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Yeast As a Chassis for Developing Functional Assays to Study Human P53

Yeast As a Chassis for Developing Functional Assays to Study Human P53

Published on: August 4, 2019

The impact of p53 on the early stage replication of retrovirus

Michaela Kinnetz¹, Faris Alghamdi¹, Michael Racz¹

¹Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences, 106 New Scotland Ave, Albany, NY, 12208, USA.

Virology Journal

|August 11, 2017

Abstract

Background:

The function of p53 in cancer biology has been studied extensively, but its role in anti-retrovirus infection has been elusive for many years. The restriction of retrovirus early stage replication by p53 was investigated in this study.

Method:

VSV-G pseudotyped retrovirus with GFP reporter gene was used to infect both HCT116 p53^+/+ cells and its isogenic p53 knockout HCT116 p53^-/- cells. The infection was detected by flow cytometry. Reverse transcription products were quantified by real time PCR. Mutation analysis was performed after 1-LTR cycle and 2-LTR cycle DNA were amplified and PCR products were sequenced. Transcription and translation of cyclin-dependent kinase inhibitor 1 (p21^Cip1) and SAM domain and HD domain-containing protein 1 (SAMHD1) were analyzed by TaqMan PCR and Western blot experiments. siRNA experiment was applied to study the role of p53 downstream gene p21^Cip1 in the restriction of retrovirus infection.

Results:

It was found that the block of retrovirus infection in non-cycling cells was significantly attenuated in HCT116 p53^-/- cells when compared to HCT116 p53^+/+ cells. It was found that both late reverse transcription products and viral 2-LTR cycle DNA were significantly increased in infected non-cycling HCT116 p53^-/- cells. Furthermore, the mutation frequency detected in 1-LTR DNA from HCT116 p53^+/+ cells were significantly decreased in comparison to HCT116 p53^-/- cells. A higher number of insertion and deletion mutations were detected in the joint region of 2-LTR cycle DNA in infected p53^+/+ cells. Cell cycle analysis showed retrovirus infection promoted host cell replication. Higher levels of mRNA and protein of p21^Cip1 were found in HCT116 p53^+/+ cells in comparison to the HCT116 p53^-/- cells. Furthermore, knockdown of p21^Cip1 in non-cycling HCT116 p53^+/+ cells significantly increased the infection.

Conclusions:

The results of this study showed that p53 is an important restriction factor that interferes with retrovirus infection in its early stage of replication. Our results suggested that p53 mediates the inhibition of retrovirus infection in non-cycling cells through it downstream gene p21^Cip1, and p53 also functions to influence formation of 1-LTR cycle and 2-LTR cycle DNA.

Keywords:

Cell cycle LTR cycles Mutation Retrovirus Reverse transcription p21Cip1 p53

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