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Comparative Study
. 2012 Jan;86(1):373-81.
doi: 10.1128/JVI.05411-11. Epub 2011 Oct 19.

Targeted entry via somatostatin receptors using a novel modified retrovirus glycoprotein that delivers genes at levels comparable to those of wild-type viral glycoproteins

Fang Li  1 , Byoung Y Ryu, Robin L Krueger, Scott A Heldt, Lorraine M Albritton
Affiliations
Comparative Study

Targeted entry via somatostatin receptors using a novel modified retrovirus glycoprotein that delivers genes at levels comparable to those of wild-type viral glycoproteins

Fang Li et al. J Virol. 2012 Jan.

Abstract

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.

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Figures

Fig 1
Fig 1
Design of the Sst-RBS and control glycoproteins. (A) Left panel, location of the putative ecotropic receptor binding sequences (shown in gray) on the predicted molecular surface of wild-type ecotropic Moloney MLV glycoprotein. Right panel, predicted location of the somatostatin Sst-14 ligand sequences (shown in gray) on the molecular surface of the Sst-RBS model structure. Arrows point to the positions of ligand insertion in traditional designs for chimeric Env. (B) Schematic diagrams of the SU proteins of Env chimeras. Black triangles indicate sites of insertion of the Sst ligand in control chimeras. RBS, putative ecotropic receptor binding sequences. RBD, receptor binding domain. PRR, proline-rich region. CTD, carboxy-terminal domain. Sst, somatostatin.
Fig 2
Fig 2
Sst-RBS gave transduction of human 293 cells transiently transfected with an SSTR2a cDNA. (A) Cells were exposed to unconcentrated MLV vectors coated with the indicated Env and then fixed and stained for transduced β-galactosidase (β-Gal+) activity 48 h later. The mean percentage of transduced cells ± standard deviation is shown for the Sst-RBS virus from an experiment that represents three that were performed; there were no lacZ-positive cells in the other cell samples in any of the experiments. (B) Quantification of SSTR-positive cells. The mean percentage of cells immunostained with anti-SSTR2a antiserum ± standard deviation is shown between representative fluorescence and phase micrographs from one of three experiments performed. (C) Quadruplicate wells of parent 293 cells or 293 cells transiently transfected with SSTR2a cDNA were exposed to serial 10-fold dilutions of vectors, and the endpoint dilution titration was calculated after staining for transduction of lacZ. Values shown represent the means ± standard deviations of the results of four independent titration experiments.
Fig 3
Fig 3
Dose-dependent inhibition by somatostatin peptides is consistent with entry through the target SSTR. Human 293 cells transiently transfected with SSTR2a cDNA were exposed to Sst-RBS pseudovirions at 22°C (left panel) or 37°C (right panel) in the presence or absence of synthetic somatostatin-14 peptide at the indicated concentrations. Values shown represent the means ± standard deviations of the results of three independent experiments.
Fig 4
Fig 4
Sst-RBS loses the use of the ecotropic receptor and shows a stable virion association. (A) Murine NIH 3T3 cells expressing endogenous ecotropic receptors were exposed to serial 10-fold dilutions of the indicated virus. Values shown represent the means ± standard deviations of the results of four independent titrations. Sst-RBS gave 0 to 8 lacZ TU/ml in the four experiments. No infection was seen in cells exposed to vectors lacking Env (No Env). (B) Assembly and subunit association of chimeric Env into pseudovirions. Viral proteins in vectors pelleted from supernatants by ultracentrifugation in the absence or presence of a sucrose cushion were separated by SDS-PAGE, and the top portion of the membrane was immunoblotted to antiserum to the surface subunit (SU) of Env and the bottom portion to antiserum to the capsid (CA). (C) Equal amounts (50 μg) of whole-cell lysates from producer cells were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-SU antiserum. Values shown to the left of the immunoblots represent molecular mass markers in kilodaltons. Pr85, 85-kDa Env precursor.
Fig 5
Fig 5
(A) 293 cells transiently transfected with SSTR2a cDNA, 293 cells selected for stable expression of the G418-resistance gene linked to SSTR2a in pcDNA-hSSTR2a, and untreated 293 cells were exposed to serial dilutions of Sst-RBS or Moloney MLV pseudovirions and then fixed and stained for lacZ transduction. Values shown represent the means ± standard deviations of the results of four independent titrations. (B) Plasma membrane proteins were subjected to affinity purification using avidin-agarose after biotinylation of the proteins on the surface of live cells and then separated by SDS-PAGE. Replicate immunoblots of the samples were probed with anti-SSTR2a antiserum (top panel) or with anti-Na+K+ ATPase, a ubiquitous membrane protein, as an internal control for recovery and loading of total plasma membrane proteins (bottom panel). (C) Cells grown on glass coverslips were fixed, permeabilized, and then incubated with anti-SSTR2a antiserum. Micrographs representative of each cell type are shown.
Fig 6
Fig 6
Generation of a host cell line stably expressing levels of target receptor SSTR comparable to the levels of ecotropic receptor on NIH 3T3 cells. 293 cells were transfected with a cDNA-encoding human SSTR5 with an amino-terminal HA epitope tag (SSTR-HA), selected for G418 resistance, and then further selected for surface expression of the target receptor by three rounds of FACS experiments using anti-HA antibody, yielding a population of cells designated SSTR-HA/293. Left panel, aliquots of this cell line were incubated at 37°C with Sst-RBS pseudovirions or mock incubated (No virus), and then both samples were incubated with goat anti-SU antiserum followed by a mouse anti-goat FITC-conjugated secondary antibody plus propidium iodide and analyzed by flow cytometry. Values shown represent FITC intensities determined with live (propidium iodide-negative) cells. Right panel, aliquots of murine NIH 3T3 cells were incubated with MoMLV Env-pseudotyped virus or mock infected (No Virus), and virus binding was performed as described for the left panel. The histograms shown are representative of the results of three independent binding experiments.
Fig 7
Fig 7
Sst-RBS glycoprotein targets entry at levels comparable to those of entry mediated by wild-type Moloney murine leukemia virus glycoprotein and approaching those of VSV G pseudovirions. Quadruplicate wells of the host cells indicated on the x axes were exposed to 10-fold serial dilutions of independently produced vectors pseudotyped with the envelope glycoprotein indicated over each graph, and the titers were calculated from the endpoint dilutions. Values shown represent the numbers of mean lacZ-transducing units per milliliter ± standard deviations for four Sst-RBS (left panel), three wild-type MoMLV Env (middle panel), and four VSV G (right panel) pseudovirion stock titers.
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