Using Co-Immunoprecipitation to Assay Binding Between Vpx and APOBEC3A
Location
CSU 202
Start Date
10-4-2018 1:05 PM
End Date
10-4-2018 2:05 PM
Student's Major
Biological Sciences
Student's College
Science, Engineering and Technology
Mentor's Name
Allison Land
Mentor's Department
Biological Sciences
Mentor's College
Science, Engineering and Technology
Description
APOBEC3A is a catalytically active DNA cytosine deaminase expressed in monocyte immune cells. This function allows APOBEC3A to mutate and restrict viruses, potentially including HIV. HIV-1, the causative agent of the major HIV/AIDS pandemic, is incapable of infecting monocytes. HIV-2, a less common variant, is capable of infecting monocytes. The unique protein Vpx, produced by HIV-2, but not HIV-1, is thought to be responsible for allowing HIV-2 infection in this immune cell. The objective of this study is to determine whether HIV-2 Vpx can counteract APOBEC3A. We hypothesize that HIV-2 Vpx binds directly to APOBEC3A, and then targets APOBEC3A for proteasomal degradation. Co-immunoprecipitation was utilized to test for APOBEC3A-Vif binding. Human 293T cells were transfected with both Vpx and APOBEC3A. After allowing time for protein expression, the cells were lysed and Vpx was immunoprecipitated using antibody bound to magnetic beads. A known Vpx binding protein, DCAF-1, was probed for to ensure that our co-immunoprecipitation protocol was working. Additionally, we probed for APOBEC3A to determine if it was co-immunoprecipitated with Vpx, which would indicate that Vpx binds APOBEC3A in cells. This project will contribute to our understanding of the innate immune response to lentiviral infection.
Using Co-Immunoprecipitation to Assay Binding Between Vpx and APOBEC3A
CSU 202
APOBEC3A is a catalytically active DNA cytosine deaminase expressed in monocyte immune cells. This function allows APOBEC3A to mutate and restrict viruses, potentially including HIV. HIV-1, the causative agent of the major HIV/AIDS pandemic, is incapable of infecting monocytes. HIV-2, a less common variant, is capable of infecting monocytes. The unique protein Vpx, produced by HIV-2, but not HIV-1, is thought to be responsible for allowing HIV-2 infection in this immune cell. The objective of this study is to determine whether HIV-2 Vpx can counteract APOBEC3A. We hypothesize that HIV-2 Vpx binds directly to APOBEC3A, and then targets APOBEC3A for proteasomal degradation. Co-immunoprecipitation was utilized to test for APOBEC3A-Vif binding. Human 293T cells were transfected with both Vpx and APOBEC3A. After allowing time for protein expression, the cells were lysed and Vpx was immunoprecipitated using antibody bound to magnetic beads. A known Vpx binding protein, DCAF-1, was probed for to ensure that our co-immunoprecipitation protocol was working. Additionally, we probed for APOBEC3A to determine if it was co-immunoprecipitated with Vpx, which would indicate that Vpx binds APOBEC3A in cells. This project will contribute to our understanding of the innate immune response to lentiviral infection.
Recommended Citation
Rachuy, Jacob. "Using Co-Immunoprecipitation to Assay Binding Between Vpx and APOBEC3A." Undergraduate Research Symposium, Mankato, MN, April 10, 2018.
https://cornerstone.lib.mnsu.edu/urs/2018/oral-session-10/4
