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Updated: Aug 13, 2026
Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein
Published on: September 24, 2010
Two point mutations produce infectious retrovirus bearing a green fluorescent protein-SU fusion protein
K Kizhatil1, A Gromley, L M Albritton
1Department of Molecular Sciences, University of Tennessee Health Sciences Center, Memphis, Tennessee 38163, USA.
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.
Insights
Second-site mutations rescue Moloney murine leukemia virus (MMLV) envelope protein defects. These mutations stabilize the green fluorescent protein-tagged envelope structure, restoring MMLV infectivity and prolonged expression.
Area of Science:
- Virology
- Molecular Biology
- Protein Engineering
Background:
- Moloney murine leukemia virus (MMLV) envelope surface protein (SU) is crucial for viral entry.
- Previous studies identified second-site mutations that rescue specific SU mutants.
- Understanding SU protein function and stability is key to MMLV replication.
Purpose of the Study:
- To investigate the effect of inserting green fluorescent protein (GFP) into the MMLV SU protein.
- To determine if second-site mutations can rescue the infectivity of GFP-tagged SU.
- To elucidate the mechanism by which second-site mutations restore MMLV infectivity.
Main Methods:
- Construction and characterization of MMLV with GFP inserted into the SU protein.
- Analysis of virion assembly, SU-transmembrane protein (TM) association, and infectivity.
- Assessment of GFP-SU expression stability in infected cells.
- Introduction of second-site mutations into GFP-SU MMLV.
Main Results:
- GFP-SU MMLV assembled into virions and associated with TM but was noninfectious.
- GFP-SU expression was unstable and potentially toxic.
- Second-site mutations restored GFP-SU MMLV infectivity and prolonged expression.
- The modified virus showed minor reductions in receptor binding and no change in protein processing.
Conclusions:
- Insertion of GFP into MMLV SU causes structural instability, leading to non-infectious virions.
- Second-site mutations rescue GFP-SU MMLV by stabilizing the envelope protein structure.
- This suggests that envelope protein folding is critical for MMLV infectivity.
