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Published on: July 24, 2011
Change of tropism of SL3-2 murine leukemia virus, using random mutational libraries
Shervin Bahrami1, Mogens Duch, Finn Skou Pedersen
1Department of Molecular Biology, University of Aarhus, Aarhus C, Denmark.
Abstract:
SL3-2 is a polytropic murine leukemia virus with a limited species tropism. We cloned the envelope gene of this virus, inserted it into a bicistronic vector, and found that the envelope protein differs from other, similar envelope proteins that also utilize the polytropic receptor (Xpr1) in that it is severely impaired in mediating infection of human and mink cells. We found that two adjacent amino acid mutations (G212R and I213T), located in a previously functionally uncharacterized segment of the surface subunit, are responsible for the restricted tropism of the SL3-2 wild-type envelope. By selection from a two-codon library, several hydrophobic amino acids at these positions were found to enable the SL3-2 envelope to infect human TE 671 cells. In particular, an M212/V213 mutant had a titer at least 6 orders of magnitude higher than that of the wild-type envelope for human TE 671 cells and infected human, mink, and murine cells with equal efficiencies. Notably, these two amino acids are not found at homologous positions in known murine leukemia virus isolates. Functional analysis and library selection were done on the basis of sequence and tropism analyses of the SL3-2 envelope gene. Similar approaches may be valuable in the design and optimization of retroviral envelopes with altered tropisms for biotechnological purposes.
Insights
Two amino acid mutations restrict the tropism of SL3-2 murine leukemia virus. Modifying these sites enhances infection of human cells, offering insights into retroviral envelope engineering for biotechnology.
Area of Science:
- Retroviral research
- Molecular biology
- Virology
Background:
- Murine leukemia viruses (MLVs) are retroviruses with varying host ranges.
- Polytropic MLVs utilize the polytropic receptor (Xpr1) for cell entry.
- The SL3-2 MLV strain exhibits limited tropism, particularly for human and mink cells.
Purpose of the Study:
- To investigate the molecular basis for the restricted tropism of the SL3-2 MLV envelope protein.
- To identify specific mutations responsible for the observed tropism limitations.
- To engineer SL3-2 envelope variants with altered tropism for biotechnological applications.
Main Methods:
- Cloning and expression of the SL3-2 MLV envelope gene in a bicistronic vector.
- Site-directed mutagenesis to introduce specific amino acid substitutions (G212R and I213T).
- Infectivity assays using human, mink, and murine cell lines.
- Selection from a two-codon library to identify beneficial mutations.
Main Results:
- Two adjacent amino acid mutations (G212R and I213T) in the SL3-2 envelope surface subunit were identified as responsible for its restricted tropism.
- Mutations at these positions, particularly hydrophobic amino acids like M212/V213, significantly enhanced infectivity in human TE 671 cells (over 6-log increase).
- Engineered SL3-2 envelope variants demonstrated broad tropism, infecting human, mink, and murine cells efficiently.
Conclusions:
- The tropism of the SL3-2 MLV envelope is determined by specific amino acid residues in a previously uncharacterized region.
- Targeted modification of these residues can overcome species tropism barriers.
- This study provides a foundation for designing retroviral envelopes with tailored tropisms for gene therapy and other biotechnological uses.
