Role of receptor polymorphism and glycosylation in syncytium induction and host range variation of ecotropic mouse gammaretroviruses.
ABSTRACT: We previously identified unusual variants of Moloney and Friend ecotropic mouse gammaretroviruses that have altered host range and are cytopathic in cells of the wild mouse species Mus dunni. Cytopathicity was attributed to different amino acid substitutions at the same critical env residue involved in receptor interaction: S82F in the Moloney variant Spl574, and S84A in the Friend mouse leukemia virus F-S MLV. Because M. dunni cells carry a variant CAT-1 cell surface virus receptor (dCAT-1), we examined the role of this receptor variant in cytopathicity and host range.We expressed dCAT-1 or mCAT-1 of NIH 3T3 origin in cells that are not normally infectible with ecotropic MLVs and evaluated the transfectants for susceptibility to virus infection and to virus-induced syncytium formation. The dCAT-1 transfectants, but not the mCAT-1 transfectants, were susceptible to virus-induced cytopathicity, and this cytopathic response was accompanied by the accumulation of unintegrated viral DNA. The dCAT-1 transfectants, however, did not also reproduce the relative resistance of M. dunni cells to Moloney MLV, and the mCAT-1 transfectants did not show the relative resistance of NIH 3T3 cells to Spl574. Western analysis, use of glycosylation inhibitors and mutagenesis to remove receptor glycosylation sites identified a possible role for cell-specific glycosylation in the modulation of virus entry.Virus entry and virus-induced syncytium formation using the CAT-1 receptor are mediated by a small number of critical amino acid residues in receptor and virus Env. Virus entry is modulated by glycosylation of cellular proteins, and this effect is cell and virus-specific.
Project description:Three N-linked glycosylation sites were removed from the envelope glycoproteins of Friend, Moloney, and AKV mouse ecotropic gammaretroviruses: gs1 and gs2, in the receptor binding domain; and gs8, in a region implicated in post-binding cell fusion. Mutants were tested for their ability to infect rodent cells expressing 4 CAT-1 receptor variants. Three mutants (Mo-gs1, Mo-gs2, and Fr-gs1) infect NIH 3T3 and rat XC cells, but are severely restricted in Mus dunni cells and Lec8, a Chinese hamster cell line susceptible to ecotropic virus. This restriction is reproduced in ferret cells expressing M. dunni dCAT-1, but not in cells expressing NIH 3T3 mCAT-1. Virus binding assays, pseudotype assays, and the use of glycosylation inhibitors further suggest that restriction is primarily due to receptor polymorphism and, in M. dunni cells, to glycosylation of cellular proteins. Virus envelope glycan size or type does not affect infectivity. Thus, host range variation due to N-glycan deletion is receptor variant-specific, cell-specific, virus type-specific, and glycan site-specific.
Project description:Spl574 MLV (murine leukemia virus) is a variant of Moloney ecotropic MLV (MoMLV) that is cytopathic in Mus dunni cells and restricted by other mouse cells. Its host range and cytopathicity are due to a mutation, S82F, at a site critical for binding to the CAT-1 receptor. To identify residues that affect affinity for receptor variants, virus with S82F was passed in restrictive cells. The env genes of the adapted viruses contained 18 novel mutations, including one, E114G, present in 6 of 30 sequenced envs. MoMLV-E114G efficiently infected all mouse cells as well as ecotropic MLV resistant Chinese hamster cells. Virus with E114G and S82F induced large multinucleated syncytia in NIH 3T3 and SC-1 cells as well as M. dunni cells. Inoculation of Mo-S82F,E114G into mice produced lymphomas typical of MoMLV. Residues at env position 114 are thus important determinants of host range, and E114G suppresses host range restriction due to S82F, but does not affect S82F-governed cytopathicity.
Project description:A variant ecotropic Friend murine leukemia virus, F-S MLV, is capable of inducing the formation of large multinucleated syncytia in Mus dunni cells. This cytopathicity resembles that of Spl574 MLV, a novel variant recently isolated from the spleen of a Mus spicilegus mouse neonatally inoculated with Moloney MLV. F-S MLV is an N-tropic Friend MLV that also has the unusual ability to infect hamster cells, which are normally resistant to mouse ecotropic MLVs. Syncytium induction by both F-S MLV and Spl574 is accompanied by the accumulation of large amounts of unintegrated viral DNA, a hallmark of pathogenic retroviruses, but not previously reported for mouse ecotropic gammaretroviruses. Sequencing and site-specific mutagenesis determined that the syncytium-inducing phenotype of F-S MLV can be attributed to a single amino acid substitution (S84A) in the VRA region of the viral env gene. This site corresponds to that of the single substitution previously shown to be responsible for the cytopathicity of Spl574, S82F. The S84A substitution in F-S MLV also contributes to the ability of this virus to infect hamster cells, but Spl574 MLV is unable to infect hamster cells. Because this serine residue is one of the critical amino acids that form the CAT-1 receptor binding site, and because M. dunni and hamster cells have variant CAT-1 receptors, these results suggest that syncytium formation as well as altered host range may be a consequence of altered interaction between virus and receptor.
Project description:The cell surface receptor for ecotropic host-range (infection limited to mice or rats) murine leukemia viruses (MuLVs) is the widely expressed system y+ transporter for cationic amino acids (CAT-1). Like other retroviruses, ecotropic MuLV infection eliminates virus-binding sites from cell surfaces and results in complete interference to superinfection. Surprisingly, infection causes only partial (ca 40 to 60%) loss of mouse CAT-1 transporter activity. The NIH/Swiss mouse CAT-1 (mCAT-1) contains 622 amino acids with 14 hydrophobic potential membrane-spanning sequences, and it is known that the third extracellular loop from the amino terminus is required for virus binding. Although loop 3 is hypervariable in different species and mouse strains, consistent with its proposed role in virus-host coevolution, loop 3 sequences of both susceptible and resistant species contain consensus sites for N-linked glycosylation. Both of the consensus sites in loop 3 of mCAT-1 are known to be glycosylated and to contain oligosaccharides with diverse sizes (J. W. Kim and J. M. Cunningham, J. Biol. Chem. 268:16316-16320, 1993). We confirmed by several lines of evidence that N-linked glycosylation occludes a potentially functional virus-binding site in the CAT-1 protein of hamsters, thus contributing to resistance of that species. To study the role of receptor glycosylation in animals susceptible to infection, we eliminated loop 3 glycosylation sites by mutagenesis of an mCAT-1 cDNA clone, and we expressed wild-type and mutant receptors in mink fibroblasts and Xenopus oocytes. These receptors had indistinguishable transport properties, as determined by kinetic and voltage-jump electrophysiological studies of arginine uptake in oocytes and by analyses Of L-[3H]arginine uptake in mink cells. Bindings of ecotropic envelope glycoprotein gp7O to the accessible receptor sites on surfaces of mink cells expressing wild-type or mutant mCAT-1 were not significantly different in kinetics or in equilibrium affinities (i.e., K(D) approximately 3.7 X 10(-10) to 7.5 X 10(-10) M). However, when values were normalized to the same levels of mCAT-1 transporter expression, cells with wild-type glycosylated mCAT-1 had only approximately 50% as many sites for gp70 binding as cells with unglycosylated mCAT-1. Although infection with ecotropic MuLV had no effect on activity of the mink CAT-1 transporter that does not bind virus, it caused partial down-modulation of wild-type mCAT-1 and complete down-modulation of unglycosylated mutant mCAT-1. These results suggest that N-linked glycosylation causes wild-type mCAT-1 heterogeneity and that a significant proportion is inaccessible to virus. In part because only the interactive fraction of mCAT-1 can be down-modulated, infected murine cells conserve an amino acid transport capability that supports their viability.
Project description:Cells of Mus minutoides, an African pygmy mouse of the subgenus Nannomys, are susceptible to ecotropic Moloney and Friend mouse leukemia viruses (MLVs) but not to AKV-type MLVs. Transfected MA139 ferret cells expressing the mCAT-1 cell surface receptor, with the minCAT-1 substitutions K222Q and V233L, did not restrict AKV MLV. The resistance of M. minutoides cells to AKV MLV was not relieved by inhibitors of glycosylation or by the introduction of NIH 3T3 mCAT-1. Resistance is thus not mediated by receptor sequence variation, expression level, or glycosylation. M. minutoides cells are also infectible with LacZ pseudotypes having AKV Env and Moloney MLV (MoMLV) Gag proteins, further indicating that AKV Env sequence variations do not contribute to the observed block. The pattern of virus resistance in M. minutoides differs from that of the known variants of the Fv1 postentry resistance gene; M. minutoides is equally resistant to N-, B-, and NR-tropic AKV viruses and is equally susceptible to NR- and NB-tropic Friend MLVs. This novel resistance blocks replication before reverse transcription, whereas Fv1 generally restricts replication after reverse transcription; M. minutoides cells produce 2-long-terminal-repeat viral DNA circles and linear viral DNA after infection with MoMLV but not with AKV MLV. Analysis of MoMLV-AKV MLV chimeras determined that the target of resistance is in the virus capsid gene. Mutagenesis demonstrated that restriction is mediated by two amino acid substitutions, H117L and A110R; substitutions at these sites can also be targeted by the resistance genes Fv1 and TRIM5alpha. M. minutoides cells thus have a novel postentry resistance to AKV MLVs.
Project description:Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 10(6) transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins.
Project description:An amino-terminal portion of the Friend murine leukemia virus (MLV) envelope surface protein [SU, residues 1 to 236 [SU:(1-236)]] and its receptor, MCAT-1, were each purified from insect cells after expression by using recombinant baculoviruses. Friend SU:(1-236) bound specifically to Xenopus oocytes that expressed MCAT-1 with an affinity (Kd, 55 nM) similar to that of viral SU binding to permissive cells. Direct binding of Friend SU:(1-236) to purified MCAT-1 was observed in detergent and after reconstitution into liposomes. Analysis of binding demonstrated that MCAT-1 and Friend SU:(1-236) interact with a stoichiometry of near 1:1. These findings demonstrate that the amino-terminal domain from the SU of ecotropic murine retroviruses contains an MCAT-1 binding domain.
Project description:CasBrE is a neurovirulent murine leukemia virus (MLV) capable of inducing paralytic disease with associated spongiform neurodegeneration. The neurovirulence of this virus has been genetically mapped to the surface expressed subunit (SU) of the env gene. However, CasBrE SU synthesized in the absence of the transmembrane subunit (TM) does not retain ecotropic receptor binding activity, indicating that folding of the receptor binding domain (RBD) requires this domain. Using a neural stem cell (NSC) based viral trans complementation approach to examine whether misfolded CasBrE SU retained neurovirulence, we observed CasBrE SU interaction with the "non-neurovirulent" amphotropic helper virus, 4070A which restored functional activity of CasBrE SU.Herein, we show that infection of NSCs expressing CasBrE SU with 4070A (CasES+4070A-NSCs) resulted in the redistribution of CasBrE SU from a strictly secreted product to include retention on the plasma membrane. Cell surface cross-linking analysis suggested that CasBrE SU membrane localization was due to interactions with 4070A Env. Viral particles produced from CasES+4070A-NSCS contained both CasBrE and 4070A gp70 Env proteins. These particles displayed ecotropic receptor-mediated infection, but were still 100-fold less efficient than CasE+4070A-NSC virus. Infectious center analysis showed CasBrE SU ecotropic transduction efficiencies approaching those of NSCs expressing full length CasBrE Env (CasE; SU+TM). In addition, CasBrE SU-4070A Env interactions resulted in robust ecotropic superinfection interference indicating near native intracellular SU interaction with its receptor, mCAT-1.In this report we provided evidence that 4070A Env and CasBrE SU physically interact within NSCs leading to CasBrE SU retention on the plasma membrane, incorporation into viral particles, restoration of mCAT-1 binding, and capacity for initiation of TM-mediated fusion events. Thus, heterotropic Env-SU interactions facilitates CasBrE SU folding events that restore Env activity. These findings are consistent with the idea that one protein conformation acts as a folding scaffold or nucleus for a second protein of similar primary structure, a process reminiscent of prion formation. The implication is that template-based protein folding may represent an inherent feature of neuropathogenic proteins that extends to retroviral Envs.
Project description:The wild mouse species most closely related to the common laboratory strains contain proviral env genes of the xenotropic/polytropic subgroup of mouse leukemia viruses (MLVs). To determine if the polytropic proviruses of Mus spretus contain functional genes, we inoculated neonates with Moloney MLV (MoMLV) or amphotropic MLV (A-MLV) and screened for viral recombinants with altered host ranges. Thymus and spleen cells from MoMLV-inoculated mice were plated on Mus dunni cells and mink cells, since these cells do not support the replication of MoMLV, and cells from A-MLV-inoculated mice were plated on ferret cells. All MoMLV-inoculated mice produced ecotropic viruses that resembled their MoMLV progenitor, although some isolates, unlike MoMLV, grew to high titers in M. dunni cells. All of the MoMLV-inoculated mice also produced nonecotropic virus that was infectious for mink cells. Sequencing of three MoMLV- and two A-MLV-derived nonecotropic recombinants confirmed that these viruses contained substantial substitutions that included the regions of env encoding the surface (SU) protein and the 5' end of the transmembrane (TM) protein. The 5' recombination breakpoint for one of the A-MLV recombinants was identified in RNase H. The M. spretus-derived env substitutions were nearly identical to the corresponding regions in prototypical laboratory mouse polytropic proviruses, but the wild mouse infectious viruses had a more restricted host range. The M. spretus proviruses contributing to these recombinants were also sequenced. The seven sequenced proviruses were 99% identical to one another and to the recombinants; only two of the seven had obvious fatal defects. We conclude that the M. spretus proviruses are likely to be recent germ line acquisitions and that they contain functional genes that can contribute to the production of replication-competent virus.
Project description:Identification of new techniques to express proteins into mammal cells is of particular interest for both research and medical purposes. The present study describes the use of engineered vesicles to deliver exogenous proteins into human cells. We show that overexpression of the spike glycoprotein of the vesicular stomatitis virus (VSV-G) in human cells induces the release of fusogenic vesicles named gesicles. Biochemical and functional studies revealed that gesicles incorporated proteins from producer cells and could deliver them to recipient cells. This protein-transduction method allows the direct transport of cytoplasmic, nuclear or surface proteins in target cells. This was demonstrated by showing that the TetR transactivator and the receptor for the murine leukemia virus (MLV) envelope [murine cationic amino acid transporter-1 (mCAT-1)] were efficiently delivered by gesicles in various cell types. We further shows that gesicle-mediated transfer of mCAT-1 confers to human fibroblasts a robust permissiveness to ecotropic vectors, allowing the generation of human-induced pluripotent stem cells in level 2 biosafety facilities. This highlights the great potential of mCAT-1 gesicles to increase the safety of experiments using retro/lentivectors. Besides this, gesicles is a versatile tool highly valuable for the nongenetic delivery of functions such as transcription factors or genome engineering agents.