LDL receptor and its family members serve as the
`cellular receptors for vesicular stomatitis virus
`
`Danit Finkelshtein1, Ariel Werman1, Daniela Novick, Sara Barak, and Menachem Rubinstein2
`
`Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel
`
`Edited by Robert A. Lamb, Northwestern University, Evanston, IL, and approved March 21, 2013 (received for review August 23, 2012)
`
`Vesicular stomatitis virus (VSV) exhibits a remarkably robust and
`pantropic infectivity, mediated by its coat protein, VSV-G. Using this
`property, recombinant forms of VSV and VSV-G-pseudotyped viral
`vectors are being developed for gene therapy, vaccination, and viral
`oncolysis and are extensively used for gene transduction in vivo and
`in vitro. The broad tropism of VSV suggests that it enters cells
`through a highly ubiquitous receptor, whose identity has so far
`remained elusive. Here we show that the LDL receptor (LDLR) serves
`as the major entry port of VSV and of VSV-G-pseudotyped lentiviral
`vectors in human and mouse cells, whereas other LDLR family
`members serve as alternative receptors. The widespread expres-
`sion of LDLR family members accounts for the pantropism of VSV
`and for the broad applicability of VSV-G-pseudotyped viral vectors
`for gene transduction.
`receptor-associated protein | virus entry | sLDLR
`The enveloped RNA virus vesicular stomatitis virus (VSV) has
`
`been extensively studied and characterized (1, 2). This virus
`exhibits a remarkably robust and pantropic infectivity, mediated
`by its surface glycoprotein, VSV-G. VSV-G has been widely used
`for pseudotyping other viruses and viral vectors (1, 3–5). VSV-
`G-pseudoyped lentiviruses exhibit the same broad tropism as VSV,
`excellent stability, and high transduction efficiency, rendering them
`the gold standard for experimental gene transfer procedures. These
`and other VSV-G pseudotyped vectors are currently enabling ef-
`fective gene therapy protocols for many human tissues (6–8).
`The versatility of the VSV-G coat protein is not only exploited as
`a pseudotype gate opener for other viruses and viral vectors, but
`also in direct clinical applications of VSV in its native or engineered
`forms. The fact that VSV infects and lyses all transformed cell lines
`tested to date has been translated into protocols designed to target
`tumor cells for viral oncolysis. Unlike transformed cells, the innate
`intracellular antiviral state elicited by VSV in nontransformed cells
`leaves them unharmed (9). WT or engineered VSV has been shown
`to be efficacious in preclinical models against malignant glioma,
`melanoma, hepatocellular carcinoma, breast adenocarcinoma, se-
`lected leukemias, prostate cancer-based tumors, osteosarcoma, and
`others (10–14). The attributes of VSV-G have also been used to
`develop VSV-based vaccination protocols for tumor antigens, as
`well as for a range of pathogens (15), including influenza (1) and
`HIV, for which experiments with monkeys showed a great deal of
`promise (4, 16). Recently, recombinant VSV-based vaccination
`against tumor antigens was shown to cure established tumors (17).
`To date, attempts to identify the VSV receptor on the cell
`membrane have been unsuccessful, and this has been a source of
`significant controversy. Genetic, biochemical, and immunochem-
`ical studies have shown that VSV-G is necessary for VSV binding
`to its putative receptor, its internalization, and its fusion with the
`target cell membrane (18–20). After binding, VSV undergoes
`clathrin-mediated endocytosis (21), indicating that it gains access
`to cells through binding of VSV-G to an as yet unidentified cellular
`receptor. Early studies reported that proteolytic digestion of the
`cell surface proteins did not affect VSV binding, suggesting that
`the cellular binding site of VSV is not a membrane protein (22). In
`line with these observations and with the wide tropism of VSV, its
`receptor was suggested to be a ubiquitous plasma membrane lipid
`
`component, such as phosphatidylserine, phosphatidylinositol, or
`the ganglioside GM3 (23-25). Whereas many publications refer to
`phosphatidylserine as the VSV receptor, more recent studies
`demonstrated that this membrane component is not the cell sur-
`face receptor for VSV (26, 27).
`Previously we reported that IFN-treated cells secrete a soluble
`form of the LDL receptor (sLDLR), contributing to inhibition of
`VSV infectivity (28). We further demonstrated that this receptor
`fragment is found naturally in body fluids (29). Here we show that
`the cell surface LDLR serves as the major cellular entry port of
`VSV and that other LDLR family members serve as alternative,
`albeit less effective, entry routes in human and mouse cells.
`
`Results
`Soluble LDLR Inhibits VSV Infectivity by Binding to VSV. Initially we
`confirmed our previously reported observation that sLDLR
`inhibits VSV infectivity (28); to this end, we used highly purified
`(Fig. 1A, Inset) recombinant human sLDLR, consisting of seven
`cysteine-rich repeats, which correspond to the ligand-binding do-
`main of LDLR (30). Recombinant sLDLR inhibited the VSV-
`triggered cytopathic effect in human epithelial WISH cells in
`a dose-dependent manner, with an IC50 of 55 ng/mL (∼0.4 nM;
`Fig. 1A). Similar results were obtained with mandin darby bovine
`kidney (MDBK) cells, and mouse L cells (Fig. 1B). Exposure of
`cells to as little as 0.1 multiplicity of infection (MOI) of VSV for
`only 5 min was sufficient to trigger a complete cytopathic effect at
`17 h after infection (Fig. 1C, well “V”), indicating that the majority
`of the cell lysis was due to secondary infection by the VSV progeny.
`Addition of sLDLR before or concomitantly with VSV completely
`blocked the VSV-triggered cytopathic effect, whereas its addition
`5–10 min after VSV challenge partly inhibited only the sec-
`ondary infection, resulting in a plaque-like appearance (Fig. 1C).
`In contrast, removal of sLDLR before virus challenge resulted in
`a near complete cytopathic effect (Fig. 1C, well “R”). These
`results indicated that to exert its antiviral effects, sLDLR must be
`present both at the early stages of the viral infection and at later
`stages, to also inhibit secondary infection by viral progeny. To test
`whether sLDLR inhibits the initial binding of VSV to cells, we
`exposed WISH cells to VSV for 15 min in the absence or presence
`of sLDLR, then washed the cells and measured cell-associated
`VSV by quantitative and by semiquantitative RT-PCR of VSV
`RNA. We found that sLDLR inhibited VSV binding to cells in
`a dose-dependent manner, at both 4 °C and 37 °C (Fig. 1D, Inset).
`The inhibition of virus–cell binding mediated by sLDLR sug-
`gested that sLDLR inhibits VSV infectivity by binding to either
`the virus or to a putative cellular VSV receptor. To test the
`
`Author contributions: D.F., A.W., D.N., and M.R. designed research; D.F., D.N., and S.B.
`performed research; D.F., D.N., and M.R. analyzed data; and M.R. wrote the paper.
`
`The authors declare no conflict of interest.
`
`This article is a PNAS Direct Submission.
`1D.F. and A.W. contributed equally to this work.
`2To whom correspondence should be addressed. E-mail: menachem.rubinstein@weizmann.
`ac.il.
`
`This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
`1073/pnas.1214441110/-/DCSupplemental.
`
`7306–7311 | PNAS | April 30, 2013 | vol. 110 | no. 18
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`CELLBIOLOGY
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`reflects avidity rather than affinity. Dose–response binding of VSV
`to immobilized sLDLR gave a dissociation constant (Kd) of 10−11 M,
`indicating a very high avidity (Fig. S1). VSV-G-pseudotyped
`lentiviral vectors (VSV-G-LV) share with VSV only their re-
`ceptor-interacting component, VSV-G, and hence can be used
`for measuring the affinity of VSV-G to sLDLR. To this end we
`immobilized VSV-G-LV to the sensor chip and analyzed binding
`of increasing sLDLR concentrations in the presence of Ca2+. As
`expected, the affinity of a single sLDLR molecule interacting
`with VSV-G (Kd = 10−8 M; Fig. 1F) was lower than the avidity
`measured by VSV binding to immobilized sLDLR. In a control
`experiment we tested binding of sLDLR to immobilized lym-
`phocytic choriomeningitis virus-pseudotyped lentiviral vector
`(LCMV-LV), which differs from VSV-G-LV only in its coat
`protein. sLDLR did not bind to the immobilized LCMV-LV. The
`high affinity of the VSV binding to sLDLR and the dependence
`of the binding on Ca2+ strongly supported the specificity and
`physiological relevance of this in vitro interaction. Further evi-
`dence for the interaction between the ligand-binding domain of
`LDLR and VSV-G was obtained by coimmunoprecipitation.
`sLDLR was added to a suspension of VSV and then immuno-
`precipitated with protein-G-bound anti-LDLR mAb 28.28 (32),
`anti-LDLR mAb C7, an isotype-matched control mAb, or no
`antibody. SDS/PAGE and immunoblotting with anti-VSV-G and
`anti-LDLR antibodies revealed that sLDLR was specifically
`bound to VSV-G (Fig. 1G).
`We also evaluated the impact of sLDLR on EGFP expression
`after transduction of cells with an EGFP-encoding VSV-G-LV.
`Figs. 1 H and I show that sLDLR completely blocked transduction
`of newborn human FS-11 foreskin fibroblasts by EGFP-encoding
`VSV-G-LV. In contrast, sLDLR did not inhibit transduction of the
`cells with an EGFP-encoding LCMV-LV, which differs from VSV-
`G-LV only by its coat protein. Taken together, these results in-
`dicate that sLDLR inhibits VSV infectivity by binding to VSV-G.
`
`LDLR Is the Major VSV Receptor in Human Cells. The fact that sLDLR
`bound VSV at high affinity and inhibited its infectivity indicated
`that sLDLR masked VSV constituents essential for its interaction
`with a cellular receptor, prompting us to examine whether LDLR
`serves as the VSV entry port. On the basis of increased binding of
`radiolabeled VSV to trypsin-treated cells, earlier studies con-
`cluded that the VSV receptor was unlikely to be a protein (22, 33).
`To examine this conclusion more rigorously, we tested trypsin-
`treated cells for their resistance to VSV infection. We exposed
`these cells in suspension to trypsin/EDTA or to EDTA alone for
`30 min, then washed the cells three times with medium containing
`10% (vol/vol) FBS to block residual trypsin activity, as described
`previously (22). We then challenged the cell suspensions with
`VSV, washed the cells, plated them, and incubated them for 17 h.
`The EDTA-treated cells were completely lysed by VSV, whereas
`the trypsin-treated cells were fully resistant to VSV infection (Fig.
`2A, Upper). Plaque assays of the culture supernatants revealed
`∼500-fold lower VSV yields in the trypsin-treated cultures (Fig.
`2A, Lower). These results indicate that a cell surface protein is
`essential for VSV infectivity, probably serving as a VSV receptor.
`We then examined whether VSV and LDL, the physiological
`LDLR ligand, compete for binding to LDLR. FS-11 fibroblasts
`were incubated with increasing concentrations of VSV, followed
`by fluorescently labeled LDL (Dil-LDL) (4 h, 4 °C). The cultures
`were then washed and brought to 37 °C for 1 h to allow in-
`ternalization of the bound Dil-LDL. VSV inhibited binding of Dil-
`LDL to the FS-11 fibroblasts in a dose-dependent manner (Fig.
`2B). No uptake was seen when Dil-LDL alone was similarly in-
`cubated with the LDLR-deficient (34) GM701 fibroblasts (Fig.
`2C). Similarly, VSV inhibited Dil-LDL binding to FS-11 fibro-
`blasts, as determined by flow cytometry (Fig. 2D). These results
`indicate that VSV and LDL share LDLR as their common re-
`ceptor. However, as we reported previously (28), LDLR-deficient
`
`Soluble LDLR binds VSV and inhibits infection by VSV and trans-
`Fig. 1.
`duction by a VSV-G-pseudotyped lentiviral vector. (A) Survival ± SD of WISH
`cells as determined by Neutral red staining after treatment with sLDLR and
`challenge by VSV at the indicated MOI. n = 3. (Inset) SDS/PAGE of sLDLR (10
`μg). Molecular mass markers (kDa) are shown on the right lane. (B) Surviving
`WISH cells, bovine MDBK cells, and murine L cells after treatment with se-
`rially twofold-diluted sLDLR (starting at 8 μg/mL) followed by VSV (MOI = 1
`for WISH and MDBK cells, MOI = 0.07 for L cells. C, no virus; V, VSV without
`sLDLR. (C) Surviving WISH cells after addition of sLDLR (1 μg/mL) at the in-
`dicated times relative to the time of VSV (MOI = 0.1) addition. In well
`R, sLDLR was added for 120 min and removed before VSV challenge. C and V
`are as in B. (D) Quantitative RT-PCR of VSV RNA after attachment of VSV
`(MOI = 10) at 4 °C for 4 h to WISH cells in the presence of the indicated sLDLR
`concentrations. VSV RNA ± SE is normalized to TATA binding protein mRNA;
`*P < 0.02, **P < 0.002, compared with the leftmost bar, n = 3. (Inset) RT-PCR
`products of VSV RNA, isolated after similar experiments, performed at 4 °C
`and at 37 °C. (E) Surface plasmon resonance analysis of VSV binding to
`immobilized sLDLR in PBS with or without CaCl2 (1 mM). (F) Surface plasmon
`resonance analysis of sLDLR binding to immobilized VSV-G-LV in PBS + 1 mM
`CaCl2. (G) (Upper) Immunoblotting of VSV-G after coimmunoprecipitation of
`a solubilized VSV-sLDLR complex with the following antibodies (lanes):
`1, mAb 28.28 anti-LDLR; 2, mAb C7 anti-LDLR; 3, isotype control mAb; 4, no
`antibody. A VSV-G marker is shown in lane 5. (Lower) Reblotting of the
`membrane with anti-LDLR mAb 29.8. (H) EGFP expression (green) after
`transduction of FS-11 fibroblasts with either EGFP-encoding VSV-G-LV or
`EGFP-encoding LCMV-LV in the presence or absence of sLDLR (5 μg/mL).
`Nuclei were counterstained with Hoechst 33258 (blue). (Insets) Enlarged
`magnifications. (I) Average ± SD EGFP expression in cultures transfected as
`shown in H. ***P < 0.003, n = 4. N.S., not significant (P = 0.525), n = 4.
`
`possible binding of sLDLR to VSV, we used surface plasmon
`resonance. Binding of LDL to LDLR is Ca2+ dependent (31).
`Similarly, we found that VSV effectively bound to immobilized
`sLDLR in PBS, but only in the presence of Ca2+ (Fig. 1E). Be-
`cause the VSV envelope contains 400–500 trimeric VSV-G
`spikes, quantitative analysis of its binding to immobilized sLDLR
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`but not in LDLR-expressing WT FS-11 fibroblasts (Fig. 3C).
`Similarly, measuring virus yields 7 h after infection revealed that
`LDLR-deficient GM701 fibroblasts were significantly less sus-
`ceptible to VSV infection compared with WT fibroblasts (Fig.
`3D). Importantly, RAP further attenuated VSV expression in the
`LDLR-deficient fibroblasts but not in the WT cells (Fig. 3D).
`We then studied the impact of blocking all LDLR family
`members on VSV infectivity by combining RAP and anti-LDLR
`antibodies. We preincubated WISH cells either with the neutral-
`izing or the nonneutralizing anti-LDLR mAbs, 29.8 and 28.28, in
`the absence or presence of RAP at 37 °C and then challenged the
`cells with VSV. RAP alone provided little protection from VSV
`infection, and nonneutralizing mAb 28.28 provided no protection,
`whereas anti-LDLR mAb 29.8 provided limited but significant
`protection. However, the combination of RAP and mAb 29.8,
`which blocks all LDLR family members, completely inhibited VSV
`infection (Fig. 3E).
`We then studied the role of the LDLR family members in VSV
`uptake. WT and LDLR-deficient fibroblasts were incubated with
`VSV at conditions leading to internalization of at least two-thirds
`
`Fig. 3. LDLR and its family members are the major and the alternative VSV
`receptors, respectively. (A) Crystal violet-stained WISH cells, untreated (Ctrl.) or
`treated with anti-LDLR mAbs (30 min, 4 °C) and then subjected to limited in-
`fection by VSV (MOI = 0.05, 4 °C, 1 h). (B) Crystal violet-stained cultures of WT
`(FS-11) and LDLR-deficient (GM701) fibroblasts, either untreated (Control) or
`treated with isotype control mAb or anti-LDLR mAb 29.8 (12.5 μg/mL each),
`followed by VSV as in A. (C) Crystal violet-stained cultures of WT FS-11 fibro-
`blasts and LDLR-deficient GM701 fibroblasts, treated with RAP (100 nM,
`30 min, 37 °C) alone, VSV (MOI = 1) alone, or RAP followed by VSV. (D) Plaque
`assay of culture supernatants from WT FS-11 fibroblasts and LDLR-deficient
`GM701 fibroblasts (50,000 cells per well) preincubated (30 min, 37 °C) in
`DMEM-10 or in DMEM-10 + RAP (100 nM), then challenged with VSV (0.5 MOI,
`30 min, 37 °C), washed three times, and incubated in DMEM-10 (0.1 mL, 37 °C,
`7 h). ***P < 0.001, n = 4. (E) Crystal violet-stained WISH cells grown to con-
`fluence in 96-well plates, incubated (30 min, 37 °C) with the indicated combi-
`nations of RAP (200 nM), neutralizing anti-LDLR mAb 29.8, and nonneutralizing
`anti-LDLR mAb 28.28 (50 μg/mL each); cells were then challenged with VSV at
`the indicated MOI. Cell viability (bar plot) was determined by reading the OD540
`of cultures treated with VSV at MOI = 0.06. ***P < 0.002, n = 4.
`
`Fig. 2. VSV and LDL share a common cell surface receptor. (A) Surviving WISH
`epithelial cells, pretreated with trypsin-EDTA or EDTA, washed and challenged
`with VSV (0.015 MOI, 15 min). Figure is representative of six replicates. VSV
`yield (Lower) was determined by a plaque assay of the culture supernatants.
`*P < 0.03, n = 3. (B) Internalized Dil-LDL (red) in FS-11 fibroblasts after binding
`(1.67 μg/mL, 4 h, 4 °C) in the presence of the indicated VSV MOI. The cultures
`were then washed, and bound Dil-LDL was allowed to internalize (1 h, 37 °C).
`(Insets) Higher magnifications. (C) (Upper) Immunoblot of LDLR in WT FS-11
`fibroblasts and LDLR-deficient GM701 fibroblasts. (Lower) Lack of Dil-LDL
`uptake by LDLR-deficient GM701 fibroblasts. (D) Flow cytometry of FS-11
`fibroblasts treated with Dil-LDL as in A in the absence or presence of VSV
`(MOI = 2000). n = 3. (E) LDLR-deficient GM701 fibroblasts untreated or treated
`with sLDLR (1 μg/mL) and challenged with VSV (MOI = 1).
`
`fibroblasts were not resistant to VSV infection, suggesting the
`existence of additional VSV receptors (Fig. 2E).
`To obtain further evidence that LDLR is a VSV receptor, we
`used mAbs raised against epitopes within the ligand-binding do-
`main of human LDLR (32). Because LDLR-deficient cells were
`still susceptible to VSV infection (Fig. 2E), we resorted to limited
`infection, thereby rendering the cell surface receptor the rate-
`limiting component. We incubated WISH cells with anti-LDLR
`mAbs for 30 min at 4 °C, followed by VSV challenge (MOI = 0.05,
`4 °C, 1 h). The cultures were washed and then incubated for 17 h at
`37 °C in the presence of the same antibodies. mAb 29.8, directed
`against class A cysteine-rich repeat 3 of the LDLR ligand-binding
`domain, almost completely inhibited the VSV-triggered cytopathic
`effect in WISH cells, whereas mAb 28.28, directed against repeat
`6, did not inhibit VSV infectivity (Fig. 3A). Using the same in-
`fection protocol revealed that mAb 29.8 almost completely
`inhibited the VSV-triggered cytopathic effect in WT FS-11 fibro-
`blasts but not in the LDLR-deficient GM701 fibroblasts (Fig. 3B).
`These experiments indicate that LDLR is the major VSV receptor
`in human cells, and VSV requires cysteine-rich repeat 3 of the
`LDLR ligand-binding domain to infect human cells; furthermore,
`it is likely that VSV uses alternative entry port(s) in the LDLR-
`deficient cells.
`
`Other LDLR Family Members Serve as Alternative VSV Entry Ports.
`The ligand-binding domain of all LDLR family members contains
`multiple, class A cysteine-rich repeats, structurally homologous to
`those of the LDLR (35). Because sLDLR completely blocked
`VSV infectivity even in LDLR-deficient cells (Fig. 2E), we hy-
`pothesized that such additional family members could serve as the
`alternative VSV entry routes. Receptor-associated protein (RAP)
`is a common chaperone of all LDLR family members (35). When
`added exogenously, RAP completely blocks ligand binding to all
`LDLR family members with the exception of LDLR itself (36).
`Indeed, preincubation of cells with RAP inhibited the VSV-
`triggered cytopathic effect in LDLR-deficient GM701 fibroblasts
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`Fig. 5. LDLR is the main entry port of VSV-G-LV. (A) EGFP expression in WT
`FS-11 fibroblasts and LDLR-deficient GM701 fibroblasts, 72 h posttransduction
`with either EGFP-encoding VSV-G-LV in the absence or presence of polybrene,
`or with EGFP-encoding LCMV-LV in the absence of polybrene. (Insets) Higher
`magnifications. (B) Average ± SD of the relative EGFP expression (Rel. expr.)
`after transduction with VSV-G-LV in the absence (open bars) or presence (filled
`bars) of polybrene. ***P < 0.0001, n = 3. (C) Average ± SD of the relative EGFP
`expression after transduction with LCMV-LV. N.S., not significant (P = 0.78),
`n = 3. (D) Immunoblot of LDLR after either mock transduction of GM701
`fibroblasts with polybrene alone (Ctrl.) or their transduction with VSV-G-LV
`encoding LDLR in the presence of polybrene (LV-LDLR). (E) EGFP expression in
`cultures of LDLR-reconstituted or mock-transduced GM701 fibroblasts, trans-
`duced for 48 h with EGFP-encoding VSV-G-LV. (Insets) Higher magnifications.
`(F) Average ± SD of the relative EGFP expression shown in E. **P < 0.01, n = 3.
`
`To further confirm the role of LDLR in VSV-G-LV entry to
`cells, we rescued LDLR expression in the LDLR-deficient GM701
`fibroblasts by polybrene-assisted transduction with an LDLR-
`encoding VSV-G-LV. After rescue, the GM701 cells expressed
`LDLR, as determined by immunoblotting (Fig. 5D), and became
`significantly more responsive to transduction with the EGFP-
`encoding VSV-G-LV in the absence of polybrene (Fig. 5 E and F).
`In a reciprocal experiment, knockdown of LDLR by specific
`siRNA and not by scrambled, nontargeting control siRNA signif-
`icantly attenuated the transduction of FS-11 fibroblasts by VSV-G-
`LV, whereas it had no significant effect on transduction of the cells
`by LCMV-LV (Fig. S2). This study further confirmed that the
`reduced transduction by VSV-G-LV observed in the LDLR-
`deficient cells was due to lack of LDLR and not due to other
`inherent differences between the WT FS-11 fibroblasts and the
`LDLR-deficient GM701 cells.
`We then studied whether other LDLR family members enable
`transduction of cells by VSV-G-LV. As was the case with VSV
`infection (Fig. 3 C–E), RAP further attenuated the transduction of
`the LDLR-deficient GM701 fibroblasts by VSV-G-LV, indicating
`that in addition to LDLR, other LDLR family members enabled
`the residual transduction observed in the LDLR-deficient fibro-
`blasts (Fig. 6 A and B). In parallel, we found that similarly to hu-
`man cells, LDLR-deficient murine embryonic fibroblasts (MEFs)
`were significantly less susceptible to transduction by VSV-G-LV
`compared with their WT counterparts, and RAP further attenu-
`ated the VSV-G-LV-mediated transduction of the LDLR-
`deficient MEFs. Unlike human fibroblasts, RAP significantly re-
`duced VSV infectivity of WT MEFs (Fig. 6 C and D), suggesting
`a more substantial role of the other LDLR family members in
`VSV infection of mouse cells.
`Taken together, our results demonstrate that LDLR is the
`major entry port of both VSV and VSV-G-LVs in human and
`mouse cells, whereas other LDLR family members serve as
`
`of the bound VSV (37). The cultures were then washed, immu-
`nostained with anti-VSV-G, and VSV foci were counted. Com-
`pared with the WT FS-11 fibroblasts, the LDLR-deficient GM701
`fibroblasts internalized significantly less VSV (Figs. 4 A and C).
`This result confirmed that LDLR has a major role in VSV in-
`ternalization. Furthermore, neutralizing mAb 29.8 but not the
`nonneutralizing mAb 28.28 significantly inhibited VSV binding
`and subsequent internalization into the WT fibroblasts (P < 0.05),
`whereas the combination of mAb 29.8 and RAP, which blocks all
`LDLR family members, completely abolished VSV binding and
`subsequent internalization to these cells (Figs. 4 B and C). Hence,
`we concluded that LDLR and its other family members mediate
`VSV entry into human cells.
`
`LDLR and Its Family Members Mediate Transduction by VSV-G-
`Pseudotyped Lentiviral Vectors. VSV and the frequently used
`VSV-G-LVs share VSV-G as their common coat protein,
`prompting us to study the role of LDLR and its family members in
`cell transduction by an EGFP-encoding VSV-G-LV. After trans-
`duction, WT FS-11 fibroblasts expressed significantly higher levels
`of EGFP compared with LDLR-deficient fibroblasts (Fig. 5 A
`and B). To demonstrate that the reduced EGFP expression in the
`LDLR-deficient fibroblasts was due to lack of LDLR and not due
`to other inherent difference between these two cell types, we per-
`formed two control experiments. First we transduced both the WT
`and the LDLR-deficient fibroblasts with EGFP-encoding VSV-G-
`LV in the presence of polybrene, an agent rendering virus entry
`receptor-independent (38). Under these conditions, the level of
`EGFP expression in the WT and the LDLR-deficient GM701
`fibroblasts was comparable (Fig. 5 A and B). Furthermore, trans-
`duction with another lentiviral vector, EGFP-encoding LCMV-LV,
`which differs from VSV-G-LV only in its coat protein, gave very
`similar levels of EGFP expression in the WT and LDLR-deficient
`fibroblasts (Fig. 5 A and C). These two control experiments con-
`firmed that the reduced level of EGFP expression observed in the
`GM701 fibroblasts after transduction with VSV-G-LV was due to
`their lack of LDLR expression.
`
`LDLR and its family members mediate VSV internalization by human
`Fig. 4.
`fibroblasts. (A) Internalized VSV in WT FS-11 fibroblasts and LDLR-deficient
`GM701 fibroblasts after incubation with VSV (MOI = 500, 4 min, 37 °C) and
`washing three times with PBS. The cultures were then fixed and stained with
`anti-VSV-G (red). (B) Internalized VSV in WT FS-11 fibroblasts preincubated
`with the indicated combinations of RAP and anti-LDLR mAbs (30 min, 37 °C),
`followed by VSV as in A. (C) VSV foci in A and B were counted in fields
`containing at least 30 cells. **P < 0.01; *P < 0.05 (compared with FS-11
`challenged with VSV only, leftmost bar); n = 3.
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`sLDLR did not inhibit infection of insect SF6 cells. Although the
`insect lipophorin receptor and mammalian LDLR are structurally
`highly similar, their mode of action is quite different. Whereas
`LDLR releases its cargo in the endosome, lipophorin remains as-
`sociated with its receptor and is eventually resecreted (43). Hence
`VSV probably infects insect cells by other means.
`LDLR family proteins are endocytosed and recycle back to the
`membrane every 10 min, irrespective of ligand binding (44), and
`hence are ideal virus entry ports. It is therefore not surprising that
`in addition to VSV, several other unrelated viruses have been
`suggested to use these receptors as their ports of cellular entry (45–
`47). Of particular interest are the minor group common cold virus
`(46) and hepatitis C virus (48), which much like VSV use LDLR as
`well as other LDLR family members for cell entry. Similar to any
`other ligand, once internalized, VSV must dissociate from its re-
`ceptor. The endosomal lumen is characterized by low pH and low
`concentration of calcium ions; both these features are required for
`β-VLDL release from LDLR (49). Our finding that Ca2+ is es-
`sential for binding of VSV to immobilized sLDLR in vitro sug-
`gests that calcium ion depletion might also facilitate VSV release
`from its receptor after internalization.
`In recent years high-throughput genome-wide screens became
`the method of choice for deciphering gene function. However,
`such screens may fail in cases of genetic redundancy, and the VSV
`receptor is a good case in point. A recent study using genome-wide
`RNAi screen identified 173 host genes essential for completion of
`the VSV replication cycle, but it did not detect the VSV receptor
`despite its obviously essential role (50). Recently it was demon-
`strated that the endoplasmic reticulum chaperone gp96 (endo-
`plasmin or GRP94) is essential for VSV binding to cells and for
`their subsequent infection (27). This chaperone is a constituent of
`a multiprotein complex, required for protein folding in the endo-
`plasmic reticulum (51). Grp78, another component of this multi-
`protein complex, was reported to interact with LDLR (52). In
`preliminary studies we found that knockdown of gp96 disrupted
`the glycosylation of LDLR, manifested by reduced apparent mo-
`lecular mass in SDS/PAGE. It is therefore likely that processing of
`other LDLR family members, which serve as VSV receptors, also
`requires gp96, thereby explaining its critical role in VSV infectivity.
`The identification of the VSV receptor is of significant clinical
`importance because recombinant VSV and VSV-G-pseudotyped
`viral vectors are being developed for viral oncolysis, for vaccination,
`and for gene therapy. Up-regulation of LDLR in vivo [e.g., by
`pretreatment with statins (53)] might increase the efficacy of such
`vectors. Furthermore, liver cells and certain tumor cells, which
`express high levels of LDLR (54), might be the preferred targets of
`VSV-G-based gene therapy as well as VSV-G-based viral oncolysis.
`
`Materials and Methods
`LDLR-deficient human GM701 fibroblasts were from the Coriell Institute. Human
`FS-11 foreskin fibroblasts were kindly provided by M. Revel. VSV (Indiana Strain)
`and all other cell types were from ATCC. Cells were grown in media containing
`10% (vol/vol) FBS (MEM-10 or DMEM-10). VSV was propagated in WISH cells,
`purified by gradient centrifugation, and plaque-assayed. sLDLR25–313 was pro-
`duced in CHO cells and purified to homogeneity. VSV cytopathic effects were
`evaluated 17 h after VSV challenge. Plaque assays, flow cytometry, preparation
`of lentiviral vectors, transduction of cells, RT-PCR, quantitative PCR, surface
`plasmon resonance, knockdown of LDLR mRNA, immunoblotting, and all other
`methods were performed according to published procedures or as recom-
`mended by the various manufacturers. Trypsin digestion was performed us-
`ing cell culture grade trypsin/EDTA on cells in suspension. Residual trypsin
`activity was blocked by 3× washing of the cells in DMEM-10 before VSV
`challenge. Image analysis and counting of nuclei, plaques, and VSV foci was
`performed using the ImageJ program (National Institutes of Health). Fluo-
`rescence intensities and internalized VSV foci were normalized to the number
`of nuclei/field, using fields containing at least 30 nuclei. Statistical analysis
`was performed using the unpaired Student t test of the KaleidaGraph pro-
`gram on at least three independent replicates. Details can be found in SI
`Materials and Methods.
`
`Fig. 6. Other LDLR family members are alternative entry ports of VSV-G-LV
`in human and mouse cells. (A) EGFP expression in WT FS-11 fibroblasts and
`LDLR-deficient GM701 fibroblasts, transduced with EGFP-encoding VSV-G-LV
`in the absence (Control) or presence of RAP (100 nM). (Insets) Higher mag-
`nifications. (B) Average ± SD of EGFP expression shown in A. ***P < 0.0002,
`n = 3. *P < 0.03, n = 3. (C) EGFP expression in WT murine embryonic
`fibroblasts (WT) and LDLR-deficient MEFs, transduced with EGFP-encoding
`VSV-G-LV as in A. (Insets) Higher magnifications. (D) Average ± SD of EGFP
`expression shown in C. All fluorescence intensity values were normalized to
`the nuclei counts. *P < 0.05, **P < 0.007, ***P < 0.002, n = 3.
`
`alternative receptors. The complete protection from VSV in-
`fection obtained by blocking all LDLR family members identi-
`fies these receptors as the only possible VSV entry ports into
`human cells.
`
`Discussion
`In this study we provide several lines of evidence establishing LDLR
`as the major entry port of VSV and VSV-G-LV, including the high
`affinity and calcium ion dependence of VSV binding to soluble
`LDLR, the competition between VSV and LDL for receptor bind-
`ing, the inhibition of VSV internalization and infectivity by mAbs to
`the ligand-binding domain of LDLR, and the crucial role of LDLR
`in cell transduction by a VSV-G-LV. On the basis of binding of
`radiolabeled VSV to protease-treated cells, earlier studies proposed
`that the VSV receptor is not a protein (22, 24, 33). In contrast, our
`finding that such trypsin-treated cells resist VSV infection indicates
`that the VSV receptor is a protein. Two earlier studies indirectly
`support the role of LDLR as the major VSV receptor. Binding of
`VSV to MDCK epithelial cells is 100 times more prevalent at the
`basolateral membrane compared with their apical surface (39). In-
`dependently, it was shown that LDLR is expres