Identification of the block in targeted retroviral-mediated gene transfer

Abstract

A chimeric retroviral vector (33E67) containing a CD33-specific single-chain antibody was generated in an attempt to target cells displaying the CD33 surface antigen. The chimeric envelope protein was translated, processed, and incorporated into viral particles as efficiently as wild-type envelope protein. The viral particles carrying the 33E67 envelope protein could bind efficiently to the CD33 receptor on target cells and were internalized, but no gene transfer occurred. A unique experimental approach was used to examine the basis for this postbinding block. Our data indicate that the chimeric envelope protein itself cannot participate in the fusion process, the most reasonable explanation being that this chimeric protein cannot undergo the appropriate conformational change that is thought to be triggered by receptor binding, a suggested prerequisite to subsequent fusion and core entry. These results indicate that the block to gene transfer in this system, and probably in most of the current chimeric retroviral vectors to date, is the inability of the chimeric envelope protein to undergo this obligatory conformational change.

Figure 1

Schematic diagrams of the chimeric envelope protein constructs. Mo-MuLV envelope expression vector CEE+ was engineered to contain a SfiI site (5′ end) and a NotI site (3′ end) between amino acids 6 and 7, yielding the vector named E67. A CD33 scFv was amplified by PCR and the PCR product was subcloned into E67 at the SfiI and NotI sites to obtain the chimeric envelope protein 33E67. A point mutant, D84K, that had been previously demonstrated to be a binding-defective mutant (17), was introduced into 33E67 to generate 33K67. Vertical arrows indicate proteinase cleavage sites. SP, signal peptide; SU, surface protein; TM, transmembrane protein; CEE+, wild-type ecotropic envelope protein. The approximate site of the binding mutant D84K is denoted by an asterisk. The membrane-spanning domain is denoted as a grey box. The other four constructs used in this study, CEETR, D84KTR, 33E67TR, and 33K67TR, are the R-peptide truncated forms of CEE+, D84K, 33E67, and 33K67, respectively.

Figure 2

Detection of envelope SU and p30 CA protein in virions by Western blot. The supernatants from transiently transfected cells were collected and centrifuged through 20% sucrose to pellet viral particles and remove free protein. Pellets were then analyzed on 8–16% SDS/PAGE gels for their gag (p30 CA) and envelope gp70 (SU) content. Viral SU could be detected for all the chimeric envelopes. SU containing the CD33 scFv migrates at a higher position than the wild type. Lanes: 1, mock transfection; 2, ecotropic viral particles from GPE86/LNCX producer cells (41); 3, 33E67; 4, 33E67TR; 5, D84K; 6, 33K67; 7, 33K67TR; 8, CEE+.

Figure 3

The strength of envelope/receptor binding of the retroviral envelope constructs. Supernatants containing viral particles from transient transfections were collected and applied to target cells, either NIH 3T3 or 3T3/CD33 cells, to detect the binding of chimeric envelope to the CD33 antigen. Binding of viral particles to the target cells was detected by an immunofluorescent flow cytometry assay. The binding signals of control incubations (using either D84K or mock transfection supernatant) are shown as grey control peaks.

Figure 4

Detection of preintegration complex in target cells. Supernatants containing viral particles from transient transfections were incubated with NIH 3T3 or 3T3/CD33 cells for 6 hr at 37°C. The cytoplasmic DNA was isolated and analyzed by Southern blot. The probe used in the study was a ³²P-dCTP-labeled long terminal repeat fragment (24). Lanes: 1 and 7, mock transfection (with the pHIT60 and pCnB plasmids, but no envelope protein plasmids); 2 and 8, D84K; 3 and 9, CEE+; 4 and 10, 33E67; 5 and 11, 33K67; 6 and 12, H₂O transfection. Lanes 1–6 are from 3T3 cells and lanes 7–12 are from 3T3/CD33 cells.

Figure 5

Internalization of bound viral particles. 3T3/CD33 cells were used as the target cells. After incubating target cells at 4°C with viral particles for 2 hr, cells were washed with PBS and incubated in D10 medium for 1 hr at 37°C, followed by washing three times with PBS and then incubation in trypsin/EDTA for 10 min at 37°C. Lysates of cells were analyzed by immunoprecipitation with anti-SU and anti-p30 antiserums. Lanes: 1, 33K67; 2, 33E67; 3, mock transfection (with pHIT60 and pCnB plasmids, but no envelope protein plasmids).

Figure 6

Chimeric envelope protein-mediated syncytia formation of 3T3/CD33 cells. Envelope protein expression plasmids were cotransfected (at a ratio of 1:1) into 3T3/CD33 cells. At 36 hr after transfection, the cells were stained with methylene blue, and those cells containing more than four nuclei counted as syncytia. (Upper) Examples of positive syncytia formation; (Lower) Examples of negative syncytia formation. These data are a portion of those summarized in Table 2.

Figure 7

Heterooligomer formation between chimeric envelope monomers and wild-type monomers. SDS/PAGE (14%) of the coimmunoprecipitated virion envelope protein is shown. Envelope proteins were transiently expressed in 293T cells. Metabolic labeling for 4 hr with [³⁵S]Met was performed 24 hr after transfection. The cells were lysed, and the supernatant was immunoprecipitated with 5 μ of anti-R peptide (22). The p12E protein, bottom band, that was present in 33E67TR and 33K67TR, was immunoprecipitated by R-peptide antiserum only in the presence of coexpressed D84K (lane 1, 33E67TR/D84K; lane 2, 33K67TR/D84K); or CEE+ (lane 3, 33K67TR/CEE+), but not when expressed by itself (lane 5, 33E67TR and lane 6, 33K67TR). There is no p12E band in the wild-type CEE+ (lane 4), indicating that there is no R-peptide cleavage in this 293T system.