Analysis of cysteine mutations on the transmembrane protein of Moloney murine leukemia virus
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1Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854, USA.
Abstract:
Earlier studies of murine leukemia viruses (MuLVs) have reported that a percentage of surface protein (SU) remains covalently associated with transmembrane protein (TM) through formation of disulfide bonds. Among MuLVs, there are three conserved cysteine residues within the extracellular domain of TM. These cysteine residues were substituted individually with serines to define their function and possible role in disulfide bonding with SU. Using oligonucleotide-directed mutagenesis, seven mutant constructs were generated with individual as well as multiple cysteine mutations. Transient transfection of all seven cysteine mutations resulted in nonviable virus. Analysis of intracellular proteins of producer mutant cell lines have demonstrated that precursor envelope protein (gPr80env; SU/TM) is being synthesized, but transport and processing of gPr80env is blocked in the endoplasmic reticulum. Two independent reversions of one cysteine mutation have been isolated and characterized.
Insights
Murine leukemia viruses (MuLVs) require specific cysteine residues in their transmembrane protein (TM) for proper envelope protein (SU/TM) processing and virus viability. Mutations blocking disulfide bonds prevent virus maturation.
Area of Science:
- Virology
- Molecular Biology
- Protein Biochemistry
Background:
- Murine leukemia viruses (MuLVs) are retroviruses known to cause leukemia in mice.
- Previous research indicated that the surface protein (SU) of MuLVs can covalently link to the transmembrane protein (TM) via disulfide bonds.
- Three conserved cysteine residues are present in the extracellular domain of the MuLV TM protein.
Purpose of the Study:
- To investigate the functional role of conserved cysteine residues in the TM protein of MuLVs.
- To determine the involvement of these cysteine residues in the disulfide bonding between SU and TM proteins.
- To understand their impact on viral processing and infectivity.
Main Methods:
- Site-directed mutagenesis was employed to substitute individual and multiple cysteine residues with serine.
- Seven distinct cysteine mutant constructs were generated.
- Mutant viruses were analyzed for viability and intracellular protein processing via transient transfection and analysis of producer cell lines.
Main Results:
- All seven cysteine mutations resulted in nonviable virus.
- The precursor envelope protein (gPr80env; SU/TM) was synthesized but its transport and processing were arrested in the endoplasmic reticulum.
- Two independent revertant mutants from one cysteine mutation were successfully isolated and characterized.
Conclusions:
- Conserved cysteine residues in the MuLV TM protein are essential for proper envelope protein complex processing and virus viability.
- Disulfide bond formation involving these cysteines is critical for the transport of gPr80env out of the endoplasmic reticulum.
- Mutations affecting these cysteine residues lead to a block in viral maturation at the endoplasmic reticulum stage.