Using Site Directed Mutagenesis to Describe the SIV Vif Interaction with APOBEC3
Location
CSU Ballroom
Start Date
10-4-2018 10:00 AM
End Date
10-4-2018 11:30 AM
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
Second Mentor's Name
Oumar Sanogo
Second Mentor's Department
Biological Sciences
Second Mentor's College
Science, Engineering and Technology
Description
APOBEC3B (A3B) is part of a family of cytosine deaminases found in most mammals that can fight viral infections, like HIV lentivirus, by creating mutations in the viral genome to restrict the virus. Many lentiviruses encode the protein Viral infectivity factor (Vif), which targets the A3 proteins for proteasomal degradation. A3B is upregulated in certain cancers, such as bladder, breast, and throat cancer, and causes mutations in the human genome leading to increased tumor progression and recurrence of cancer after treatment. Previous research indicates that Vif from SIVmac239, an immunodeficiency lentivirus related to HIV that infects rhesus macaques, targets human A3B for degradation. The goal of my project is to determine where Vif protein binds A3B. APOBEC3F (A3F) in rhesus macaques, closely related to human A3B, is resistant to SIVmac239 Vif. I used site-directed mutagenesis to swap coding sections of A3B DNA with coding sections of A3F DNA based on amino acid alignment. The mutagenesis was accomplished using back-to-back (non-overlapping) PCR primer pairs. One primer of each pair contained the desired substitution and 10 complementary nucleotides. The other primer was completely complementary. After PCR amplification, the plasmids were cloned into E. coli and confirmed by sequencing. These A3B and A3F chimeric constructs will be cotransfected with Vif into mammalian cells, and degradation by Vif will be assessed by immunoblot. By finding A3B constructs that become resistant to Vif degradation when the homologous region of A3F is swapped in, we will identify the region(s) on A3B that are bound by Vif.
Using Site Directed Mutagenesis to Describe the SIV Vif Interaction with APOBEC3
CSU Ballroom
APOBEC3B (A3B) is part of a family of cytosine deaminases found in most mammals that can fight viral infections, like HIV lentivirus, by creating mutations in the viral genome to restrict the virus. Many lentiviruses encode the protein Viral infectivity factor (Vif), which targets the A3 proteins for proteasomal degradation. A3B is upregulated in certain cancers, such as bladder, breast, and throat cancer, and causes mutations in the human genome leading to increased tumor progression and recurrence of cancer after treatment. Previous research indicates that Vif from SIVmac239, an immunodeficiency lentivirus related to HIV that infects rhesus macaques, targets human A3B for degradation. The goal of my project is to determine where Vif protein binds A3B. APOBEC3F (A3F) in rhesus macaques, closely related to human A3B, is resistant to SIVmac239 Vif. I used site-directed mutagenesis to swap coding sections of A3B DNA with coding sections of A3F DNA based on amino acid alignment. The mutagenesis was accomplished using back-to-back (non-overlapping) PCR primer pairs. One primer of each pair contained the desired substitution and 10 complementary nucleotides. The other primer was completely complementary. After PCR amplification, the plasmids were cloned into E. coli and confirmed by sequencing. These A3B and A3F chimeric constructs will be cotransfected with Vif into mammalian cells, and degradation by Vif will be assessed by immunoblot. By finding A3B constructs that become resistant to Vif degradation when the homologous region of A3F is swapped in, we will identify the region(s) on A3B that are bound by Vif.
Recommended Citation
Schleper, Katie. "Using Site Directed Mutagenesis to Describe the SIV Vif Interaction with APOBEC3." Undergraduate Research Symposium, Mankato, MN, April 10, 2018.
https://cornerstone.lib.mnsu.edu/urs/2018/poster-session-A/19
