Ligand-Modified Vesicular Stomatitis Virus Glycoprotein Displays a Temperature-Sensitive Intracellular Trafficking and Virus Assembly Phenotype

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doi:10.1016/j.ymthe.2003.09.018

Ligand-Modified Vesicular Stomatitis Virus Glycoprotein Displays a Temperature-Sensitive Intracellular Trafficking and Virus Assembly Phenotype Ghiabe H. Guibinga,1 Frederick L. Hall,2 Erlinda M. Gordon,2 Erkki Ruoslahti,3 and Theodore Friedmann1,* 1

Department of Pediatrics, Center for Molecular Genetics, University of California at San Diego School of Medicine, La Jolla, CA 92093-0634, USA 2 Epeius Biotechnologies Corporation, Los Angeles, CA, USA 3 Burnham Institute, La Jolla, CA, USA

*To whom correspondence and reprint requests should be addressed. Fax: (858) 534-1422. E-mail: [email protected].

The production of potentially targetable VSV-G-pseudotyped retrovirus vectors has been hampered by inadequate understanding of the structure – function relationships of the VSV-G protein. In these studies we demonstrate assembly and production of MLV-based and HIV-1based vector particles using VSV-G proteins modified by the insertion of a peptide ligand into a site corresponding to amino acid position 24 of the native VSV-G molecule. The inserted ligand represents the decapeptide encoding the collagen-binding domain of von Willebrand factor. We have used deconvolution microscopy to demonstrate that the modified VSV-G molecules sequester in perinuclear structures and are unavailable for assembly of infectious virus particles at the cell surface under standard tissue culture conditions at 37°C. In contrast, at a lower permissive temperature of 30°C, the modified VSV-G protein traffics appropriately to the cell surface and participates in useful titers. Furthermore, VSV-G-pseudotyped MLV-based and HIV-1-based vectors displaying the collagen-binding domain demonstrate a statistically significant increased attachment to a collagen matrix as indicated by an ELISA-like cell binding assay and by a focus transduction assay. Key Words: VSV-G, lentivirus, oncoretrovirus, retargeting, virus assembly, gene therapy

INTRODUCTION Gene transfer vectors derived from Moloney murine leukemia virus (MLV) have constituted one of the earliest and most useful vectors systems for preclinical gene transfer studies. This class of vectors has become even more useful through the development of methods for pseudotyping retroviruses with the G glycoprotein of vesicular stomatitis virus (VSV-G), thereby permitting the production of vectors with far higher titers and with vastly expanded host range and tissue tropism [1,2]. The more recent development of the useful lentivirus vector system has also taken advantage of VSV-G pseudotyping and has permitted efficient gene transfer into a wide host range of target cells and into nonreplicating cells such as neurons that are generally nonpermissive for infection by MLV-based vectors [3 – 5]. While the VSV-Gpseudotyped vectors are useful for many kinds of studies and even for some early clinical applications, their relative promiscuity for target cells and tissues may contribute in clinical application to toxicity and serious

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adverse events through unintended infection of nontarget cells [5 – 7]. The need for efficient and safe gene transfer not only in preclinical studies but also in clinical applications has recently led to growing interest in development of methods for targeting these and other vectors to specific target cells and tissues. Promising targeting methodology has been developed for some vector systems, particularly the adenovirus vectors [8 – 12], but progress toward retrovirus targeting has been slower and more difficult, despite enticing results in some retrovirus vector systems [13 – 17]. In the case of VSV-G-pseudotyped vectors, tissue targeting through ligand incorporation into VSV-G envelope has been made difficult by several factors, including the lack of a three-dimensional crystal structure of the envelope glycoproteins for deducing and manipulating mechanisms of virus attachment to specific cell receptors and also by the glycosaminoglycans-mediated envelope protein-independent mechanisms that drive the initial virus attachment to cells [18].

MOLECULAR THERAPY Vol. 9, No. 1, January 2004 Copyright n The American Society Of Gene Therapy 1525-0016/$30.00

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We have identified a ligand insertion site in a domain near the N-terminus of VSV-G and constructed strategic insertion sites and have introduced a potentially useful targeting ligand comprising the collagen-binding decapeptide domain N-WREPGRMELN-C derived from the von Willebrand factor [20,21]. By using deconvolution microscopy, we demonstrate that the intracellular trafficking of the ligand-modified VSV-G variant is defective at the restrictive temperature of 37jC and that it sequesters in perinuclear structures and does not traffic effectively to the cell membrane to become available for assembly into infectious virus particles. In contrast, at the lower permissive temperature of 30jC, native VSV-G along with ligand-modified VSV-G proteins traffic to the cell surface and are able to assemble efficiently into infectious virus in packaging cells. Finally, we also demonstrate that these

vector particles pseudotyped with ligand-modified VSV-G envelope glycoprotein displaying the collagen-binding domain attach significantly more efficiently to a collagen matrix than do particles containing native, unmodified VSV-G.

RESULTS AND DISCUSSION Expression of Native and Ligand-Modified VSV-G Envelope Protein at 37jC In the absence of a detailed three-dimensional structure for VSV-G, we relied on the known functional domains of the protein together with tertiary protein structure predictions based on primary amino acid sequence, such as those derived from the Kyte – Doolittle hydropathy method [19], to evaluate general structural features of

FIG. 1. Construction of ligand-modified VSV-G variant. (A) Hydropathy plot corresponding to sequences spanning the entire VSV-G protein. The native VSV-G expression plasmid (pCMV-G) was modified within the domain harboring the highest index of hydrophilicity. The hydropathy plot (mean values for a window of 17 amino acids) was elaborated using the Kyte – Doolittle (KD) hydropathic index. (B) pCMV-G was mutated by insertion of a PstI site into a strategic position near the N-terminus of VSV-G, at amino acid position 24. To provide a unique PstI cloning site, the resulting construct was further modified to remove the existing PstI site at position 262. The resulting insertless VSV-G plasmid was designated p24. Finally, plasmid p24 was modified by the incorporation of a decapeptide, N-WREPGRMELN-C, derived from the collagen-binding domain of von Willebrand factor to produce pCBD-G. Insert is flanked by flexible linkers: GHA . . . insert . . . GAA.

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the VSV-G protein [19,22,23] and to select sites that could tolerate the insertion of potentially targeting ligands and that could present such ligands on the protein surface for efficient presentation to cell receptors. Fig. 1A shows the hydropathy plot of the VSV-G sequence and reveals a suitable hydrophilic domain peaking at approximately amino acid 24. It has been demonstrated by other workers that a portion of the molecule immediately following this domain is highly conserved in VSV and rabies viruses and is important for

FIG. 2. Expression of ligand-modified VSV-G envelope protein is temperature sensitive. Western blot analysis of modified and native VSV-G molecules. (A) 293 cells were transfected with pCMV-G encoding native VSV-G or with pCBD-G encoding the collagen-binding domain-modified VSV-G. At 48 h after transfection, conditioned media were harvested and concentrated by centrifugation (see Materials and Methods), and resulting pellets were resuspended in phosphate-buffered saline (PBS). The transfected cells were lysed and centrifuged. The resulting pellet was separated from the supernatant and resuspended in PBS. The resuspended pellets and conditioned media were subjected to SDS – PAGE (15%), blotted onto nitrocellulose filters, and analyzed by Western blotting methods, using the anti-VSV-G monoclonal antibody P5D4. (B) Expression of native and ligand-modified VSV-G after transfection at 37jC and (C) at 30jC. Cultures of 293 cells were transfected with pCMV-G or pCBD-G (see Materials and methods). The conditioned media were harvested, concentrated, and subjected to Western blot analysis for VSV-G expression as described above.

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membrane destabilization and membrane fusion functions of VSV-G [24]. We used established site-specific mutation methods to introduce a PstI cloning site encoding the amino acid cassette Ala-Ala-Gly in-frame at a position corresponding to amino acid 24. The resulting construct pCMV-G is illustrated in Fig. 1B. In separate studies we have shown that such an insert does not interfere with the ability of VSV-G to assemble efficiently into fully infectious virus particles (data not shown). Into the newly created PstI site in p24, we introduced the collagen-binding decapeptide N-WREPGRMELN-C to generate the ligand-modified VSV-G CBD-G (Fig. 1B). We examined the synthesis, intracellular trafficking, and secretion of VSV-G and the collagen-modified CBD-G derivative by Western blotting, immunohistochemical, and deconvolution microscopic methods as well as by virus infectivity methods assays. The preparation of relatively pure VSV-G from transfected cells was simplified by the fact that VSV-G-expressing cells secrete the protein as pelletable vesicles of >90% purity efficiently into the conditioned medium [25 – 27]. As illustrated in Fig. 2, native VSV-G expressed from 293 cells transfected with pCMV-G is found at high levels in the pellet of conditioned medium as well as in the pelletable fraction of the cell lysate. In contrast, the ligand-modified VSV-G variant containing the collagen-binding domain (CBD-G) is found only in the pelletable fraction of the cell lysate (Fig. 2A) and is not detectable either in the suspension fraction of the cell lysate or in the conditioned medium. We interpret these data to indicate that the ligand-modified VSV-G variant pCBD-G is efficiently produced in transfected 293 cells but is not efficiently secreted out into conditioned medium. Expression of Ligand-Modified VSV-G Envelope Protein Is Temperature Sensitive Wild-type VSV-G protein is normally glycosylated in the endoplasmic reticulum by the en bloc transfer of the oligosaccharide Glc3Man9GlcNac to specific asparagine residues in positions 178 and 335 in the nascent polypeptide chain [28 – 30]. It is known that defects in VSV-G glycosylation produce a temperature-dependent deficiency of VSV-G intermolecular disulfide bonding, inefficient transport through the ER/Golgi network, and VSV-G sequestration at 37jC [31,32], but those properties revert to wild type at 30jC [31,32]. We wished to determine if the insertion of ligands into sites near the N-terminus of the VSV-G could also disrupt structurally vital glycosylation mechanisms in the molecule, and therefore we examined whether the expression and properties of ligand-modified VSV-G envelopes under our present conditions are also temperature sensitive. Along these lines, we examined the expression of the CBD-G variant at restrictive (37jC) and permissive (30jC) temperatures. Fig. 2B illustrates Western blot analysis of the expression

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in 293 cells transfected at either 37 or 30jC with plasmids expressing either native VSV-G or ligand-modified CBDG from the human CMV promoter – enhancer [2]. The conditioned media of cells expressing native VSV-G and maintained at 37jC demonstrated robust expression of VSV-G while no expression was detected under similar circumstances in cells transfected with ligand-modified envelope pCBD-G (Fig. 2B). In contrast, VSV-G protein expression could readily be detected in cells transfected with the CBD-G expression plasmid and maintained at 30jC, albeit at markedly reduced levels compared with native VSV-G (Fig. 2C). Temperature-Sensitive Intracellular Trafficking of Ligand-Modified VSV-G Envelope Protein We have used deconvolution microscopy to examine the intracellular trafficking properties of native VSV-G and the modified CBD-G in 293 cells. As expected, immunostaining of 293 cells transfected with the control pCMV-LacZ plasmid revealed no detectable VSV-G expression (Fig. 3A). On the other hand, cells transfected with plasmids expressing the native VSV-G at 37jC demonstrate expression of VSV-G protein scattered within transfected cells, as revealed by Cy3-tagged VSV-G monoclonal antibody staining. Interestingly, while cells expressing native VSV-G showed intense VSV-G staining distributed in the vicinity of the 4,6-diamido-2-phenylindole (DAPI)-stained nuclei, the bulk of protein expression was distributed more diffusely throughout the cytoplasm rather than in a perinuclear pattern (Fig. 3B). Similarly, cells expressing native VSV-G after transfection at 30jC showed a similar pattern of VSV-G expression as at 37jC (Fig. 3C). In contrast, however, cells expressing ligand-modified CBD-G showed a predominantly perinuclear distribution at 37jC with only minimal VSV-G expression throughout the rest of the cytoplasm (Fig. 3D). Conversely, cells expressing the ligand-modified CBD-G at 30jC showed a generally diffuse cytoplasmic distribution (Fig. 3E). These findings demonstrate that intracellular trafficking of ligand-modified VSV-G is temperature sensitive, and at restrictive temperature, intracellular transport is interrupted to cause a sequestration of the modified VSV-G, presumably during its passage through the endoplasmic reticulum. At the permissive temperature, intracellular transport of ligand-modified CBD-G proceeds effectively and results in transport of the protein to the plasma membrane for incorporation into budding virus particles and for secretion as vesicles into the conditioned medium [32 – 34]. Ligand-Modified VSV-G Envelopes Assemble into Infectious MLV- and HIV-1-Based Particles at Permissive Temperature To test the ability of the ligand-modified CBD-G to participate in assembly of infectious MLV-based and

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HIV-based retroviruses, we first examined CBD trafficking and virus production in the packaging cell line 293 GPLZRNL [35]. This cell line expresses MLV gag and pol genes and also contains an integrated provirus expressing the lacZ gene from the 5V viral LTR and the neomycin-resistance gene from an internal Rous sarcoma virus LTR [35]. In the presence of a functional envelope glycoprotein such as VSV-G, this cell line produces a high titer of infectious retrovirus [35]. Deconvolution microscopy of 293 GPLZRNL cells in the absence of a functional envelope glycoprotein revealed that the capsid protein (p30) was distributed around the plasma membrane and diffusely throughout the cytoplasm (Fig. 4A). After transfection of the cells with the pCMV-G plasmid at either 30 or 37jC, the expressed native VSV-G was found largely at the plasma membrane with occasional colocalization with capsid protein (see merged yellow fluorescence shown in Fig. 4B). A similar distribution was found after transfection with plasmid expressing the CBD-G variant at 30jC, with most of the CBD-G found around the plasma membrane with occasional colocalization with p30 (see merged yellow fluorescence shown in Fig. 4C). The relative paucity of colocalized p30 and VSV-G in both native and ligand-modified envelopes indicates that, at any given time, most membrane-embedded VSV-G on the plasma membrane is not at the site of active virus assembly. Nevertheless, we proceeded to evaluate the production of infectious mature MLV-based vector in packaging GPLZRNL cells and HIV-based vector in transfected 293T cells with these envelope proteins. Table 1 summarizes the infectivity assays of MLVbased and HIV-1-based vector preparations pseudotyped with native VSV-G or with ligand-modified CBD-G. For both MLV-based and HIV-based vectors, native VSV-G envelope produced infectious virus with titers of approximately 106 infectious particles/ml. In both indicator cells, the titers of MLV-based vector pseudotyped at permissive temperature with ligand-modified CBD-G VSV-G variants were approximately 100-fold lower, generally in the mid to high 103 range (Table 1), consistent with the reduced level of ligand-modified VSV-G production demonstrated in Fig. 2B. On the other hand, in the case of HIV-1-based vectors, the assembly of vector with variant VSV-G molecules was equally as efficient as with native VSV-G, with titers of vectors assembled with native VSV-G or pseudotyped with ligand-modified VSVG variants all approximately 106 infectious units/ml (Table 1). The mechanisms responsible for the apparently greater efficiency of assembly or infectivity of HIVbased vectors are not clear but are consistent with our experience of greater efficiency of virus production in transfected producer cells compared with MLV-based vectors (Miyanohara et al., unpublished results). These results indicate that the ligand-modified VSV-G protein as the sole envelope glycoprotein in virus-producing

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cells is capable of trafficking and multimerizing sufficiently well to be incorporated into mature MLV- and HIV-based virus particles and of retaining its fusogenic function to permit cell entry and infectivity at the permissive temperature. MLV- and HIV-1-Based Vectors Pseudotyped with CBD-G Exhibit Enhanced Binding to a Collagen Surface in Vitro To determine whether the ligand-modified VSV-G in the viral envelope can redirect the tropism of vector particles to a collagen matrix in vitro, we used a modified ELISA-like collagen binding assay and a focus-formation transduction assay to test the attachment and infectivity of MLV-based and HIV-1-based vectors pseudotyped with ligand-modified VSV-G envelope [14,20,21]. Fig. 5A illustrates the binding of MLV-based particles pseudotyped with either native VSV-G or CBD-G ligand-modified to a collagen surface in vitro. Although particles pseudotyped with the native VSV-G bound detectably to the collagen surface (2.06 F 0.73) arbitrary units), vector displaying the ligand-modified CBD-G vector showed an increased binding (6.26 F 0.78, P = 0.01), an approximately threefold increase in attachment over particles pseudotyped with native VSV-G, even though the adsorbed native VSV-G preparation contained approximately 2 logs

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higher titer of infectious virus than the CBD-G-modified vector (see Table 1). As a further test of the ability of ligand-modified VSVG-pseudotyped HIV-1-based vector to attach preferentially to a collagen surface, we used our previously described focus-formation transduction assay [21] to measure the effect of CBD pseudotyping of an HIV-based vector on particle adsorption to the collagen surface. Figs. 5B and 5C illustrate histochemically detectable LacZ activity in BHK cells overlaid onto adsorbed native VSV-G and CBD-modified vectors. Wells containing adsorbed vector pseudotyped with native VSV-G demonstrated only occasional LacZ-positive cells (64.8 F 3 foci). In contrast, wells overlaid with CBD-G/HIV-1 vector showed numerous LacZ-positive cells (553 F 17 foci, Fig. 5C), representing a frequency approximately eightfold higher than that of native VSV-G-pseudotyped virus particles (Fig. 5D). This report constitutes the first identification of a suitable site for the insertion of potential targeting ligands into the surrogate virus envelope protein VSV-G and the first evidence that such variants display temperature-sensitive defects in their intracellular trafficking. We further demonstrate that at a permissive temperature of 30jC, the modified VSV-G can undergo sufficiently

FIG. 3. Deconvolution microscopic examination of intracellular trafficking properties of native and ligand-modified VSV-G in 293 cells. The 293 cell lines were transfected with pCMV-G and pCBD-G. Cells were immunostained using a primary anti-VSV-G mouse monoclonal antibody, P5D4, Cy3-tagged (red) (Sigma), and nuclear DNA with DAPI. VSV-G (red) and nuclear DNA (blue) were identified in multiple optical sections. (A) Control, pCMV-LacZ-transfected 293 cell line. (B) 293 cell line transfected with pCMV-G and expressing native VSV-G envelope protein at 37jC. VSV-G staining distributed in the vicinity of the DAPI-stained nuclei; the bulk of VSV-G staining appears, however, disseminated all over the cell, away from the nucleus, and most likely in cytoplasm and plasma membrane. A similar pattern of expression was seen for the native VSV-G expressed from pCMV-G at 30jC (C). (D) Expression of ligand-modified VSV-G in transfected 293 cells from pCBD-G at 37jC. The distribution of ligand-modified VSV-G at the restrictive temperature of 37jC appears largely confined to the perinuclear region with negligible leaky expression outside of the perinuclear area. (E) Expression of ligand-modified envelopes from pCBD-G in 293 cells at the permissive temperature of 30jC. As with native envelope, ligand-modified VSV-G expression appears to have largely moved away from the perinuclear area and more into the cytoplasm and plasma membrane (original magnification 200). FIG. 4. Deconvolution microscopy examination of intracellular trafficking properties of native and ligand-modified VSV-G in the MLV-producing 293 GPLZRNL packaging cell line. The packaging cell line 293 GPLZRNL was transfected with pCMV-G or pCBD-G and cells were immunostained for (i) MLV capsid protein (p30), using a primary anti-RLV p30 goat polyclonal antibody and a secondary Cy2-conjugated donkey anti-goat antibody (green); (ii) VSV-G, using primary anti-VSV-G mouse monoclonal antibody P5D4 Cy3-tagged (red) (Sigma); and (iii) nuclear DNA with DAPI. Sites of p30 viral capsid (green), VSV-G (red), and nuclear DNA (blue) were identified in multiple optical sections. (A) Control, untransfected packaging cell line 293 GPLZRNL. (B) The packaging cell line GPLZRNL transfected with pCMV-G and expressing native VSV-G envelope protein at 30jC. Native VSV-G is found largely localized to the plasma membrane with occasional colocalization with capsid protein. (C) Expression of ligand-modified VSV-G in transfected 293 GPLZRNL cells from pCBD-G at the permissive temperature of 30jC. As with native VSV-G, ligand-modified VSV-G is found occasionally to localize with capsid protein at the plasma membrane (original magnification 200). FIG. 5. Binding of MLV-based and HIV-1-based vectors pseudotyped with ligand-modified (CBD-G) VSV-G to a collagen surface in vitro. (A) Binding of native and CBD-G-pseudotyped MLV-based vector particles. Native and ligand-modified VSV-G-pseudotyped MLV-based vectors were overlaid on collagen-coated 96-well plates and incubated at 37jC for 30 min (see Materials and methods for details). Binding is expressed as arbitrary units (OD 450 nm for adsorbed vector sample minus OD 450 nm of PBS background, multiplied by 100).The graph represents the binding of native and ligand-modified VSV-G (CBD-G) MLV vectors. Data are expressed as means F SE (standard error) and represent quadruplicate assays of two independent experiments. Statistical analyses were performed by Statistix7 (Analytical Software), using a paired t test; statistical significance was set at P < 0.05. (B and C) LacZ expression of baby hamster kidney (BHK) cells plated in 24well plates onto collagen surfaces with adsorbed HIV-1 virions pseudotyped with (B) native and (C) CBD-G-modified VSV-G. Equal amounts of infectious vector particles, as determined by infectivity assays on BHK cells, were applied to the collagen-coated wells of 24-well plates as described above. A total of 104 BHK cells in DMEM 2% FCS and Polybrene (8 Ag/ml) were then overlaid onto the bound virions in each well and the cultures were further incubated at 37jC overnight in DMEM 10% FCS. Medium was replaced with fresh DMEM 10% FCS and the cells were further incubated for an additional 24 h. Cells were then stained for LacZ activity. After adsorption of vector pseudotyped with native VSV-G, only very rare LacZ-positive cells are detectable. In C, clusters of LacZ-positive BHK cells are readily detectable after extensive washing of applied vector (original magnification 100). (D) Quantitation of transduction events after adsorption on vectors pseudotyped with native VSV-G and with the ligand-modified VSV-G-pseudotyped HIV-based vectors. Data are expressed as means F SE and represent duplicates of three independent experiments. Analyses were performed by Statistix7 (Analytical Software), using a paired t test; statistical significance was set at P < 0.05.

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FIG. 3.

effective intracellular processing and transport to the cell surface to permit assembly into infectious virus particles. To test the function of such modified VSV-G, we have chosen to introduce the von Willebrand collagen-binding domain, which has previously been reported to redi-

rect virus vector tropism to collagen-exposed vascular lesions in vivo when expressed on amphotropic and ecotropic retroviral envelope proteins [13,14,20,21]. Indeed, we have demonstrated a moderate increase in the attachment to a collagen surface of virus particles dis-

FIG. 4.

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FIG. 5.

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TABLE 1. Ligand-modified VSV-G envelops assemble into infectious MLV- and HIV-1-based particles at permissive temperature Envelope

HT1080

BHK

MLV Native-VSV-G CBD-G

8  105 9.2  103

3  105 6.2  103

HIV-1 Native-VSV-G CBD-G

1.08  106 1.04  106

6.90  105 6.08  106

Infectivity of MLV and HIV-1. Titers are expressed as either the number of G418-resistant colonies or the number of LacZ infection units per milliliter.

playing the CBD-G protein compared with native VSV-G. The introduction of ligands of larger size and greater affinities, such as single-chain antibodies, into VSV-G to pseudotype oncoretroviral and lentiviral vectors may help redirect their tropism for ex vivo and in vivo gene transfer and gene therapy applications. Toward these ends, we are currently extending these findings to the design of additional VSV-G envelope protein modifications through the incorporation of additional small peptide and larger protein ligands.

MATERIALS AND METHODS Cell lines. HT1080, HEK 293, and BHK were obtained from the American Type Culture Collection and along with 293 GPLZRNL were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS). Construction of ligand-modified envelope expression plasmid. To prepare the ligand-modified VSV-G variants, a novel PstI site in the VSV-G expression plasmid pCMV-G at a position corresponding to amino acid 24 of native VSV-G was created by mutational insertion of the sequence GCTGCAGGA encoding Ala-Ala-Gly in frame and creating a cloning site for further modification. An existing PstI site at position 3866 (GenBank JO2428) corresponding to amino acid 262 (NCBI Accession No. NP-041715) was destroyed by silent mutation. The resulting envelope construct, designated p24, encodes the small neutral amino acids Ala-Ala-Gly at the prospective insertion site designed to produce minimal disturbance of secondary structure. Transfection of p24 into 293derived packaging cell lines led to the appearance of infectious virus titers indistinguishable from unmodified native VSV-G (data not shown). Into the now unique PstI site of p24 we introduced the high-affinity collagen-binding decapeptide (WREPGRMELN) derived from human von Willebrand factor [20,21]. The peptide was flanked by short peptide linkers (GHA—GAA) as previously described [21]. The plasmid encoding the collagen-binding ligand VSV-G variant was designated pCBD-G. The insertion of the ligand into the expression plasmid was carried out by standard cloning methods as previously reported [14,20,21,36]. VSV-G envelope protein expression. The level of expression of native and ligand-modified VSV-G protein was evaluated in 293 cells. Twentyfour micrograms of native or ligand-modified VSV-G expression plasmid was transfected into 80% confluent 293 cells for 10 to 12 h in 10cm culture dishes using calcium phosphate at 37jC as previously described [1,35]. After transfection, the medium was replaced with fresh DMEM supplemented with 10% FCS and maintained at either

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30 or 37jC for another 48 to 72 h before harvesting of the conditioned medium. The conditioned medium was centrifuged at 20,000 rpm with a Beckman SW28 rotor for 90 min. The resulting VSV-G pellet was resuspended with 1 phosphate-buffered saline (PBS). Transfected cells were lysed using CelLytic-M (Sigma, St. Louis, MO). The lysate was then spun at 4000 rpm with a Sorvall RT6000B centrifuge for 10 min at 4jC, and the pellet, which contains various cell organelles and debris, was separated from the supernatant and resuspended in 1 PBS. Both resuspensions from the concentrated conditioned medium and cell lysates were subjected to 15% sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis and blotted. VSV-G protein was detected with anti-VSV-G monoclonal antibody P5D4 as previously documented [25,26]. Fluorescent immunostaining of intracellular trafficking of VSV-G envelope protein. Packaging cell line 293 GPLZRNL has been described previously [2]. 293 and 293 GPLZRNL were transfected with various VSV-G expression plasmids as described above. Forty-eight to 72 h after transfection 293 cells were immunostained with anti-VSV-G mouse monoclonal antibody P5D4 cyanine-3-tagged (red) (Sigma). 293 GPLZRNL were doubly immunostained for VSV-G as described above and MLV capsid p30 (anti-RLV, p30, NCI Antiserum 77S000087). Cyanine-2 (green)-conjugated donkey anti-goat was used as secondary antibody. Multiple optical sections by deconvolution microscopy were captured as previously described [18,37]. For nuclear visualization, cells were treated with 0.01% DAPI (blue) (Sigma) as previously reported [18]. Virus production and infectivity assay. VSV-G-pseudotyped MLV-based vectors were prepared as previously described [27,35]. Some modifications include the maintenance of the transfected cells at 30jC for 72 h instead of the standard 37jC for 24 to 48 h after transfection. Briefly, to produce temperature-sensitive VSV-G-pseudotyped MLV-based vectors, 293 GPLZRNL cells were transfected with expression plasmid encoding native and ligand-modified VSV-G envelopes as mentioned earlier and maintained at 30jC for 72 h. Virus was collected at 72 and 96 h after transfection. The conditioned medium containing virus particles was purified and centrifuged as described [27,35]. To produce temperature-sensitive VSV-Gpseudotyped HIV-1 vectors, 293 T cells were subjected to the established triplicate transfection protocols [5]. Cells were then maintained at 30jC for 72 h. The conditioned medium was collected at 72 and 96 h after transfection. Preparations were purified and centrifuged as previously reported [6,35]. The infectivity and titer assessment for MLV-based vectors was carried out on HT1080 and BHK cells as described before [27,35] and was expressed as number of G418-resistant colonies per milliliter. For HIV1-based vectors titer was expressed as number of X-gal transduction units per milliliter, where clusters of LacZ-positive cells were considered a single transduction event. ELISA-like collagen binding assay. The ELISA-like assay was carried out as previously described [21]. Briefly, equal volumes (100 Al) of infectious vectors pseudotyped with native and CBD-G, and corresponding to infectivity assays measured on BHK cells (see Table 1), were adsorbed to collagen-coated wells of 96-well plates (Biocoat, Becton – Dickinson, Bedford, MA) and incubated at 37jC for 30 min. The vectors were aspirated and discarded. The wells were washed once with abundant amounts of 1 PBS. Vectors were then reapplied for a total of three applications. The virion-coated wells were then incubated with 100 Al (1:50,000 in 1% BSA – PBS buffer) of monoclonal antibody P5D4 to VSV-G at room temperature for 45 min. Following washing (three times with PBS), wells were incubated with 100 Al (1:100,000 in 1% BSA – PBS buffer) of secondary antibody, goat anti-mouse IgG peroxidase conjugate. After intense washing (four times with PBS), 50 Al of 3,3V,5,5V-tetramethylbenzidine liquid substrate was applied to each well at room temperature; the reaction was quenched after 30 min by addition of 50 Al of 0.5 M H2SO4. The absorbance was read at 450 nm on a microplate reader equipped with SOFTmax software.

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Transduction collagen binding assay. Each tested vector (at equal titers as evaluated on BHK cells) was overlaid in each collagen-coated well of 24well plates. The wells were incubated at 37jC for 30 min. The vectors were aspirated and discarded. The wells were washed once with abundant amounts of 1 PBS. Vectors were then reapplied for a total of three applications. BHK cells (104) in DMEM 2% FCS and Polybrene (8 Ag/ml) were overlaid on bound virions in each well. The cultures were incubated at 37jC overnight. Medium was replaced with fresh DMEM 10% FCS and the cells were further incubated for another 24 h. Cells were fixed and stained for h-galactosidase activity. Statistical analysis. Data are expressed as means F SE and represent duplicates and quadruplicates of two to three independent experiments. Analyses were performed by Statistix7 (Analytical Software, Tallahassee, FL), using a paired t test; significance was set at P < 0.05.

ACKNOWLEDGMENTS This work is supported by NIH RO1 Grant HL64730. We thank Steve McMullen (UCSD Cancer Center, Digital Imaging Shared Resource) and Liqiong Liu for their technical assistance. G.H.G. is a recipient of a Fellowship Award from UNCF-Pfizer Biomedical Initiative. RECEIVED FOR PUBLICATION JULY 28, 2003; ACCEPTED SEPTEMBER 30, 2003.

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MOLECULAR THERAPY Vol. 9, No. 1, January 2004 Copyright n The American Society of Gene Therapy