Session Number
Project ID: MEDH 15
Advisor(s)
Estefany Rios Guzman, Northwestern University, Feinberg School of Medicine
Dr. Margarita Rzhetskaya, Northwestern University, Feinberg School of Medicine
Dr. Judd F. Hultquist, Northwestern University, Feinberg School of Medicine
Discipline
Medical and Health Sciences
Start Date
20-4-2022 8:50 AM
End Date
20-4-2022 9:05 AM
Abstract
With no current vaccine for Human Immunodeficiency Virus (HIV), it is crucial to study what factors influence how HIV infects host cells to develop new strategies and therapies to combat this pandemic. Cleavage and polyadenylation specificity factor 6 (CPSF6) is an HIV host factor known to influence HIV infectivity and trafficking early in the infection. We hypothesize that the overexpression of CPSF6 will increase the antiviral response and decrease HIV infection in T cells. To test the hypothesis, we require a way to overexpress CPSF6 in mammalian cells to observe the antiviral response in HIV infection. To make a CPSF6-containing vector, we prepared a pLVX-TetOne-Puro-Cter3xFlag empty vector through E. coli transformation and plasmid purification, restriction enzyme digest, and then gel electrophoresis to confirm successful plasmid linearization. Post-purification, two inserts of the CPSF6 gene were then assembled into the empty vector using Gibson Assembly reactions. With this assembled construct, repetition of all of the aforementioned steps were performed to confirm the successful isolation of CPSF6 gene-containing plasmid. Plasmid amplification was then achieved using a polymerase chain reaction (PCR) to amplify the assembled construct. Further research will consist of expressing the vector in mammalian cells. These cells will be tested for their antiviral gene expression by Western blot and infectivity for HIV by flow cytometry. Ultimately, this experiment will lead to a greater understanding of CPSF6’s role in the innate immune response in HIV infection.
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The Impact of CPSF6 on the Innate Immune Response to HIV Infection
With no current vaccine for Human Immunodeficiency Virus (HIV), it is crucial to study what factors influence how HIV infects host cells to develop new strategies and therapies to combat this pandemic. Cleavage and polyadenylation specificity factor 6 (CPSF6) is an HIV host factor known to influence HIV infectivity and trafficking early in the infection. We hypothesize that the overexpression of CPSF6 will increase the antiviral response and decrease HIV infection in T cells. To test the hypothesis, we require a way to overexpress CPSF6 in mammalian cells to observe the antiviral response in HIV infection. To make a CPSF6-containing vector, we prepared a pLVX-TetOne-Puro-Cter3xFlag empty vector through E. coli transformation and plasmid purification, restriction enzyme digest, and then gel electrophoresis to confirm successful plasmid linearization. Post-purification, two inserts of the CPSF6 gene were then assembled into the empty vector using Gibson Assembly reactions. With this assembled construct, repetition of all of the aforementioned steps were performed to confirm the successful isolation of CPSF6 gene-containing plasmid. Plasmid amplification was then achieved using a polymerase chain reaction (PCR) to amplify the assembled construct. Further research will consist of expressing the vector in mammalian cells. These cells will be tested for their antiviral gene expression by Western blot and infectivity for HIV by flow cytometry. Ultimately, this experiment will lead to a greater understanding of CPSF6’s role in the innate immune response in HIV infection.