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

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 S U that exhibits weak subunit association. We report here that they also res cue infection of a third defective SU, one modified by insertion of the gre en fluorescent protein (GFP) between serine 6 and proline 7. GFP-SU assembl ed into virions and showed a strong association with the transmembrane prot ein (TM). However, these virions were noninfectious. GFP-SU expression was not maintained within cells, suggesting that the protein was toxic. Additio n of the second-site mutations rendered the GFP-SU virus infectious and res ulted in prolonged expression of the modified envelope protein. This virus showed a slight reduction in receptor binding but not in envelope protein p rocessing, suggesting that addition of the GFP sequences results in subtle structural changes. Extrapolating these data, we see that the fundamental p roblem with the GFP-SU envelope protein appears to be a folding problem, su ggesting that the second-site mutations rescue GFP-SU primarily by a mechan ism that involves stabilizing the envelope protein structure.