Residues in the Murine Leukemia Virus Capsid That
http://www.100md.com
病菌学杂志 2005年第10期
Institut de Génétique Moléculaire de Montpellier, CNRS UMR5535, IFR122, 1919 Route de Mende, F-34293 Montpellier Cedex 5, France
ABSTRACT
We identified new residues within a 101-amino-acid stretch of the murine leukemia virus capsid that differentially modulate resistance and susceptibility to the mouse Fv1 and human Ref1 genes. Among these residues, aspartate 92 and histidine 117 are both required for Fv1b resistance, whereas the latter is sufficient to confer Ref1 resistance.
TEXT
The Friend virus susceptibility gene 1 (Fv1) restricts replication of N-tropic and B-tropic murine leukemia viruses (MLV) in laboratory mouse strains harboring the b or n allele, respectively (15, 29). Several other alleles are present in wild mice (18, 26). Fv1 products are related to retroviral capsids (CA) with closest homologies to human (HERV-L) and mouse (MuERV-L) endogenous retroviral sequences (2, 5). The Fv1 restriction does not block reverse transcription but prevents proviral DNA integration through unknown mechanisms. Restriction at early stages of retroviral replication has been described in other mammalian species, including monkeys and humans (3, 6, 8, 11, 14, 30, 32). The most studied restriction genes in primates are the human Ref1 (32) and simian Lv1 (4, 8) genes. The primate TRIM5 protein, a member of the tripartite motif protein superfamily, unrelated to Fv1, has recently been shown to be an early-stage restrictive factor of primate lentiviruses (31) associated with Ref1 and Lv1 activities (13, 17, 25, 34). Ref1 and Lv1, in contrast to Fv1, target a wide range of mammalian lentiviruses and have been shown to restrict N-tropic but not B-tropic MLV (11, 32).
MLV CA proteins are the main target of the Fv1, Ref1, and Lv1 restrictions (9, 12, 22-24). Residue 110 of MLV CA has been identified as the discriminating target determinant between N-tropic, Ref1-susceptible MLV and B-tropic, Ref1-resistant MLV in murine (18) as well as other mammalian (17, 25, 32, 34) cells. However, under certain conditions, changes at CA residues 105 (10) and 114 (16) have also been reported to modulate Fv1 restriction.
NB-tropic MLV strains, such as the prototypic Friend and Moloney MLV, are resistant to all tested Fv1 alleles. Moloney MLV has also been documented to be resistant to Ref1 and Lv1 (25, 31, 34), but the status of Friend MLV with respect to these primate restriction systems has not been described. Intriguingly, Friend MLV is NB-tropic despite the presence of the N-tropic hallmark, an arginine at position 110 (Arg110), and conserved residues at positions 105 and 114 (Fig. 1A). Therefore, evasion of the mouse restriction systems, as observed with NB-tropic Friend MLV, appears to involve a residue(s) other than residue 110. To determine the sensitivity of Friend MLV to Ref1 and more precisely map CA determinants affecting resistance to the mouse Fv1b and Fv1n alleles, we derived Friend MLV CA-based constructs and mutants. Here, we demonstrate that Friend MLV is resistant to the human Ref1 restriction and identify CA residues that condition N, B, NB, and Ref1 tropisms. We thereby describe residue combinations that differentially modulate susceptibility and resistance to Fv1 and Ref1 restrictions.
A 101-amino-acid fragment recapitulates MLV capsid susceptibilities to Fv1 and Ref1 targets. Early studies of Fv1 MLV target determinants mapped differences between N- and B-tropic MLV to a 302-bp fragment encoding a CA sequence comprised between residues 33 and 133. Residues 109 and 110 were reported to be the MLV NB determinant (9) (Fig. 1A). We constructed a Friend MLV-based Gag-Pol expression vector (pC57GP) that allowed allelic exchanges of this 302-bp MLV CA cassette between BamHI and BstXI restriction sites. These sites were either naturally present or introduced by PCR-directed mutagenesis, in prototypic MLV strains of different NB tropisms. Additional EheI and XhoI sites were also used to swap smaller fragments (Fig. 1A). Prototypic CA sequences were derived from the N-tropic Akr-623 and B-tropic WNB5 MLV clones (7, 19) (kind gifts of A. Rein) and from the NB-tropic Friend MLV 57 and Moloney MLV 8.2 clones (see reference 27 for details). We also PCR amplified and sequenced a BamHI-BstXI fragment from the historical N-tropic Tennant isolate of the Friend complex (NT1; a kind gift of S. Gisselbrecht). NT1 differs from NB-tropic Friend MLV 57 at positions 92 and 124, with glutamate and serine residues substituted for aspartate and alanine, respectively (Fig. 1A).
Single-round infectious retroviral particles with MLV cores were harvested from 293T cell supernatants 48 h after cotransfection of one of the gag-pol vectors with the pCSI-G vesicular stomatitis virus G protein expression vector (1) and the pLAPSN retroviral vector carrying the neo and human placental alkaline phosphatase reporter genes (20). Viral tropism was assayed on Fv1-nonrestrictive Mus dunni (dunni) cells or on dunni cells stably transfected with Fv1n (dunnin) or Fv1b (dunnib) expression vectors (Fv1 plasmids were a kind gift of J. Stoye). Ref1 restriction was tested on human HT1080 cells (Fig. 1B) and TE671 cells as indicated.
All reported virion preparations had similar infectious levels as measured on nonrestrictive dunni cells, with titers ranging from 105 to 106 focus-forming units (FFU)/ml. All parental MLV prototypic tropisms were reproduced when using our chimeric CA-based vector system. Thus, Fv1b and Fv1n restrictions resulted in a 20- to 400-fold drop in titers. Strong Ref1-mediated restriction, with an average 200-fold drop in titers, was also observed with N-MLV virions (Fig. 1B and 2A). The NB-tropic F-MLV construct was resistant to both Fv1b and Fv1n, as well as to Ref1. Surprisingly though, the Fv1b-susceptible NT1-derived construct was resistant to Ref1-mediated restriction (Fig. 1B). This Ref1 resistance of an N-tropic MLV was observed on HT1080 and TE671, human cell lines that exert strong Ref1 restriction on other retroviruses (32), as well as on dunni cells stably transduced with human TRIM5 expressed from the pLXSN retroviral expression vector (dunni/TRIM5) (data not shown). This is the first identified CA sequence that distinguishes between Fv1b and Ref1 susceptibilities.
MLV resistance to Fv1 is due to multiple determinants that modulate position 110. Arg110 is the discriminating determinant between N-tropic, Ref1-susceptible MLV and B-tropic, Ref1-resistant MLV (17, 18, 25, 32, 34). However, the fact that NB-tropic and N-tropic Friend MLV strains encoding Arg110 are resistant to Fv1b, Ref1, or both indicated that other residues in the 101-amino-acid-long capsid region govern Fv1b and Ref1 susceptibilities. We therefore further evaluated the bases for the Friend MLV Fv1 and Ref1 resistance with smaller domain swaps.
A domain swap introducing N-MLV residues 33 to 100, excluding the canonical 110 residue, into Friend MLV was sufficient to render the latter susceptible to Fv1b restriction (chimera NNF, Fig. 2B). Friend MLV CA residues 92 to 95 consist of a DIND motif corresponding to an EVDA motif in prototypic N-tropic MLV (Fig. 1A). The swap of this motif was sufficient to convert Friend MLV to N-tropism as shown by a full Fv1b susceptibility of the corresponding construct (FNF, Fig. 2B). Thus, the DIND motif plays a key role in suppressing the accessibility of the N-tropic target in Friend MLV. Notably though, swapping of this motif did not render Friend MLV susceptible to Ref1, and this resistance was maintained even following a larger swap from residues 33 to 100 (NNF, Fig. 2B).
We further assessed the suppressive effect of the Friend MLV DIND motif in a parental B-MLV (BFB) or N-MLV (NFN) context. The sole substitution of this DIND motif was not sufficient to modulate Fv1 or Ref1 resistance in either context (Fig. 2C). When this swap was combined with that of the upstream fragment, B-tropic virions became resistant to the Fv1n restriction. Therefore, glycine, threonine, and/or asparagine at positions 35, 46, and 82, respectively, conditions CA targeting by Fv1. Nevertheless, this contribution was not as potent in the N-tropic context, since FFN particles remained susceptible to Fv1b and Ref1 restrictions, albeit with a slightly decreased susceptibility (Fig. 2C). The prominent exposure of Asp82, according to the recently published structure of the N-AKV CA amino-terminal domain (21) (Fig. 4B), is in favor of a role for this residue in Fv1 tropism, as also suggested by others (28).
Mutation of histidine to leucine at position 117 is sufficient to render Friend MLV susceptible to Fv1b and Ref1 restrictions. In contrast to Friend MLV virions, FFN virions (Fig. 2C) were susceptible to both Fv1b and Ref1 restrictions. As the difference between these viruses was limited to amino acid changes at positions 107 and 117, we introduced individual substitutions at these positions in the Friend MLV background. Infections on restrictive cell lines showed that the presence of leucine at position 117, F(H117L), was sufficient to induce a susceptibility to Fv1 and Ref1 restrictions (Fig. 3A). Charge rather than aromatic properties was crucial for resistance, as mutation of His117 to a lysine in Friend MLV maintained this property whereas mutation to either phenylalanine or tyrosine reversed resistance (not shown).
Loss of Fv1b but not Ref1 restriction in the presence of aspartate 92. As indicated above, suppression of the canonical residue 110 as a target for Fv1b is due to the combined presence of His117 and a DIND motif. Interestingly, the N-tropic NT1 strain harbors an EIND motif. The aspartate-to-glutamate change was sufficient to confer Fv1b susceptibility with no effect on Ref1 resistance, as observed with the F(D92E) mutant (Fig. 3B). When introduced alone, the second amino acid difference between the Friend MLV and NT1 strain CA at position 124 did not influence either Fv1 or Ref1 susceptibility [F(A124S), Fig. 3B]. Therefore, Glu92 was key to the recognition of the Fv1b target in the presence of His117, while it did not modulate Ref1 restriction. Similar data were obtained using dunni/TRIM5 cells.
In conclusion, while Arg110 is required for both Fv1b and Ref1 restrictions, Leu117 is also a major target determinant for both genes (Fig. 4A, left panels). Furthermore, Asp92 is required for efficient resistance to Fv1b when combined with His117. This is in agreement with a recent report using a different model of Fv1 restriction (28). However, we found that Ref1 resistance was not affected by Asp92 (Fig. 4A, right panels). This is compatible with the recently reported crystal structure of the CA amino-terminal domain of an N-tropic MLV in which residue 92, on the one hand, and residues 110 and 117, on the other, are located in two separate neighboring helices, -helices 5 and 6, respectively (21) (Fig. 4B). Our results suggest that CA MLV recognition by Fv1 involves both -helices, which are exposed to the outside of the amino-terminal CA hexamer (21), whereas Ref1/TRIM5 recognition appears to involve mainly -helix 6. While -helices 4 and 6 appear structurally conserved between human immunodeficiency virus type 1 and N-Akv CA, the interconnecting sequence, which includes -helix 5 of N-Akv CA, displays a variable succession of loops and helices (21). Interestingly, the latter sequence comprises residue 92 of the MLV CA, which we identified as a major Fv1-modulating determinant, and the human immunodeficiency virus type 1 CA CypA-binding loop involved in Lv1 restriction (12, 23, 33), thus providing differential target patterns recognized by these restrictive cellular mechanisms.
Nucleotide sequence accession number. The GenBank accession number of the BamHI-BstXI fragment from the historical N-tropic Tennant isolate of the Friend complex is AY883167.
ACKNOWLEDGMENTS
We thank N. Taylor for helpful discussions and critical reading of the manuscript and L. Boone, F.-L. Cosset, S. Gisselbrecht, A. Rein, J. Stoye, and G. Towers for providing us with materials. We also thank Muriel Audit, Dorothée Molle, and Valérie Thibert for their participation in the preparation of some of the reagents used in this study and all members of our laboratory for their continuous input.
A.L. is funded by the Ministère de l'Education Nationale de la Recherche et de la Technologie (MENRT). M.S. and J.-L.B. are supported by the Institut National de la Santé et de la Recherche Médicale (INSERM). This work was partly supported by grants from the Agence Nationale de la Recherche sur le Sida (ANRS), to M.S. and J.-L.B., and the Association Fran?aise contre les Myopathies (AFM), to M.S.
REFERENCES
Battini, J. L., J. E. Rasko, and A. D. Miller. 1999. A human cell-surface receptor for xenotropic and polytropic murine leukemia viruses: possible role in G protein-coupled signal transduction. Proc. Natl. Acad. Sci. USA 96:1385-1390.
Benit, L., N. De Parseval, J. F. Casella, I. Callebaut, A. Cordonnier, and T. Heidmann. 1997. Cloning of a new murine endogenous retrovirus, MuERV-L, with strong similarity to the human HERV-L element and with a gag coding sequence closely related to the Fv1 restriction gene. J. Virol. 71:5652-5657.
Besnier, C., Y. Takeuchi, and G. Towers. 2002. Restriction of lentivirus in monkeys. Proc. Natl. Acad. Sci. USA 99:11920-11925.
Besnier, C., L. Ylinen, B. Strange, A. Lister, Y. Takeuchi, S. P. Goff, and G. J. Towers. 2003. Characterization of murine leukemia virus restriction in mammals. J. Virol. 77:13403-13406.
Best, S., P. Le Tissier, G. Towers, and J. P. Stoye. 1996. Positional cloning of the mouse retrovirus restriction gene Fv1. Nature 382:826-829.
Bieniasz, P. D. 2003. Restriction factors: a defense against retroviral infection. Trends Microbiol. 11:286-291.
Boone, L. R., F. E. Myer, D. M. Yang, C. Y. Ou, C. K. Koh, L. E. Roberson, R. W. Tennant, and W. K. Yang. 1983. Reversal of Fv-1 host range by in vitro restriction endonuclease fragment exchange between molecular clones of N-tropic and B-tropic murine leukemia virus genomes. J. Virol. 48:110-119.
Cowan, S., T. Hatziioannou, T. Cunningham, M. A. Muesing, H. G. Gottlinger, and P. D. Bieniasz. 2002. Cellular inhibitors with Fv1-like activity restrict human and simian immunodeficiency virus tropism. Proc. Natl. Acad. Sci. USA 99:11914-11919.
DesGroseillers, L., and P. Jolicoeur. 1983. Physical mapping of the Fv-1 tropism host range determinant of BALB/c murine leukemia viruses. J. Virol. 48:685-696.
Doi, K., A. Kawana, A. Iwamoto, H. Yoshikura, and T. Odawara. 1997. One base change is sufficient for host range conversion of murine leukemia virus from B to NB tropism. Arch. Virol. 142:1889-1894.
Hatziioannou, T., S. Cowan, S. P. Goff, P. D. Bieniasz, and G. J. Towers. 2003. Restriction of multiple divergent retroviruses by Lv1 and Ref1. EMBO J. 22:385-394.
Hatziioannou, T., S. Cowan, U. K. Von Schwedler, W. I. Sundquist, and P. D. Bieniasz. 2004. Species-specific tropism determinants in the human immunodeficiency virus type 1 capsid. J. Virol. 78:6005-6012.
Hatziioannou, T., D. Perez-Caballero, A. Yang, S. Cowan, and P. D. Bieniasz. 2004. Retrovirus resistance factors Ref1 and Lv1 are species-specific variants of TRIM5. Proc. Natl. Acad. Sci. USA 101:10774-10779.
Hofmann, W., D. Schubert, J. LaBonte, L. Munson, S. Gibson, J. Scammell, P. Ferrigno, and J. Sodroski. 1999. Species-specific, postentry barriers to primate immunodeficiency virus infection. J. Virol. 73:10020-10028.
Jolicoeur, P. 1979. The Fv-1 gene of the mouse and its control of murine leukemia virus replication. Curr. Top. Microbiol. Immunol. 86:67-122.
Jung, Y. T., and C. A. Kozak. 2000. A single amino acid change in the murine leukemia virus capsid gene responsible for the Fv1nr phenotype. J. Virol. 74:5385-5387.
Keckesova, Z., L. M. Ylinen, and G. J. Towers. 2004. The human and African green monkey TRIM5 genes encode Ref1 and Lv1 retroviral restriction factor activities. Proc. Natl. Acad. Sci. USA 101:10780-10785.
Kozak, C. A., and A. Chakraborti. 1996. Single amino acid changes in the murine leukemia virus capsid protein gene define the target of Fv1 resistance. Virology 225:300-305.
Levin, J. G., S. C. Hu, A. Rein, L. I. Messer, and B. I. Gerwin. 1984. Murine leukemia virus mutant with a frameshift in the reverse transcriptase coding region: implications for pol gene structure. J. Virol. 51:470-478.
Miller, D. G., R. H. Edwards, and A. D. Miller. 1994. Cloning of the cellular receptor for amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus. Proc. Natl. Acad. Sci. USA 91:78-82.
Mortuza, G. B., L. F. Haire, A. Stevens, S. J. Smerdon, J. P. Stoye, and I. A. Taylor. 2004. High-resolution structure of a retroviral capsid hexameric amino-terminal domain. Nature 431:481-485.
Ou, C. Y., L. R. Boone, C. K. Koh, R. W. Tennant, and W. K. Yang. 1983. Nucleotide sequences of gag-pol regions that determine the Fv-1 host range property of BALB/c N-tropic and B-tropic murine leukemia viruses. J. Virol. 48:779-784.
Owens, C. M., B. Song, M. J. Perron, P. C. Yang, M. Stremlau, and J. Sodroski. 2004. Binding and susceptibility to postentry restriction factors in monkey cells are specified by distinct regions of the human immunodeficiency virus type 1 capsid. J. Virol. 78:5423-5437.
Owens, C. M., P. C. Yang, H. Gottlinger, and J. Sodroski. 2003. Human and simian immunodeficiency virus capsid proteins are major viral determinants of early, postentry replication blocks in simian cells. J. Virol. 77:726-731.
Perron, M. J., M. Stremlau, B. Song, W. Ulm, R. C. Mulligan, and J. Sodroski. 2004. TRIM5 mediates the postentry block to N-tropic murine leukemia viruses in human cells. Proc. Natl. Acad. Sci. USA 101:11827-11832.
Qi, C. F., F. Bonhomme, A. Buckler-White, C. Buckler, A. Orth, M. R. Lander, S. K. Chattopadhyay, and H. C. Morse III. 1998. Molecular phylogeny of Fv1. Mamm. Genome 9:1049-1055.
Richardson, J., A. Corbin, F. Pozo, S. Orsoni, and M. Sitbon. 1993. Sequences responsible for the distinctive hemolytic potentials of Friend and Moloney murine leukemia viruses are dispersed but confined to the -gag-PR region. J. Virol. 67:5478-5486.
Stevens, A., M. Bock, S. Ellis, P. LeTissier, K. N. Bishop, M. W. Yap, W. Taylor, and J. P. Stoye. 2004. Retroviral capsid determinants of Fv1 NB and NR tropism. J. Virol. 78:9592-9598.
Stoye, J. P. 1998. Fv1, the mouse retrovirus resistance gene. Rev. Sci. Technol. 17:269-277.
Stoye, J. P. 2002. An intracellular block to primate lentivirus replication. Proc. Natl. Acad. Sci. USA 99:11549-11551.
Stremlau, M., C. M. Owens, M. J. Perron, M. Kiessling, P. Autissier, and J. Sodroski. 2004. The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 427:848-853.
Towers, G., M. Bock, S. Martin, Y. Takeuchi, J. P. Stoye, and O. Danos. 2000. A conserved mechanism of retrovirus restriction in mammals. Proc. Natl. Acad. Sci. USA 97:12295-12299.
Towers, G. J., T. Hatziioannou, S. Cowan, S. P. Goff, J. Luban, and P. D. Bieniasz. 2003. Cyclophilin A modulates the sensitivity of HIV-1 to host restriction factors. Nat. Med. 9:1138-1143.
Yap, M. W., S. Nisole, C. Lynch, and J. P. Stoye. 2004. Trim5 protein restricts both HIV-1 and murine leukemia virus. Proc. Natl. Acad. Sci. USA 101:10786-10791.(Adeline Lassaux, Marc Sit)
ABSTRACT
We identified new residues within a 101-amino-acid stretch of the murine leukemia virus capsid that differentially modulate resistance and susceptibility to the mouse Fv1 and human Ref1 genes. Among these residues, aspartate 92 and histidine 117 are both required for Fv1b resistance, whereas the latter is sufficient to confer Ref1 resistance.
TEXT
The Friend virus susceptibility gene 1 (Fv1) restricts replication of N-tropic and B-tropic murine leukemia viruses (MLV) in laboratory mouse strains harboring the b or n allele, respectively (15, 29). Several other alleles are present in wild mice (18, 26). Fv1 products are related to retroviral capsids (CA) with closest homologies to human (HERV-L) and mouse (MuERV-L) endogenous retroviral sequences (2, 5). The Fv1 restriction does not block reverse transcription but prevents proviral DNA integration through unknown mechanisms. Restriction at early stages of retroviral replication has been described in other mammalian species, including monkeys and humans (3, 6, 8, 11, 14, 30, 32). The most studied restriction genes in primates are the human Ref1 (32) and simian Lv1 (4, 8) genes. The primate TRIM5 protein, a member of the tripartite motif protein superfamily, unrelated to Fv1, has recently been shown to be an early-stage restrictive factor of primate lentiviruses (31) associated with Ref1 and Lv1 activities (13, 17, 25, 34). Ref1 and Lv1, in contrast to Fv1, target a wide range of mammalian lentiviruses and have been shown to restrict N-tropic but not B-tropic MLV (11, 32).
MLV CA proteins are the main target of the Fv1, Ref1, and Lv1 restrictions (9, 12, 22-24). Residue 110 of MLV CA has been identified as the discriminating target determinant between N-tropic, Ref1-susceptible MLV and B-tropic, Ref1-resistant MLV in murine (18) as well as other mammalian (17, 25, 32, 34) cells. However, under certain conditions, changes at CA residues 105 (10) and 114 (16) have also been reported to modulate Fv1 restriction.
NB-tropic MLV strains, such as the prototypic Friend and Moloney MLV, are resistant to all tested Fv1 alleles. Moloney MLV has also been documented to be resistant to Ref1 and Lv1 (25, 31, 34), but the status of Friend MLV with respect to these primate restriction systems has not been described. Intriguingly, Friend MLV is NB-tropic despite the presence of the N-tropic hallmark, an arginine at position 110 (Arg110), and conserved residues at positions 105 and 114 (Fig. 1A). Therefore, evasion of the mouse restriction systems, as observed with NB-tropic Friend MLV, appears to involve a residue(s) other than residue 110. To determine the sensitivity of Friend MLV to Ref1 and more precisely map CA determinants affecting resistance to the mouse Fv1b and Fv1n alleles, we derived Friend MLV CA-based constructs and mutants. Here, we demonstrate that Friend MLV is resistant to the human Ref1 restriction and identify CA residues that condition N, B, NB, and Ref1 tropisms. We thereby describe residue combinations that differentially modulate susceptibility and resistance to Fv1 and Ref1 restrictions.
A 101-amino-acid fragment recapitulates MLV capsid susceptibilities to Fv1 and Ref1 targets. Early studies of Fv1 MLV target determinants mapped differences between N- and B-tropic MLV to a 302-bp fragment encoding a CA sequence comprised between residues 33 and 133. Residues 109 and 110 were reported to be the MLV NB determinant (9) (Fig. 1A). We constructed a Friend MLV-based Gag-Pol expression vector (pC57GP) that allowed allelic exchanges of this 302-bp MLV CA cassette between BamHI and BstXI restriction sites. These sites were either naturally present or introduced by PCR-directed mutagenesis, in prototypic MLV strains of different NB tropisms. Additional EheI and XhoI sites were also used to swap smaller fragments (Fig. 1A). Prototypic CA sequences were derived from the N-tropic Akr-623 and B-tropic WNB5 MLV clones (7, 19) (kind gifts of A. Rein) and from the NB-tropic Friend MLV 57 and Moloney MLV 8.2 clones (see reference 27 for details). We also PCR amplified and sequenced a BamHI-BstXI fragment from the historical N-tropic Tennant isolate of the Friend complex (NT1; a kind gift of S. Gisselbrecht). NT1 differs from NB-tropic Friend MLV 57 at positions 92 and 124, with glutamate and serine residues substituted for aspartate and alanine, respectively (Fig. 1A).
Single-round infectious retroviral particles with MLV cores were harvested from 293T cell supernatants 48 h after cotransfection of one of the gag-pol vectors with the pCSI-G vesicular stomatitis virus G protein expression vector (1) and the pLAPSN retroviral vector carrying the neo and human placental alkaline phosphatase reporter genes (20). Viral tropism was assayed on Fv1-nonrestrictive Mus dunni (dunni) cells or on dunni cells stably transfected with Fv1n (dunnin) or Fv1b (dunnib) expression vectors (Fv1 plasmids were a kind gift of J. Stoye). Ref1 restriction was tested on human HT1080 cells (Fig. 1B) and TE671 cells as indicated.
All reported virion preparations had similar infectious levels as measured on nonrestrictive dunni cells, with titers ranging from 105 to 106 focus-forming units (FFU)/ml. All parental MLV prototypic tropisms were reproduced when using our chimeric CA-based vector system. Thus, Fv1b and Fv1n restrictions resulted in a 20- to 400-fold drop in titers. Strong Ref1-mediated restriction, with an average 200-fold drop in titers, was also observed with N-MLV virions (Fig. 1B and 2A). The NB-tropic F-MLV construct was resistant to both Fv1b and Fv1n, as well as to Ref1. Surprisingly though, the Fv1b-susceptible NT1-derived construct was resistant to Ref1-mediated restriction (Fig. 1B). This Ref1 resistance of an N-tropic MLV was observed on HT1080 and TE671, human cell lines that exert strong Ref1 restriction on other retroviruses (32), as well as on dunni cells stably transduced with human TRIM5 expressed from the pLXSN retroviral expression vector (dunni/TRIM5) (data not shown). This is the first identified CA sequence that distinguishes between Fv1b and Ref1 susceptibilities.
MLV resistance to Fv1 is due to multiple determinants that modulate position 110. Arg110 is the discriminating determinant between N-tropic, Ref1-susceptible MLV and B-tropic, Ref1-resistant MLV (17, 18, 25, 32, 34). However, the fact that NB-tropic and N-tropic Friend MLV strains encoding Arg110 are resistant to Fv1b, Ref1, or both indicated that other residues in the 101-amino-acid-long capsid region govern Fv1b and Ref1 susceptibilities. We therefore further evaluated the bases for the Friend MLV Fv1 and Ref1 resistance with smaller domain swaps.
A domain swap introducing N-MLV residues 33 to 100, excluding the canonical 110 residue, into Friend MLV was sufficient to render the latter susceptible to Fv1b restriction (chimera NNF, Fig. 2B). Friend MLV CA residues 92 to 95 consist of a DIND motif corresponding to an EVDA motif in prototypic N-tropic MLV (Fig. 1A). The swap of this motif was sufficient to convert Friend MLV to N-tropism as shown by a full Fv1b susceptibility of the corresponding construct (FNF, Fig. 2B). Thus, the DIND motif plays a key role in suppressing the accessibility of the N-tropic target in Friend MLV. Notably though, swapping of this motif did not render Friend MLV susceptible to Ref1, and this resistance was maintained even following a larger swap from residues 33 to 100 (NNF, Fig. 2B).
We further assessed the suppressive effect of the Friend MLV DIND motif in a parental B-MLV (BFB) or N-MLV (NFN) context. The sole substitution of this DIND motif was not sufficient to modulate Fv1 or Ref1 resistance in either context (Fig. 2C). When this swap was combined with that of the upstream fragment, B-tropic virions became resistant to the Fv1n restriction. Therefore, glycine, threonine, and/or asparagine at positions 35, 46, and 82, respectively, conditions CA targeting by Fv1. Nevertheless, this contribution was not as potent in the N-tropic context, since FFN particles remained susceptible to Fv1b and Ref1 restrictions, albeit with a slightly decreased susceptibility (Fig. 2C). The prominent exposure of Asp82, according to the recently published structure of the N-AKV CA amino-terminal domain (21) (Fig. 4B), is in favor of a role for this residue in Fv1 tropism, as also suggested by others (28).
Mutation of histidine to leucine at position 117 is sufficient to render Friend MLV susceptible to Fv1b and Ref1 restrictions. In contrast to Friend MLV virions, FFN virions (Fig. 2C) were susceptible to both Fv1b and Ref1 restrictions. As the difference between these viruses was limited to amino acid changes at positions 107 and 117, we introduced individual substitutions at these positions in the Friend MLV background. Infections on restrictive cell lines showed that the presence of leucine at position 117, F(H117L), was sufficient to induce a susceptibility to Fv1 and Ref1 restrictions (Fig. 3A). Charge rather than aromatic properties was crucial for resistance, as mutation of His117 to a lysine in Friend MLV maintained this property whereas mutation to either phenylalanine or tyrosine reversed resistance (not shown).
Loss of Fv1b but not Ref1 restriction in the presence of aspartate 92. As indicated above, suppression of the canonical residue 110 as a target for Fv1b is due to the combined presence of His117 and a DIND motif. Interestingly, the N-tropic NT1 strain harbors an EIND motif. The aspartate-to-glutamate change was sufficient to confer Fv1b susceptibility with no effect on Ref1 resistance, as observed with the F(D92E) mutant (Fig. 3B). When introduced alone, the second amino acid difference between the Friend MLV and NT1 strain CA at position 124 did not influence either Fv1 or Ref1 susceptibility [F(A124S), Fig. 3B]. Therefore, Glu92 was key to the recognition of the Fv1b target in the presence of His117, while it did not modulate Ref1 restriction. Similar data were obtained using dunni/TRIM5 cells.
In conclusion, while Arg110 is required for both Fv1b and Ref1 restrictions, Leu117 is also a major target determinant for both genes (Fig. 4A, left panels). Furthermore, Asp92 is required for efficient resistance to Fv1b when combined with His117. This is in agreement with a recent report using a different model of Fv1 restriction (28). However, we found that Ref1 resistance was not affected by Asp92 (Fig. 4A, right panels). This is compatible with the recently reported crystal structure of the CA amino-terminal domain of an N-tropic MLV in which residue 92, on the one hand, and residues 110 and 117, on the other, are located in two separate neighboring helices, -helices 5 and 6, respectively (21) (Fig. 4B). Our results suggest that CA MLV recognition by Fv1 involves both -helices, which are exposed to the outside of the amino-terminal CA hexamer (21), whereas Ref1/TRIM5 recognition appears to involve mainly -helix 6. While -helices 4 and 6 appear structurally conserved between human immunodeficiency virus type 1 and N-Akv CA, the interconnecting sequence, which includes -helix 5 of N-Akv CA, displays a variable succession of loops and helices (21). Interestingly, the latter sequence comprises residue 92 of the MLV CA, which we identified as a major Fv1-modulating determinant, and the human immunodeficiency virus type 1 CA CypA-binding loop involved in Lv1 restriction (12, 23, 33), thus providing differential target patterns recognized by these restrictive cellular mechanisms.
Nucleotide sequence accession number. The GenBank accession number of the BamHI-BstXI fragment from the historical N-tropic Tennant isolate of the Friend complex is AY883167.
ACKNOWLEDGMENTS
We thank N. Taylor for helpful discussions and critical reading of the manuscript and L. Boone, F.-L. Cosset, S. Gisselbrecht, A. Rein, J. Stoye, and G. Towers for providing us with materials. We also thank Muriel Audit, Dorothée Molle, and Valérie Thibert for their participation in the preparation of some of the reagents used in this study and all members of our laboratory for their continuous input.
A.L. is funded by the Ministère de l'Education Nationale de la Recherche et de la Technologie (MENRT). M.S. and J.-L.B. are supported by the Institut National de la Santé et de la Recherche Médicale (INSERM). This work was partly supported by grants from the Agence Nationale de la Recherche sur le Sida (ANRS), to M.S. and J.-L.B., and the Association Fran?aise contre les Myopathies (AFM), to M.S.
REFERENCES
Battini, J. L., J. E. Rasko, and A. D. Miller. 1999. A human cell-surface receptor for xenotropic and polytropic murine leukemia viruses: possible role in G protein-coupled signal transduction. Proc. Natl. Acad. Sci. USA 96:1385-1390.
Benit, L., N. De Parseval, J. F. Casella, I. Callebaut, A. Cordonnier, and T. Heidmann. 1997. Cloning of a new murine endogenous retrovirus, MuERV-L, with strong similarity to the human HERV-L element and with a gag coding sequence closely related to the Fv1 restriction gene. J. Virol. 71:5652-5657.
Besnier, C., Y. Takeuchi, and G. Towers. 2002. Restriction of lentivirus in monkeys. Proc. Natl. Acad. Sci. USA 99:11920-11925.
Besnier, C., L. Ylinen, B. Strange, A. Lister, Y. Takeuchi, S. P. Goff, and G. J. Towers. 2003. Characterization of murine leukemia virus restriction in mammals. J. Virol. 77:13403-13406.
Best, S., P. Le Tissier, G. Towers, and J. P. Stoye. 1996. Positional cloning of the mouse retrovirus restriction gene Fv1. Nature 382:826-829.
Bieniasz, P. D. 2003. Restriction factors: a defense against retroviral infection. Trends Microbiol. 11:286-291.
Boone, L. R., F. E. Myer, D. M. Yang, C. Y. Ou, C. K. Koh, L. E. Roberson, R. W. Tennant, and W. K. Yang. 1983. Reversal of Fv-1 host range by in vitro restriction endonuclease fragment exchange between molecular clones of N-tropic and B-tropic murine leukemia virus genomes. J. Virol. 48:110-119.
Cowan, S., T. Hatziioannou, T. Cunningham, M. A. Muesing, H. G. Gottlinger, and P. D. Bieniasz. 2002. Cellular inhibitors with Fv1-like activity restrict human and simian immunodeficiency virus tropism. Proc. Natl. Acad. Sci. USA 99:11914-11919.
DesGroseillers, L., and P. Jolicoeur. 1983. Physical mapping of the Fv-1 tropism host range determinant of BALB/c murine leukemia viruses. J. Virol. 48:685-696.
Doi, K., A. Kawana, A. Iwamoto, H. Yoshikura, and T. Odawara. 1997. One base change is sufficient for host range conversion of murine leukemia virus from B to NB tropism. Arch. Virol. 142:1889-1894.
Hatziioannou, T., S. Cowan, S. P. Goff, P. D. Bieniasz, and G. J. Towers. 2003. Restriction of multiple divergent retroviruses by Lv1 and Ref1. EMBO J. 22:385-394.
Hatziioannou, T., S. Cowan, U. K. Von Schwedler, W. I. Sundquist, and P. D. Bieniasz. 2004. Species-specific tropism determinants in the human immunodeficiency virus type 1 capsid. J. Virol. 78:6005-6012.
Hatziioannou, T., D. Perez-Caballero, A. Yang, S. Cowan, and P. D. Bieniasz. 2004. Retrovirus resistance factors Ref1 and Lv1 are species-specific variants of TRIM5. Proc. Natl. Acad. Sci. USA 101:10774-10779.
Hofmann, W., D. Schubert, J. LaBonte, L. Munson, S. Gibson, J. Scammell, P. Ferrigno, and J. Sodroski. 1999. Species-specific, postentry barriers to primate immunodeficiency virus infection. J. Virol. 73:10020-10028.
Jolicoeur, P. 1979. The Fv-1 gene of the mouse and its control of murine leukemia virus replication. Curr. Top. Microbiol. Immunol. 86:67-122.
Jung, Y. T., and C. A. Kozak. 2000. A single amino acid change in the murine leukemia virus capsid gene responsible for the Fv1nr phenotype. J. Virol. 74:5385-5387.
Keckesova, Z., L. M. Ylinen, and G. J. Towers. 2004. The human and African green monkey TRIM5 genes encode Ref1 and Lv1 retroviral restriction factor activities. Proc. Natl. Acad. Sci. USA 101:10780-10785.
Kozak, C. A., and A. Chakraborti. 1996. Single amino acid changes in the murine leukemia virus capsid protein gene define the target of Fv1 resistance. Virology 225:300-305.
Levin, J. G., S. C. Hu, A. Rein, L. I. Messer, and B. I. Gerwin. 1984. Murine leukemia virus mutant with a frameshift in the reverse transcriptase coding region: implications for pol gene structure. J. Virol. 51:470-478.
Miller, D. G., R. H. Edwards, and A. D. Miller. 1994. Cloning of the cellular receptor for amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus. Proc. Natl. Acad. Sci. USA 91:78-82.
Mortuza, G. B., L. F. Haire, A. Stevens, S. J. Smerdon, J. P. Stoye, and I. A. Taylor. 2004. High-resolution structure of a retroviral capsid hexameric amino-terminal domain. Nature 431:481-485.
Ou, C. Y., L. R. Boone, C. K. Koh, R. W. Tennant, and W. K. Yang. 1983. Nucleotide sequences of gag-pol regions that determine the Fv-1 host range property of BALB/c N-tropic and B-tropic murine leukemia viruses. J. Virol. 48:779-784.
Owens, C. M., B. Song, M. J. Perron, P. C. Yang, M. Stremlau, and J. Sodroski. 2004. Binding and susceptibility to postentry restriction factors in monkey cells are specified by distinct regions of the human immunodeficiency virus type 1 capsid. J. Virol. 78:5423-5437.
Owens, C. M., P. C. Yang, H. Gottlinger, and J. Sodroski. 2003. Human and simian immunodeficiency virus capsid proteins are major viral determinants of early, postentry replication blocks in simian cells. J. Virol. 77:726-731.
Perron, M. J., M. Stremlau, B. Song, W. Ulm, R. C. Mulligan, and J. Sodroski. 2004. TRIM5 mediates the postentry block to N-tropic murine leukemia viruses in human cells. Proc. Natl. Acad. Sci. USA 101:11827-11832.
Qi, C. F., F. Bonhomme, A. Buckler-White, C. Buckler, A. Orth, M. R. Lander, S. K. Chattopadhyay, and H. C. Morse III. 1998. Molecular phylogeny of Fv1. Mamm. Genome 9:1049-1055.
Richardson, J., A. Corbin, F. Pozo, S. Orsoni, and M. Sitbon. 1993. Sequences responsible for the distinctive hemolytic potentials of Friend and Moloney murine leukemia viruses are dispersed but confined to the -gag-PR region. J. Virol. 67:5478-5486.
Stevens, A., M. Bock, S. Ellis, P. LeTissier, K. N. Bishop, M. W. Yap, W. Taylor, and J. P. Stoye. 2004. Retroviral capsid determinants of Fv1 NB and NR tropism. J. Virol. 78:9592-9598.
Stoye, J. P. 1998. Fv1, the mouse retrovirus resistance gene. Rev. Sci. Technol. 17:269-277.
Stoye, J. P. 2002. An intracellular block to primate lentivirus replication. Proc. Natl. Acad. Sci. USA 99:11549-11551.
Stremlau, M., C. M. Owens, M. J. Perron, M. Kiessling, P. Autissier, and J. Sodroski. 2004. The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 427:848-853.
Towers, G., M. Bock, S. Martin, Y. Takeuchi, J. P. Stoye, and O. Danos. 2000. A conserved mechanism of retrovirus restriction in mammals. Proc. Natl. Acad. Sci. USA 97:12295-12299.
Towers, G. J., T. Hatziioannou, S. Cowan, S. P. Goff, J. Luban, and P. D. Bieniasz. 2003. Cyclophilin A modulates the sensitivity of HIV-1 to host restriction factors. Nat. Med. 9:1138-1143.
Yap, M. W., S. Nisole, C. Lynch, and J. P. Stoye. 2004. Trim5 protein restricts both HIV-1 and murine leukemia virus. Proc. Natl. Acad. Sci. USA 101:10786-10791.(Adeline Lassaux, Marc Sit)