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Updated: Aug 15, 2026
Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay
Published on: September 14, 2014
Comprehensive mutational analysis of the Moloney murine leukemia virus envelope protein
S M Rothenberg1, M N Olsen, L C Laurent
1Program in Cancer Biology, Stanford University Medical Center, Palo Alto, California 94305, USA.
Abstract:
The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the env gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues. The mutants with residual infectivity had insertions either in the amino-terminal signal sequence region, two disulfide-bonded loops in the receptor binding domain, discrete regions of the carboxy-terminal region of the surface subunit (SU), or the cytoplasmic tail. Insertions that allowed the mutants to reach the cell surface but not to mediate detectable infection were located within the amino-terminal sequence of the mature Env, within the SU carboxy-terminal region, near putative receptor binding residues, and throughout the fusion peptide. Independent analysis of select mutants in this group allowed more precise identification of the defect in Env function. Mapping of mutant phenotypes to a structural model of the receptor-binding domain provides insights into the protein's functional organization. The high-resolution functional map reported here will be valuable for the engineering of the Env protein for a variety of uses, including gene therapy.
Insights
Moloney murine leukemia virus envelope (Env) protein mutations reveal critical regions for cell surface expression and viral infectivity. This functional map aids in engineering Env for gene therapy applications.
Area of Science:
- Virology
- Molecular Biology
- Protein Engineering
Background:
- The envelope (Env) protein of Moloney murine leukemia virus is essential for viral entry into host cells.
- Understanding Env protein function is crucial for controlling viral spread and developing gene therapy vectors.
Purpose of the Study:
- To construct and analyze a comprehensive library of insertion mutations within the Moloney murine leukemia virus env gene.
- To determine the impact of these mutations on Env protein expression, cell surface delivery, and viral infectivity.
- To generate a high-resolution functional map of the Env protein.
Main Methods:
- Creation of a large pool of insertion mutants in the env gene.
- Analysis of Env protein expression and cell surface localization.
- Assessment of viral infectivity mediated by mutant Env proteins.
- Mapping of mutant phenotypes onto a structural model of the Env receptor-binding domain.
Main Results:
- Most env mutants exhibited poor cell surface expression, indicating folding defects.
- Mutants with residual infectivity were identified with insertions in specific regions, including the signal sequence, receptor-binding domain, SU carboxy-terminal region, and cytoplasmic tail.
- Mutants reaching the cell surface but lacking infectivity were localized to the mature Env amino-terminal sequence, SU carboxy-terminal region, near receptor-binding sites, and the fusion peptide.
Conclusions:
- The study provides a detailed functional map of the Moloney murine leukemia virus Env protein.
- Specific regions of the Env protein are critical for distinct steps in the viral life cycle.
- This functional map can inform the engineering of Env proteins for gene therapy and other applications.
