Murine Leukemia Virus Envelope Glycoprotein Research Articles

Efficient Gene Delivery to Quiescent Interleukin-2 (IL-2)–Dependent Cells by Murine Leukemia Virus-Derived Vectors Harboring IL-2 Chimeric Envelopes Glycoproteins

AbstractInterleukin-2 (IL-2) is a cytokine that induces the proliferation of certain IL-2 receptor expressing quiescent cells. Human IL-2 was fused to the amino-terminus of amphotropic murine leukemia virus (MLV) envelope glycoproteins. Retroviral vectors were pseudotyped with both the IL-2 chimeric envelope and the wild-type amphotropic MLV envelope. The chimeric IL-2 glycoproteins were incorporated on retroviral vectors and the IL-2–displaying vector particles could bind specifically to cell surface IL-2 receptors. In addition, the IL-2–displaying vectors could infect proliferating cells through amphotropic receptors irrespective of whether the cells expressed the IL-2 receptor. IL-2–displaying vector particles could also transiently stimulate the cell cycle entry and proliferation of several IL-2–dependent cell lines. Finally, retroviral vectors displaying IL-2 could efficiently transduce G0/G1-arrested cells expressing the IL-2 receptor at a 34-fold higher efficiency compared with vectors with unmodified envelopes. This new strategy, whereby C-type retroviral vector particles display a ligand that activates the cell cycle of the target cells at the time of virus entry, may represent an alternative to lentivirus-derived retroviral vectors for the infection of quiescent cells. In addition, upon infection of an heterogeneous population of nonproliferating cells, MLV-retroviral vectors that display cytokines/growth factors will allow the transgene of interest to be integrated specifically in quiescent cells expressing the corresponding cytokine/growth factor receptor.

In Vivo Analysis of Infectivity, Fusogenicity, and Incorporation of a Mutagenic Viral Glycoprotein Library Reveals Determinants for Virus Incorporation.

Enveloped viruses utilize transmembrane surface glycoproteins to gain entry into target cells. Glycoproteins from diverse viral families can be incorporated into nonnative viral particles in a process termed pseudotyping; however, the molecular mechanisms governing acquisition of these glycoproteins are poorly understood. For murine leukemia virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles has been shown to be an active process, but it does not appear to be caused by direct interactions among viral proteins. In this study, we coupled in vivo selection systems with Illumina next-generation sequencing (NGS) to test hundreds of thousands of MLV Env mutants for the ability to be enriched in viral particles and to perform other glycoprotein functions. NGS analyses on a subset of these mutants predicted that the residues important for incorporation are in the membrane-proximal external region (MPER), particularly W127 and W137, and the residues in the membrane-spanning domain (MSD) and also immediately flanking it (T140 to L163). These predictions were validated by directly measuring the impact of mutations in these regions on fusogenicity, infectivity, and incorporation. We suggest that these two regions dictate pseudotyping through interactions with specific lipid environments formed during viral assembly. Researchers from numerous fields routinely exploit the ability to manipulate viral tropism by swapping viral surface proteins. However, this process, termed pseudotyping, is poorly understood at the molecular level. For murine leukemia virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles is an active process, but it does not appear to occur through direct viral protein-protein interactions. In this study, we tested hundreds of thousands of MLV Env mutants for the ability to be enriched in viral particles as well as perform other glycoprotein functions. Our analyses on a subset of these mutants predict that the glycoprotein regions embedded in and immediately flanking the viral membrane dictate active incorporation into viral particles. We suggest that pseudotyping occurs through specific lipid-protein interactions at the viral assembly site.

Characterizing the Murine Leukemia Virus Envelope Glycoprotein Membrane-Spanning Domain for Its Roles in Interface Alignment and Fusogenicity.

The membrane-proximal region of murine leukemia virus envelope (Env) is a critical modulator of its functionality. We have previously shown that the insertion of one amino acid (+1 leucine) within the membrane-spanning domain (MSD) abolished protein functionality in infectivity assays. However, functionality could be restored to this +1 leucine mutant by either inserting two additional amino acids (+3 leucine) or by deleting the cytoplasmic tail domain (CTD) in the +1 leucine background. We inferred that the ectodomain and CTD have protein interfaces that have to be in alignment for Env to be functional. Here, we made single residue deletions to the Env mutant with the +1 leucine insertion to restore the interface alignment (gain of functionality) and therefore define the boundaries of the two interfaces. We identified the glycine-proline pairs near the N terminus (positions 147 and 148) and the C terminus (positions 159 and 160) of the MSD as being the boundaries of the two interfaces. Deletions between these pairs restored function, but deletions outside of them did not. In addition, the vast majority of the single residue deletions regained function if the CTD was deleted. The exceptions were four hydroxyl-containing amino acid residues (T139, T140, S143, and T144) that reside in the ectodomain interface and the proline at position 148, which were all indispensable for functionality. We hypothesize that the hydroxyl-containing residues at positions T139 and S143 could be a driving force for stabilizing the ectodomain interface through formation of a hydrogen-bonding network. The membrane-proximal external region (MPER) and membrane-spanning domains (MSDs) of viral glycoproteins have been shown to be critical for regulating glycoprotein fusogenicity. However, the roles of these two domains are poorly understood. We report here that point deletions and insertions within the MPER or MSD result in functionally inactive proteins. However, when the C-terminal tail domain (CTD) is deleted, the majority of the proteins remain functional. The only residues that were found to be critical for function regardless of the CTD were four hydroxyl-containing amino acids located at the C terminus of the MPER (T139 and T140) and at the N terminus of the MSD (S143 and T144) and a proline near the beginning of the MSD (P148). We demonstrate that hydrogen-bonding at positions T139 and S143 is critical for protein function. Our findings provide novel insights into the role of the MPER in regulating fusogenic activity of viral glycoproteins.

Targeted Entry via Somatostatin Receptors Using a Novel Modified Retrovirus Glycoprotein That Delivers Genes at Levels Comparable to Those of Wild-Type Viral Glycoproteins

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.

Optimized Lentiviral Transduction of Mouse Bone Marrow-Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have attracted much attention as potential platforms for transgene delivery and cell-based therapy for human disease. MSCs have the capability to self-renew and retain multipotency after extensive expansion in vitro, making them attractive targets for ex vivo modification and autologous transplantation. Viral vectors, including lentiviral vectors, provide an efficient means for transgene delivery into human MSCs. In contrast, mouse MSCs have proven more difficult to transduce with lentiviral vectors than their human counterparts, and because many studies use mouse models of human disease, an improved method of transduction would facilitate studies using ex vivo-modified mouse MSCs. We have worked toward improving the production of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors and optimizing transduction conditions for mouse MSCs using lentivirus vectors pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G), the ecotropic murine leukemia virus envelope glycoprotein (MLV-E), and the glycoproteins derived from the Armstrong and WE strains of lymphocytic choriomeningitis virus (LCMV-Arm, LCMV-WE). Mouse MSCs were readily transduced following overnight incubation using a multiplicity of infection of at least 40. Alternatively, mouse MSCs in suspension were readily transduced after a 1-h exposure to lentiviral pseudotypes immediately following trypsin treatment or retrieval from storage in liquid nitrogen. LCMV-WE pseudotypes resulted in efficient transduction of mouse MSCs with less toxicity than VSV-G pseudotypes. In conclusion, our improved production and transduction conditions for lentiviral vectors resulted in efficient transduction of mouse MSCs, and these improvements should facilitate the application of such cells in the context of mouse models of human disease.

Membrane activity of an amphiphilic α-Helical membrane-proximal cytoplasmic domain of the MoMuLV envelope glycoprotein

In the Moloney murine leukemia virus (MoMuLV) envelope glycoprotein (Env) we identified a membrane-proximal cytoplasmic domain (residues 598–616) that facilitates the Env incorporation into virions and Env-mediated fusion [Rozenberg, Y., Conner, J., Aguilar-Carreno, H., Chakraborti, S., Dimiter, D.S., Anderson, W.F., 2008. Viral entry: membrane-proximal cytoplasmic domain of MoMuLV envelope tail facilitates fusion. In the same issue. (accompanying paper)]. By biophysical methods (CD, EPR) a corresponding peptide (membrane-proximal peptide, 598–616) was demonstrated to form a membrane-parallel amphiphilic α-helix in the presence of membranes. Electrophysiological studies with planar bilayers and liposomes indicate that the membrane-proximal peptide is membrane destabilizing. This peptide and the fusion peptide from the MoMuLV transmembrane (TM) ectodomain were tested for their effect on the bilayer for hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine (T H). Importantly, the external fusion peptide and the internal membrane-proximal peptides of MoMuLV env exert opposite effects on membrane curvature. The fusion peptide lowers T H while the membrane proximal peptide raises it. These effects on T H correlate with the ability of these peptides to induce lipid mixing in large unilamellar vesicles composed of dioleoylphosphatidylethanolamine: dioleoylphosphatidylcholine:cholesterol (1:1:1 mol). When added externally to preformed liposomes, the N-terminal fusion peptide promotes lipid mixing while the cytoplasmic membrane-proximal peptide inhibits this effect. These finding indicate a possible mechanism by which the membrane-proximal domain in MoMuLV Env may affect the formation of membrane fusion intermediates.

Characterization of R peptide of murine leukemia virus envelope glycoproteins in syncytium formation and entry

The C-terminal R peptide of ecotropic murine leukemia virus (MLV) envelope protein (Env) negatively controls membrane fusion activity. The R peptide cleavage during virion maturation activates its fusogenicity and is required for viral entry. We analyzed fusogenicity and transduction efficiency of mutant Env proteins of ecotropic, amphotropic, polytropic, and xenotropic MLVs. As the result, we found that the hydrophobic amino acid residues around the R peptide cleavage site are important for membrane fusion inhibition by the R peptide. In addition, we found that Env complexes with R peptide-truncated and -containing Env proteins have lower fusogenicity and transduction efficiency than those with the R-peptide-truncated Env alone, suggesting that efficient R peptide cleavage is required for efficient MLV vector transduction. The role of R peptide cleavage in amphotropic, polytropic, and xenotropic MLV infection has not been investigated. We found in this study that the R peptide cleavage is required for amphotropic, xenotropic, and polytropic MLV vector transduction, like with ecotropic MLV. The R-peptide-truncated Env proteins of the xenotropic and polytropic MLVs, however, had much lower fusogenicity than those of the ecotropic and amphotropic MLVs. These results provide valuable information for construction of efficient MLV vectors and for understanding the retroviral entry mechanism.

Retroviral targeting of proliferating endothelial cells.

Tumor growth requires the formation of new blood vessels by endothelial cells. Thus, surface molecules -- such as angiogenin receptors -- that are selectively expressed on growing endothelium represent an attractive target for directed delivery of compounds to tumor tissue. We attempted to obtain genetically engineered retroviral vectors targeted to the endothelium by inserting the human angiogenin sequence into Moloney murine leukemia virus envelope glycoprotein. Abundant expression of the chimeric protein could be verified. However, while being selective for proliferating human endothelial cells, the recombinant retroviral particles displayed low transduction efficiencies and thus have to be further improved.

Intracellular trafficking of Gag and Env proteins and their interactions modulate pseudotyping of retroviruses.

Glycoproteins derived from most retroviruses and from several families of enveloped viruses can form infectious pseudotypes with murine leukemia virus (MLV) and lentiviral core particles, like the MLV envelope glycoproteins (Env) that are incorporated on either virus type. However, coexpression of a given glycoprotein with heterologous core proteins does not always give rise to highly infectious viral particles, and restrictions on pseudotype formation have been reported. To understand the mechanisms that control the recruitment of viral surface glycoproteins on lentiviral and retroviral cores, we exploited the fact that the feline endogenous retrovirus RD114 glycoprotein does not efficiently pseudotype lentiviral cores derived from simian immunodeficiency virus, whereas it is readily incorporated onto MLV particles. Our results indicate that recruitment of glycoproteins by the MLV and lentiviral core proteins occurs in intracellular compartments and not at the cell surface. We found that Env and core protein colocalization in intracytoplasmic vesicles is required for pseudotype formation. By investigating MLV/RD114 Env chimeras, we show that signals in the cytoplasmic tail of either glycoprotein differentially influenced their intracellular localization; that of MLV allows endosomal localization and hence recruitment by both lentiviral and MLV cores. Furthermore, we found that upon membrane binding, MLV core proteins could relocalize Env glycoproteins in late endosomes and allow their incorporation on viral particles. Thus, intracellular colocalization, as well as interactions between Env and core proteins, may influence the recruitment of the glycoprotein onto viral particles and generate infectious pseudotyped viruses.

The tyrosine-based YXXØ targeting motif of murine leukemia virus envelope glycoprotein affects pathogenesis

Retroviruses, such as human and simian immunodeficiency viruses (HIV and SIV), and murine leukemia viruses (MuLV), harbor a tyrosine-based motif in the intracytoplasmic domain of their envelope glycoprotein. This motif can act as an endocytosis signal or as a targeting signal, restricting viral budding at specific cell surface membrane domains. In the present study, proviral DNA of the ecotropic Cas-Br-E strain of MuLV was modified by substitution or deletion of the critical tyrosine residue. Mutant viruses lost basolateral targeting in polarized MDCK epithelial cells while expression level of the glycoprotein at the cell surface was not affected. This suggests that the tyrosine-based motif in MuLV does not act as an endocytosis signal. Only a small delay in the appearance of disease was observed in inoculated mice. In contrast, a striking change in the pathology was observed with enlarged thymus and lymph nodes in animals inoculated with mutant viruses.

Transduction of human islets with pseudotyped lentiviral vectors.

Type I diabetes is caused by an autoimmune-mediated elimination of insulin-secreting pancreatic islets. Genetic modification of islets offers a powerful molecular tool for improving our understanding of islet biology. Moreover, efficient genetic engineering of islets could allow for evaluation of new strategies aimed at preventing islet destruction. The present study evaluated the ability of a human immunodeficiency virus (HIV)-based lentiviral vector pseudotyped with various viral envelopes to target human islets ex vivo, with the goal of improving efficiency while minimizing toxicity. Transfer of the enhanced green fluorescent protein reporter gene in human islets was first evaluated with an HIV-based vector pseudotyped with the vesicular stomatitis virus (VSV), murine leukemia virus, Ebola, rabies, Mokola, or lymphocytic choriomeningitis virus (LCMV) envelope glycoprotein to optimize transduction efficiency. Results indicated that LCMV-pseudotyped vector transduced insulin-secreting beta cells with the highest efficiency. Moreover, toxicity associated with transduction of islets was found to be lower with LCMV-pseudotyped vector than with VSV-G-pseudotyped vector, the second most efficient vector for islet transduction. Overall, our study describes an improved methodology for achieving safe and efficient gene transfer into cells of human islets.

Organ distribution of gene expression after intravenous infusion of targeted and untargeted lentiviral vectors.

Lentiviral vectors represent an attractive technology platform from which to develop a targetable injectable gene delivery system for transduction of specific cell populations in vivo, irrespective of their cell cycle status. Targeted HIV-1-based lentiviral vectors were generated by pseudotyping them with chimeric murine leukemia virus (MLV) envelope glycoproteins displaying N-terminal targeting polypeptides. Vectors displaying an EGF polypeptide were fully infectious on EGF receptor-negative cells, but were inactive on cells with abundant EGF receptors (inverse targeting). Receptor-mediated inactivation of gene transfer was overcome by competing the EGF receptors on the target cells with soluble EGF or by removing the displayed EGF domain from the surface of the vector particles by factor Xa cleavage of a specific protease substrate engineered into its tethering linker (protease targeting). Intravenous infusion of nontargeted HIV-1 vectors led to maximal luciferase activity in liver and spleen with moderate or minimal activity in heart, skeletal muscle, lung, brain, kidney, ovaries and bone marrow. In contrast, intravenous EGF-displaying vectors were expressed maximally in spleen with very low level luciferase expression detectable in liver (EGF-receptor rich). Liver transduction by the EGF-displaying vector was restored by pretreating the animals with soluble EGF suggesting that these vectors are inversely targeted to spleen.

Evidence for nonspecific adsorption of targeted retrovirus vector particles to cells.

The ability to specifically target a cell-type is important for the development of vectors for in vivo gene therapy. In order to produce retrovirus vectors targeting ovarian cancer cells, which specifically overexpress alpha folate receptor (alphaFR), a single chain antibody was fused as an N-terminal extension of the ecotropic and amphotropic murine leukemia virus (MLV) envelope glycoproteins. Vector particles bearing the modified glycoproteins were produced and analysed. Although conventional FACS studies indicated that viral particles bearing the modified Env could bind to ovarian cancer cells, targeted infection was not achieved. The initial step of virus-cell interaction was further studied using an immunofluorescence technique, which allows visualisation of single retrovirus particles. Vectors bearing chimeric or wild-type glycoproteins bound equally well to cells with or without the targeted receptor, although soluble chimeric glycoproteins bound specifically to FBP. Our results indicate that the incorporation of specific ligands to the virus envelope does not necessarily result in significant enhancement of vector particle binding. A similar interaction was also observed using Env-defective virus particles, suggesting that cellular factors incorporated into the lipid envelope play a dominant role in promoting initial adsorption of virus particles to cells. Significant implications arise from these observations on the interpretation of previous reports on 'targeted' vectors, and for the development of vectors for in vivo gene therapy protocols.

Identification of two intracellular mechanisms leading to reduced expression of oncoretrovirus envelope glycoproteins at the cell surface.

All retrovirus glycoproteins have a cytoplasmic domain that plays several roles in virus replication. We have determined whether and how the cytoplasmic domains of oncoretrovirus glycoproteins modulate their intracellular trafficking, by using chimeric proteins that combined the alpha-chain of the interleukin-2 receptor with the glycoprotein cytoplasmic domains of five oncoretroviruses: human T-cell leukemia virus type 1 (HTLV-1), Rous sarcoma virus (RSV), bovine leukemia virus (BLV), murine leukemia virus (MuLV), and Mason-Pfizer monkey virus (MPMV). All of these proteins were synthesized and matured in the same way as a control protein with no retrovirus cytoplasmic domain. However, the amounts of all chimeric proteins at the cell surface were smaller than that of the control protein. The protein appearing at and leaving the cell surface and endocytosis were measured in stable transfectants expressing the chimera. We identified two groups of proteins which followed distinct intracellular pathways. Group 1 included chimeric proteins that reached the cell surface normally but were rapidly endocytosed afterwards. This group included the chimeric proteins with HTLV-1, RSV, and BLV cytoplasmic domains. Group 2 included chimeric proteins that were not detected at the cell surface, despite normal intracellular concentrations, and were accumulated in the Golgi complex. This group included the chimeric proteins with MuLV and MPMV cytoplasmic domains. Finally, we verified that the MuLV envelope glycoproteins behaved in the same way as the corresponding chimeras. These results indicate that retroviruses have evolved two distinct mechanisms to ensure a similar biological feature: low concentrations of their glycoproteins at the cell surface.

Cleavage of the murine leukemia virus transmembrane env protein by human immunodeficiency virus type 1 protease: transdominant inhibition by matrix mutations.

We have identified mutations in the human immunodeficiency virus type 1 (HIV-1) matrix protein (MA) which block infectivity of virions pseudotyped with murine leukemia virus (MuLV) envelope (Env) glycoproteins without affecting infectivity conferred by HIV-1 Env or vesicular stomatitis virus G glycoproteins. This inhibition is very potent and displays a strong transdominant effect; infectivity is reduced more than 100-fold when wild-type and mutant molecular clones are cotransfected at a 1:1 ratio. This phenomenon is observed with both ecotropic and amphotropic MuLV Env. The MA mutations do not affect the incorporation of MuLV Env into virions. We demonstrate that in HIV-1 virions pseudotyped with MuLV Env, the HIV-1 protease (PR) efficiently catalyzes the cleavage of the p15(E) transmembrane (TM) protein to p12(E). Immunoprecipitation analysis of pseudotyped virions reveals that the mutant MA blocks this HIV-1 PR-mediated cleavage of MuLV TM. Furthermore, the transdominant inhibition exerted by the mutant MA on wild-type infectivity correlates with the relative level of p15(E) cleavage. Consistent with the hypothesis that abrogation of infectivity imposed by the mutant MA is due to inhibition of p15(E) cleavage, mutant virions are significantly more infectious when pseudotyped with a truncated p12(E) form of MuLV Env. These results indicate that HIV-1 Gag sequences can influence the viral PR-mediated processing of the MuLV TM Env protein p15(E). These findings have implications for the development of HIV-1-based retroviral vectors pseudotyped with MuLV Env, since p15(E) cleavage is essential for activating membrane fusion and virus infectivity.

MuLV-based vectors pseudotyped with truncated HIV glycoproteins mediate specific gene transfer in CD4+ peripheral blood lymphocytes.

Human immunodeficiency virus (HIV) infection ultimately leads to the destruction of the CD4+ lymphocyte subset and the onset of AIDS. In recent years, several gene therapy procedures making use of retroviral vectors that selectively target HIV susceptible cells have been proposed in order to interfere with HIV productive infection. However, the HIV glycoproteins' inability to be incorporated in other heterologous retroviruses considerably limits true HIV cell tropism of such vectors. We now report the use of murine leukemia virus (MuLV) viral particles harboring a truncated form of the HIV glycoprotein for specific gene delivery. Reporter lacZ gene transfer was determined to be appropriately specific to CD4+ cells when HeLaCD4 cells or peripheral blood lymphocytes (PBLs) were infected with these pseudotyped MuLV virus vectors. In contrast, MuLV viruses harboring amphotropic MuLV envelope glycoproteins displayed a broad and nonspecific infection of PBL subpopulations. This new approach, taking advantage of the ability of truncated HIV envelope glycoproteins to be incorporated into heterologous retroviral particles, may foreseeably be used in future interventions based on the coordinated delivery of therapeutic gene products specifically to cell types susceptible to HIV infection.

Definition of a 14-amino-acid peptide essential for the interaction between the murine leukemia virus amphotropic envelope glycoprotein and its receptor.

Hydrophilic loops in the receptor binding domain of the amphotropic murine leukemia virus (MLV) envelope glycoprotein (SU) are predicted and may participate in SU-receptor interactions. We have replaced five segments of 6 to 15 amino acids located in each of these regions with an 11-amino-acid tag from the vesicular stomatitis virus glycoprotein (VSV-G). Substitution was compatible with envelope processing, transport, and incorporation into virions. However, three substitution mutants showed a temperature-dependent phenotype, suggesting structural unstability. Accessibility of the tagging epitope for a monoclonal anti-VSV-G antibody was greater in oligomeric than in monomeric SUs when insertion was done in VRA, a domain essential for receptor recognition. In contrast, accessibility was independent of structural constraints when insertion was done in VRB, a domain playing an accessory role in receptor binding. Interaction with the amphotropic receptor was investigated by interference assay and study of binding and infection of target cells with MLV particles coated with the substituted envelopes. Envelope-receptor interaction was abolished when substitution was performed in a potential loop-forming segment located at the N-terminal half of VRA. Although interaction was affected to variable extents, depending on the substituted segment, other mutants conserved the ability to interact with the amphotropic receptor. These experiments indicate the 14-amino-acid segment between positions 50 and 64 of SU as an essential determinant of amphotropic-receptor recognition. They also show that a foreign linear epitope can be tolerated in several locations of the amphotropic SU receptor binding site, and this result has implications for the design of targeted retroviral vectors.

Two distinct oncornaviruses harbor an intracytoplasmic tyrosine-based basolateral targeting signal in their viral envelope glycoprotein.

It has been clearly established that the budding of the human immunodeficiency virus (HIV-1), a lentivirus, occurs specifically through the basolateral membrane in polarized epithelial cells. More recently, the signal was assigned to a tyrosine-based motif located in the intracytoplasmic domain of the envelope glycoprotein, as previously observed on various other viral and cellular basolateral proteins. In the present study, expression of human T-cell leukemia virus type 1 (HTLV-1) or Moloney murine leukemia virus envelope glycoproteins was used for trans-complementation of an envelope-negative HIV-1. This demonstrated the potential of oncornaviral retrovirus envelope glycoproteins to confer polarized basolateral budding in epithelial Madin-Darby canine kidney cells (MDCK cells). Site-directed mutagenesis confirmed the importance of a common motif encompassing at least one crucial membrane-proximal intracytoplasmic tyrosine residue. The conservation of a similar basolateral maturation signal in different retroviruses further supports its importance in the biology of this group of viruses.

A gene delivery system activatable by disease-associated matrix metalloproteinases.

We are developing protease-activatable gene delivery vehicles for selective gene delivery to protease-expressing cells. Angiogenesis, inflammation, and cancer invasion are linked to the overexpression of matrix metalloproteinases (MMPs), which destroy the extracellular matrix. Therefore, the MMPs are promising targets for therapy. We have displayed epidermal growth factor (EGF) on retroviral vector particles as an MMP-cleavable amino-terminal extension of the 4070A murine leukemia virus (MLV) envelope glycoprotein. This was achieved by engineering an MMP-cleavage signal (PLGLWA) into the linker between the EGF domain and the 4070A SU. The chimeric envelope was expressed and incorporated into viral particles, and the EGF domain could be cleaved from the surface of the viral particles by gelatinase A (MMP-2). The MMP-sensitive vector and control MMP-insensitive vectors could bind, via their displayed EGF domains, to EGF receptors on A431 cells but were unable to infect them because the EGF receptor (EGFR) does not support postbinding steps required for retroviral entry. In the presence of exogenous MMPs, the infectivity of the MMP-sensitive vector, but not of the MMP-insensitive vectors, was restored on A431 cells, and this cleavage activation could be partially blocked by MMP inhibitors. Endogenous MMPs produced by EGFR-positive HT 1080 cells could selectively activate the MMP-sensitive vector giving rise to a titer that was 1,000-fold higher on HT 1080 cells than on MMP-negative A431 cells. Inhibitor studies and gelatin zymograms indicated that the membrane-associated MT-MMP expressed on the HT 1080 cells played an important role in cleavage activation of the vector. When presented simultaneously with both EGFR-positive cell lines A431 and HT 1080, the vector could efficiently discriminate between the two different cell types, infecting the MMP-positive HT 1080 cells in preference over the A431 cells.

Receptor-binding properties of a purified fragment of the 4070A amphotropic murine leukemia virus envelope glycoprotein.

A 208-amino-acid amino-terminal fragment of the 4070A amphotropic murine leukemia virus envelope glycoprotein contains all of the determinants required to recognize cell surface amphotropic receptors. This fragment was fused with a streptavidin-binding tag, expressed in Sf9 insect cells by using a baculovirus vector, and purified to homogeneity. The (125)I-labeled purified fragment (AS208) specifically bound various cell lines susceptible to amphotropic murine leukemia virus infection. The number of AS208-binding sites was in the range of 7 X 10(4) to 17 X 10(4) per cell. Quantitative analysis of binding revealed that AS208-binding sites are heterogeneous with regard to ligand binding affinity or that cooperativity exists between receptors. Competition experiments showed that the concentration of AS208 required to inhibit virus entry was lower than that required to inhibit the binding of virus particles at the cell surface. Taken together, these data suggested that amphotropic envelope-binding sites present at the cell surface do not act independently and do not participate equally in virus infection.