide range of retrovirus packaging cell lines Target that are available for production of retroviral vectors with cells Ecotropic Amphotropic GALV RD114 1OAl Table 6. Hamster sera inhibit amphotropic vector infection of Mouse + + - - + tunicamycin-treated CHO cells but not HeLa cells Rat + + + - + Hamster - +/_ + - + Target Vector titer, Inhibition, Rabbit - + + cells Additional serum CFU/ml % Mink - + + + CHO None 1 x 103 Cow - +/_ + Chinese hamster (5%) <10 >99 Cat - + + + + Syrian hamster (12.5%) 35 97 Dog - + + + + Fetal bovine (25%) 2 x 103 Monkey - + + + + HeLa None 3 x 105 Human - + + + + Chinese hamster (5%) 2 x 105 Chicken - +/- + Syrian hamster (12.5%) 3 X 105 The ability ofvectors from the indicated packaging cells to transduce The indicated target cells were plated at 105 per 6-cm dish on day target cells from the indicated species is shown as + if the cells can be 1, infected with an amphotropic-pseudotype vector carrying the neo transduced, - if the cells cannot be transduced, and as +/- if there gene on day 2 in the presence of culture medium containing 5% FBS is poor transduction or if there is variable transduction of different (no additional serum), 5% FBS plus 5% Chinese hamster serum, 5% cells from the indicated species. These evaluations are only intended FBS plus 12.5% Syrian hamster serum, or 5% FBS plus 25% additional as a general guide because there are many factors that can influence FBS, and G418-resistant colony formation was measured. Inhibition is transduction rates, including the particular animals from within each reported only when >50%. Data are from Miller and Miller (43). species that are the source of the target cells. Downloaded by guest on October 6, 2021 Colloquium Paper: Miller Proc. Natl. Acad. Sci. USA 93 (1996) 11413 from other virus families, such as the vesicular stomatitis virus 18. Albritton, L. M., Tseng, L., Scadden, D. & Cunningham, J. M. G protein (3, 50) and efforts to alter the receptor specificity of (1989) Cell 57, 659-666. existing retroviral Env proteins by the incorporation ofpeptide 19. Kim, J. W., Closs, E. I., Albritton, L. M. & Cunningham, J. M. or antibody domains that can bind to other cell-surface pro- (1991) Nature (London) 352, 725-728. 20. Wang, H., Kavanaugh, M. P., North, R. A. & Kabat, D. (1991) teins (51, 52). An understanding of the principles governing Nature (London) 352, 729-731. cell entry by naturally occurring retroviruses will help in the 21. Miller, D. G., Edwards, R. H. & Miller, A. D. (1994) Proc. Natl. design and application of these strategies. Acad. Sci. USA 91, 78-82. A fascinating aspect of retroviruses is their utilization of 22. van Zeijl, M., Johann, S. V., Closs, E., Cunningham, J., Eddy, R., diverse proteins for cell entry. The analysis presented here Shows, T. B. & O'Hara, B. (1994) Proc. Natl. Acad. Sci. USA 91, favors a mutational basis for retrovirus evolution to utilize new 1168-1172. receptors, rather than acquisition and expression of cellular 23. Kavanaugh, M. P., Miller, D. G., Zhang, W., Law, W., Kozak, proteins that naturally bind to cell-surface receptors, but more S. L., Kabat, D. & Miller, A. D. (1994) Proc. Natl. Acad. Sci. USA information is needed to resolve this issue. 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