Factors affecting the direct targeting of murine leukemia virus vectors containing peptide ligands in the envelope protein

To develop a reliable strategy for cell-specific delivery of retroviral vectors, we genetically modified the envelope (Env) protein of the ecotropic Moloney murine leukemia virus. We found a site in the variable region A, where the insertion of ligands, epidermal growth factor (EGF) and stromal-derived factor-1α (SDF-1α), was possible without abolishing virion incorporation of the Env protein and its ecotropic entry function. The vector containing the EGF–Env did not show the EGF receptor-dependent transduction. The vector containing the SDF-1α–Env, however, specifically transduced human cells expressing CXCR4, the receptor for SDF-1α, at titers of 10³–10⁴ c.f.u./ml. Further experiments showed that the CXCR4-dependent transduction was based on the specific interaction between the SDF-1α moiety of the SDF-1α–Env and CXCR4 and was independent of the ecotropic entry function. The direct targeting of the retroviral vector may be possible if the proper chimeric Env structure and the appropriate ligand–receptor system are employed.     

A transient three-plasmid expression system for the production of hepatocytes targeting retroviral vectors

Targeting of retroviral vectors to specific cells was attempted through modifying the surface protein of the murine leukemia viruses （MLVs）, but in many cases the protein function was affected, and it is difficult to achieve the targeted delivery. In this study, we have tried to engineer ecotropic Moloney murine leukemia viruses （MoMLV）-based retroviral vectors to transduce hepatocytes. A chimeric envelope （Env） expression plasmid was constructed containing the hepatitis B virus PreS2 peptide fused to aa ＋1 at the Nterminus of Env. Following simultaneous transfection of pgag-pol, pLEGFP and chimeric env plasmids into 293T cells, helper-free retrovirus stocks with the titer of approximately 104 infectious units/ml were achieved at 48 h post-transfection. These pseudotype vectors showed the normal host range of retrovirus, infecting host NIH 3T3 cells, although the efficiency was reduced compared with that of virions carrying wild-type ecotropic MoMLV envelope. In addition, the resultant pseudotype viruses could transduce human hepatoma cells mediated by polymerized human serum albumin with relatively high titers in comparison with those transductions without polymerized human serum albumin. This approach can be used to target hepatocytes selectively.     

Redirecting retroviral tropism by insertion of short, nondisruptive peptide ligands into envelope

A potentially powerful approach for in vivo gene delivery is to target retrovirus to specific cells through interactions between cell surface receptors and appropriately modified viral envelope proteins. Previously, relatively large (>100 residues) protein ligands to cell surface receptors have been inserted at or near the N terminus of retroviral envelope proteins. Although viral tropism could be altered, the chimeric envelope proteins lacked full activity, and coexpression of wild-type envelope was required for production of transducing virus. Here we analyze more than 40 derivatives of ecotropic Moloney murine leukemia virus (MLV) envelope, containing insertions of short RGD-containing peptides, which are ligands for integrin receptors. In many cases pseudotyped viruses containing only the chimeric envelope protein could transduce human cells. The precise location, size, and flanking sequences of the ligand affected transduction specificity and efficiency. We conclude that retroviral tropism can be rationally reengineered by insertion of short peptide ligands and without the need to coexpress wild-type envelope.     

Gene transfer into mammalian cells by a Rous sarcoma virus-based retroviral vector with the host range of the amphotropic murine leukemia virus.

We have constructed and characterized a Rous sarcoma virus-based retroviral vector with the host range of the amphotropic murine leukemia virus (MLV). The chimeric retroviral genome was created by replacing the env coding region in the replication-competent retroviral vector RCASBP(A) with the env region from an amphotropic MLV. The recombinant vector RCASBP-M(4070A) forms particles containing MLV Env glycoproteins. The vector replicates efficiently in chicken embryo fibroblasts and is able to transfer genes into mammalian cells. Vector stocks with titers exceeding 10(6) CFU/ml on mammalian cells can be easily prepared by passaging transfected chicken embryo fibroblasts. Since the vector is inherently defective in mammalian cells, it appears to have the safety features required for gene therapy.     

Host cell cathepsins potentiate Moloney murine leukemia virus infection

The roles of cellular proteases in Moloney murine leukemia virus (MLV) infection were investigated using MLV particles pseudotyped with vesicular stomatitis virus (VSV) G glycoprotein as a control for effects on core MLV particles versus effects specific to Moloney MLV envelope protein (Env). The broad-spectrum inhibitors cathepsin inhibitor III and E-64d gave comparable dose-dependent inhibition of Moloney MLV Env and VSV G pseudotypes, suggesting that the decrease did not involve the envelope protein. Whereas, CA-074 Me gave a biphasic response that differentiated between Moloney MLV Env and VSV G at low concentrations, at which the drug is highly selective for cathepsin B, but was similar for both glycoproteins at higher concentrations, at which CA-074 Me inhibits other cathepsins. Moloney MLV infection was lower on cathepsin B knockout fibroblasts than wild-type cells, whereas VSV G infection was not reduced on the B-/- cells. Taken together, these results support the notion that cathepsin B acts at an envelope-dependent step while another cathepsin acts at an envelope-independent step, such as uncoating or viral-DNA synthesis. Virus binding was not affected by CA-074 Me, whereas syncytium induction was inhibited in a dose-dependent manner, consistent with cathepsin B involvement in membrane fusion. Western blot analysis revealed specific cathepsin B cleavage of SU in vitro, while TM and CA remained intact. Infection could be enhanced by preincubation of Moloney MLV with cathepsin B, consistent with SU cleavage potentiating infection. These data suggested that during infection of NIH 3T3 cells, endocytosis brings Moloney MLV to early lysosomes, where the virus encounters cellular proteases, including cathepsin B, that cleave SU.     

Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer

Retroviral vectors have become an important tool for gene transfer in vitro and in vivo. Classical Moloney murine leukemia virus (MLV) based retroviral vectors have been used for over 20 years to transfer genes into dividing cells. Cell lines for production of retroviral vectors have become commonly available and modifications in retroviral vector design and use of envelope proteins have made the production of high titer, helper-free, infectious virus stocks relatively easy. More recently, lentiviral vectors, another class of retroviruses, have been modified for in vitro and in vivo gene transfer. The ability of lentiviral vectors to transduce non-dividing cells has made them especially attractive for in vivo gene transfer into differentiated, non-dividing tissues. Several improvements in helper plasmids and vectors have made lentivirus a safe vector system for ex vivo and in vivo gene transfer. This review will briefly summarize the background of these vector systems and provide some common protocols available for the preparation of MLV based retroviral vectors and HIV-1 based lentiviral vectors.     

Retroviral vector targeting to human immunodeficiency virus type 1-infected cells by receptor pseudotyping

We report the generation of retroviral vectors based on Moloney murine leukemia virus that specifically transduce cells infected with T-cell-tropic human immunodeficiency virus type 1 (HIV-1). This vector was pseudotyped with T-cell-tropic HIV-1 receptors CD4 and CXCR4. We demonstrate that transduction is contingent upon HIV-1 gp120 and gp41 expression.     

Functional Complementation of a Model Target to Study Vpu Sensitivity

HIV-1 forms infectious particles with Murine Leukemia virus (MLV) Env, but not with the closely related Gibbon ape Leukemia Virus (GaLV) Env. We have determined that the incompatibility between HIV-1 and GaLV Env is primarily caused by the HIV-1 accessory protein Vpu, which prevents GaLV Env from being incorporated into particles. We have characterized the ‘Vpu sensitivity sequence’ in the cytoplasmic tail domain (CTD) of GaLV Env using a chimeric MLV Env with the GaLV Env CTD (MLV/GaLV Env). Vpu sensitivity is dependent on an alpha helix with a positively charged face containing at least one Lysine. In the present study, we utilized functional complementation to address whether all the three helices in the CTD of an Env trimer have to contain the Vpu sensitivity motif for the trimer to be modulated by Vpu. Taking advantage of the functional complementation of the binding defective (D84K) and fusion defective (L493V) MLV and MLV/GaLV Env mutants, we were able to assay the activity of mixed trimers containing both MLV and GaLV CTDs. Mixed trimers containing both MLV and GaLV CTDs were functionally active and remained sensitive to Vpu. However, trimers containing an Env with the GaLV CTD and an Env with no CTD remained functional but were resistant to Vpu. Together these data suggest that the presence of at least one GaLV CTD is sufficient to make an Env trimer sensitive to Vpu, but only if it is part of a trimeric CTD complex.     

Complementation of a binding-defective retrovirus by a host cell receptor mutant

The entry of ecotropic murine leukemia virus (MLV) into cells requires the interaction of the envelope protein (Env) with its receptor, mouse cationic amino acid transporter 1 (mATRC1). An aspartic acid-to-lysine change at position 84 (D84K) of ecotropic Moloney MLV Env abolishes virus binding and infection. We recently identified lysine 234 (rK234) in mATRC1 as a residue that influences virus binding and infection. Here we show that D84K virus infection increased 3,000-fold on cells expressing receptor with an rK234A change and 100,000-fold on cells expressing an rK234D change. The stronger complementation of D84K virus infection by rK234D than by the rK234A receptor suggests that although the major reason for loss of infection of D84K and D84R virus is due to steric hindrance and charge repulsion, the loss of an interaction of D84 with receptor appears to contribute as well. Taken together, these results indicate that D84 is very close to rK234 of mATRC1 in the bound complex and there is likely an interaction between them. The definitive localization of the receptor binding site on SU should facilitate the design of chimeric envelope proteins that target infection to new receptors by replacing the receptor binding site with an exogenous ligand sequence.     

Inhibition of infectious murine leukemia virus production by Fv-4 env gene products exerting dominant negative effect on viral envelope glycoprotein

Fv-4 is a mouse gene that confers resistance against ecotropic murine leukemia virus (MLV) infection on mice. While receptor interference by the Fv-4 env gene product, Fv-4 Env, that can bind to the ecotropic MLV receptor has been shown to play an important role in the resistance, other mechanisms have also been suggested because it confers extremely efficient, complete resistance in vivo. Here, we have examined the effect of Fv-4 Env on infectious MLV production. Infectious MLV titers in supernatants obtained after transfection with a Friend MLV (FMLV) Env-expressing plasmid from MLV gag-pol producer cells harboring a retroviral vector were largely reduced by coexpression of Fv-4 Env. Syncytia formation mediated by R-peptide-deleted FMLV Env in NIH 3T3 cells was impaired by Fv-4 Env coexpression. Similarly, Fv-4 Env inhibited infectious amphotropic MLV production and syncytia formation mediated by R-peptide-deleted amphotropic MLV Env. Immunoprecipitation analysis revealed interaction of Fv-4 Env with amphotropic MLV Env as well as FMLV Env. These results indicate that Fv-4 Env inhibits infectious ecotropic and amphotropic MLV production by exerting dominant negative effect on MLV Env, suggesting contribution of this inhibitory effect to the resistance against ecotropic MLV infection in Fv-4-expressing mice.     

Inhibition of infectious murine leukemia virus production by Fv-4 env gene products exerting dominant negative effect on viral envelope glycoprotein

Fv-4 is a mouse gene that confers resistance against ecotropic murine leukemia virus (MLV) infection on mice. While receptor interference by the Fv-4 env gene product, Fv-4 Env, that can bind to the ecotropic MLV receptor has been shown to play an important role in the resistance, other mechanisms have also been suggested because it confers extremely efficient, complete resistance in vivo. Here, we have examined the effect of Fv-4 Env on infectious MLV production. Infectious MLV titers in supernatants obtained after transfection with a Friend MLV (FMLV) Env-expressing plasmid from MLV gag–pol producer cells harboring a retroviral vector were largely reduced by coexpression of Fv-4 Env. Syncytia formation mediated by R-peptide-deleted FMLV Env in NIH 3T3 cells was impaired by Fv-4 Env coexpression. Similarly, Fv-4 Env inhibited infectious amphotropic MLV production and syncytia formation mediated by R-peptide-deleted amphotropic MLV Env. Immunoprecipitation analysis revealed interaction of Fv-4 Env with amphotropic MLV Env as well as FMLV Env. These results indicate that Fv-4 Env inhibits infectious ecotropic and amphotropic MLV production by exerting dominant negative effect on MLV Env, suggesting contribution of this inhibitory effect to the resistance against ecotropic MLV infection in Fv-4-expressing mice.     

N-Linked glycosylation is required for XC cell-specific syncytium formation by the R peptide-containing envelope protein of ecotropic murine leukemia viruses

The XC cell line undergoes extensive syncytium formation after infection with ecotropic murine leukemia viruses (MLVs) and is frequently used to titrate these viruses. This cell line is unique in its response to the ecotropic MLV envelope protein (Env) in that it undergoes syncytium formation with cells expressing Env protein containing R peptide (R(+) Env), which is known to suppress the fusogenic potential of the Env protein in other susceptible cells. To analyze the ecotropic receptor, CAT1, in XC cells, a mouse CAT1 tagged with the influenza virus hemagglutinin epitope (mCAT1-HA)-expressing retroviral vector was inoculated into XC and NIH 3T3 cells. The molecular size of the mCAT1-HA protein expressed in XC cells was smaller than that in NIH 3T3 cells due to altered N glycosylation in XC cells. Treatment of XC cells with tunicamycin significantly suppressed the formation of XC cell syncytia induced by the R(+) Env protein but not that induced by the R(-) Env protein. This result indicates that N glycosylation is required for XC cell-specific syncytium formation by the R(+) Env protein. The R(+) Env protein induced syncytia in XC cells expressing a mutant mCAT1 lacking both of two N glycosylation sites, and tunicamycin treatment suppressed syncytium formation by R(+) Env in those cells. This suggests that N glycosylation of a molecule(s) other than the receptor is required for the induction of XC cell syncytia by the R(+) Env protein.     

trans-dominant interference with virus infection at two different stages by a mutant envelope protein of Friend murine leukemia virus.

A dominant negative mutant Friend murine leukemia virus (FMLV) env gene was cloned from an immunoselected Friend erythroleukemia cell. The mutant env had a point mutation which resulted in a Cys-to-Arg substitution at the 361st amino acid in the FMLV envelope protein (Env). The mutant Env was retained in the endoplasmic reticulum (ER) and accumulated because of its slow degradation. The NIH 3T3 cells expressing the mutant env were resistant to ecotropic Moloney MLV (MoMLV) penetration, suggesting that the mutant Env traps the ecotropic MLV receptors in the ER. When the mutant env gene was transfected into and expressed in the cells persistently infected with MoMLV, the wild-type Env was trapped in the ER, and the MoMLV production was suppressed. Thus, the mutant Env accumulating in the ER trans-dominantly and efficiently interfered with the ecotropic MLV infection at both the early and the late stages.     

Selective targeting and inducible destruction of human cancer cells by retroviruses with envelope proteins bearing short peptide ligands

In the accompanying study, we show how retroviral tropism can be redirected by insertion of short peptide ligands at multiple locations in envelope. Here we use this approach to selectively target and destroy human cancer cells. Many cancer cells overexpress specific cell surface receptors. We have generated Moloney murine leukemia virus (MLV) envelope derivatives bearing short peptide ligands for gastrin-releasing protein (GRP) and human epidermal growth factor receptors. Pseudotyped viruses containing these chimeric envelope derivatives selectively transduce human cancer cell lines that overexpress the cognate receptor. A retrovirus targeting the GRP receptor can deliver the thymidine kinase gene to human melanoma and breast cancer cells, which are killed by the subsequent addition of ganciclovir. Collectively, our results demonstrate that short peptide ligands inserted at appropriate locations in MLV envelope can selectively target retroviruses to human cancer cells and deliver a therapeutically relevant gene.     

Expression and functional characterization of mutant human CXCR4 in insect cells: role of cysteinyl and negatively charged residues in ligand binding

Human CXCR4 was expressed in Sf9 insect cells using the Bac-to-Bac baculovirus expression system. The recombinant receptor exhibited ligand binding activities with a K(d) value (3.3 nM) comparable to that of the native receptor. The role of four conserved cysteinyl residues was explored by site-directed mutagenesis. Each cysteine was individually changed to an alanine residue. All of the four mutants showed decreased ligand binding activity with increased K(d) values although comparable levels of receptor expression were observed. These results suggest that each of these four cysteinyl residues may be important for the ligand binding of the receptor. Evidence suggests that the ionic interaction may be involved in ligand binding. Point mutation of several relatively conserved acidic residues (Asp-10, Asp-262, Glu-275, and Glu-277) to an alanine residue greatly decreased the ligand binding activity and affinity. Since SDF-1alpha is a highly basic protein, these acidic residues may interact with the basic residues of SDF-1alpha by ionic pairing in addition to other molecular interactions and play an important role in ligand binding.     

Intracellular versus cell surface assembly of retroviral pseudotypes is determined by the cellular localization of the viral glycoprotein, its capacity to interact with Gag, and the expression of the Nef protein

Retroviral Gag and Env glycoproteins (GPs) are expressed from distinct cellular areas and need to encounter to interact and assemble infectious particles. Retroviral particles may also incorporate GPs derived from other enveloped viruses via active or passive mechanisms, a process known as "pseudotyping." To further understand the mechanisms of pseudotyping, we have investigated the capacity of murine leukemia virus (MLV) or lentivirus core particles to recruit GPs derived from different virus families: the G protein of vesicular stomatitis virus (VSV-G), the hemagglutinin from an influenza virus, the E1E2 glycoproteins of hepatitis C virus (HCV-E1E2), and the retroviral Env glycoproteins of MLV and RD114 cat endogenous virus. The parameters that influenced the incorporation of viral GPs onto retroviral core particles were (i) the intrinsic cell localization properties of both viral GP and retroviral core proteins, (ii) the ability of the viral GP to interact with the retroviral core, and (iii) the expression of the lentiviral Nef protein. Whereas the hemagglutinin and VSV-G glycoproteins were recruited by MLV and lentivirus core proteins at the cell surface, the HCV and MLV GPs were most likely recruited in late endosomes. In addition, whereas these glycoproteins could be passively incorporated on either retrovirus type, the MLV GP was also actively recruited by MLV core proteins, which, through interactions with the cytoplasmic tail of the latter GP, induced its localization to late endosomal vesicles. Finally, the expression of Nef proteins specifically enhanced the incorporation of the retroviral GPs by increasing their localization in late endosomes.     

An acidic cluster of the cytoplasmic tail of the RD114 virus glycoprotein controls assembly of retroviral envelopes

Retroviral core proteins, Gag and envelope (Env) glycoproteins are expressed from distinct cellular areas and therefore need to encounter to assemble infectious particles. The intrinsic cell localisation properties of either viral component or their capacity to mutually interact determines the assembly of infectious particles. Here, we address how Env determinants and cellular sorting proteins allow the Env derived from gamma retroviruses, murine leukemia virus (MLV) and RD114, to travel to or from late endosomes (LE), which may represent the Env assembly site of retroviruses in some cells. The individual expression of MLV Env resulted in its accumulation in LE in contrast to RD114 Env that required the presence of gamma retroviral Gag proteins. To discriminate between intrinsic intracellular Env localisation and gamma retroviral Gag/Env interactions in influencing Env viral incorporation, we studied Env assembly on heterologous lentiviral particles on which they are passively recruited. We found that an acidic cluster present at the C-terminus of the RD114 Env cytoplasmic tail determines its sub-cellular localisation and retrograde transport. Mutation of this motif induced late endosomal concentration of the RD114 Env, correlating with increased viral incorporation and infectivity. Reciprocally, the reinforcement of a poorly functional acidic motif in the MLV Env resulted in a marked decrease of its late endosomal localisation, leading to weakly infectious lentiviral particles with low Env densities. Finally, through upregulation versus downregulation of its cellular expression, we show that phosphofurin acidic-cluster-sorting protein 1 (PACS-1) controls the function of the RD114 Env acidic cluster, assigning to this cellular effector a crucial role in modulation of Env assembly of some retroviruses.     

Transient gene expression mediated by integrase-defective retroviral vectors

Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.