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 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.
Kizhatil
K
K
Department of Molecular Sciences, University of Tennessee Health Sciences Center, Memphis, Tennessee 38163, USA.
Gromley
A
A
Albritton
L M
LM
D002460:Cell Line; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D014774:Virulence
D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins