Two point mutations produce infectious retrovirus bearing a green fluorescent protein-SU fusion protein

Abstract

Two second-site mutations in Moloney murine leukemia virus envelope surface protein (SU) were previously shown to rescue infection of two different SU mutants, a fusion-defective point mutant and a fusion-defective modified SU that exhibits weak subunit association. We report here that they also rescue infection of a third defective SU, one modified by insertion of the green fluorescent protein (GFP) between serine 6 and proline 7. GFP-SU assembled into virions and showed a strong association with the transmembrane protein (TM). However, these virions were noninfectious. GFP-SU expression was not maintained within cells, suggesting that the protein was toxic. Addition of the second-site mutations rendered the GFP-SU virus infectious and resulted in prolonged expression of the modified envelope protein. This virus showed a slight reduction in receptor binding but not in envelope protein processing, suggesting that addition of the GFP sequences results in subtle structural changes. Extrapolating these data, we see that the fundamental problem with the GFP-SU envelope protein appears to be a folding problem, suggesting that the second-site mutations rescue GFP-SU primarily by a mechanism that involves stabilizing the envelope protein structure.

FIG. 1

Addition of the second-site changes (Q227R and D243Y) to the GFP-SU fusion protein renders the virus infectious. (A) H1BAG prepackaging cells were transfected with pcDNA MoMLV encoding a wild-type envelope protein or with pcDNA MoMLV GFP-SU or pcDNA MoMLV GFP-SU Q227R D243Y, each encoding modified SU. Replicate sets of quadruplicate wells of ecotropic MoMLV receptor-positive mouse NIH 3T3 fibroblasts or receptor-negative HEK 293 cells were exposed to 10-fold serial dilutions of a 3-ml aliquot of each cell culture supernatant of the H1BAG cells taken 24 h posttransfection. The titer was calculated from the end point dilution. No infection of HEK 293 cells was observed. i.f.u., infection- forming units. (B) The subunit association of the envelope protein does not appear to be weakened by insertion of the GFP sequences. A 7-ml aliquot of each of the virus supernatants described for panel A was then subjected to ultracentrifugation at 30,000 rpm in a Beckman SW41 rotor at 4°C for 2 h through a 25% sucrose cushion to purify virions. Viral proteins were resolved by sodium dodecyl sulfate-PAGE and transferred to nitrocellulose membranes. For the left panels, membranes were cut into two pieces at the position of the 45-kDa molecular mass marker and the upper portion was probed with anti-SU antiserum (1:100 dilution of serum ID 80S000018; Quality Biotech, Inc.), while the lower portion was probed with anti-capsid (CA) antibodies (1:10,000 of serum ID 81S000263; Quality Biotech, Inc.). All primary antibodies were detected by horseradish peroxidase-conjugated secondary antibodies at a 1:10,000 dilution. (C) Transient expression of envelope proteins in transfected H1BAG cells. After the 48-h harvest of supernatant, transfected H1BAG cells were lysed and the cell lysates were subjected to sodium dodecyl sulfate-PAGE and immunoblotting with anti-SU antiserum. WT SU, virus containing wild-type MoMLV envelope protein; GFP-SU, virus containing the GFP-SU fusion protein; GFP-SU Q227R D243Y, virus containing GFP-SU fusion protein with the second-site mutations added.

FIG. 2

Stable production of pseudotype virions. (A and B) Cells transfected with plasmid encoding GFP-SU without (A) or with (B) the second-site mutations fluoresced green during the first 24 h posttransfection, but only cells transfected with GFP-SU plus the second-site mutations continued to show robust green fluorescence at later times posttransfection. HEK 293/DHFR cells were transfected with 15 μg of pcDNA MoMLV GFP-SU Q227R D243Y or pcDNA MoMLV GFP-SU and then grown in medium containing 1 mg of G418/ml to select for the plasmids. Micrographs were taken at 24 h posttransfection and after 2 and 12 weeks of selection. The fluorescence emission of GFP (left panels) and phase-contrast images (right panels) of live cells were visualized using a 40× water-immersible objective on an epifluorescent microscope (Zeiss Axiophot). Images were captured using a Kodak digital camera. (C) Western blot analysis of lysates from transfected cell populations at 5 weeks posttransfection using anti-SU antiserum (1:100 dilution of serum ID 80S000018; Quality Biotech, Inc.). For comparison, lysates from cells transfected with wild-type SU were also analyzed. (D) Assembly of GFP-SU Q227R D243Y into virus particles and infectivity of pseudotype virions. Two clonal cell lines, clones 1 and 2, expressing the highest levels of green fluorescence were isolated from the population of HEK 293/DHFR cells stably expressing pcDNA MoMLV GFP-SU Q227R D243Y after 5 weeks of selection with G418. Virions were purified by ultracentrifugation (30,000 rpm in a Beckman SW41 rotor at 4°C for 2 h) through a 25% sucrose density cushion from the cell culture supernatant of each clonal line and from control cells producing wild-type envelope pseudotype of MoMLV DHFR. Replicate sets of HEK 293 cells stably expressing ecotropic receptor cDNA were exposed to 10-fold serial dilutions of virus supernatant. Forty-eight hours later, infected cells were selected by growth in methotrexate-containing medium which required expression of the mutant dhfr gene transduced by the virus, and the titer was calculated from the end point dilution after 21 days of selection. For the left panels, the upper portion of the membrane was probed with anti-SU antiserum, while the lower portion was probed with anti-CA antibodies as described in the legend to Fig. 1. Values under each lane show the titer in infectious units per milliliter. For the right panel, the upper portion of the membrane was stripped of anti-SU antibodies and reprobed with rabbit anti-GFP antiserum (1:1,000 dilution; Clontech). All primary antibodies were detected by horseradish peroxidase-conjugated secondary antibodies at a 1:10,000 dilution. WT SU, recombinant MoMLV DHFR particles containing wild-type SU; GFP-SU Q227R D243Y, virus from the two clonal HEK 293 DHFR cell lines stably expressing pcDNA MoMLV GFP-SU Q227R D243Y.