Title

The Effects of Single Point Mutagenesis in the SSP Subunit of the Junin Virus Envelope Glyco-protein on Membrane Fusion

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Presentation

Abstract

Arenaviruses are RNA-based viruses commonly found in rodents, and can cause severe hemorrhagic fever in humans, often resulting in death. In order to infect, the virus must enter the host cell by fusing its membrane with that of the cells. The viral envelope glycoprotein (GPC) which retains its stable signal peptide is necessary for this fusion activity to occur. For my studies, I wanted to understand how the three GPC subunits (SSP, G1, and G2) work together by looking specifically at how the stable signal peptide (SSP) subunit interacts with G2. This can be done by examining how they interact in the membrane because the G2 subunit spans the membrane and is thus in a position to interact with the membrane region of SSP. Cysteine-scanning mutagenesis was conducted to replace three important polar residues on the hydrophilic face of the membrane-spanning region of SSP (T13C, E17C, and N20C) with cysteine. Cells expressing GPC were then metabolically labeled with radioactive amino acids and GPC was immunoprecipitated. This method is generally referred to as a radioactive immunoprecipitation and was conducted to determine if the mutations affected cleavage of the G1G2 precursor or SSP association with the G2 subunit. The immunoprecipitations showed that cleavage and SSP association did in fact occur. I then wanted to know if the mutants supported fusion and thus performed a cell-cell fusion assay. The results indicate that fusion did occur in all of the mutants. These results suggest that the mutant SSPs are functional, and further studies are being pursued to crosslink the cysteine mutations introduced in SSP with cysteine mutations in G2 to determine how the subunits interact in the GPC complex.

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The Effects of Single Point Mutagenesis in the SSP Subunit of the Junin Virus Envelope Glyco-protein on Membrane Fusion

UC 333

Arenaviruses are RNA-based viruses commonly found in rodents, and can cause severe hemorrhagic fever in humans, often resulting in death. In order to infect, the virus must enter the host cell by fusing its membrane with that of the cells. The viral envelope glycoprotein (GPC) which retains its stable signal peptide is necessary for this fusion activity to occur. For my studies, I wanted to understand how the three GPC subunits (SSP, G1, and G2) work together by looking specifically at how the stable signal peptide (SSP) subunit interacts with G2. This can be done by examining how they interact in the membrane because the G2 subunit spans the membrane and is thus in a position to interact with the membrane region of SSP. Cysteine-scanning mutagenesis was conducted to replace three important polar residues on the hydrophilic face of the membrane-spanning region of SSP (T13C, E17C, and N20C) with cysteine. Cells expressing GPC were then metabolically labeled with radioactive amino acids and GPC was immunoprecipitated. This method is generally referred to as a radioactive immunoprecipitation and was conducted to determine if the mutations affected cleavage of the G1G2 precursor or SSP association with the G2 subunit. The immunoprecipitations showed that cleavage and SSP association did in fact occur. I then wanted to know if the mutants supported fusion and thus performed a cell-cell fusion assay. The results indicate that fusion did occur in all of the mutants. These results suggest that the mutant SSPs are functional, and further studies are being pursued to crosslink the cysteine mutations introduced in SSP with cysteine mutations in G2 to determine how the subunits interact in the GPC complex.