Abstract
BackgroundThe 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.MethodVSV-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 (p21Cip1) 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 p21Cip1 in the restriction of retrovirus infection.ResultsIt 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 p21Cip1 were found in HCT116 p53+/+ cells in comparison to the HCT116 p53−/− cells. Furthermore, knockdown of p21Cip1 in non-cycling HCT116 p53+/+ cells significantly increased the infection.ConclusionsThe 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 p21Cip1, and p53 also functions to influence formation of 1-LTR cycle and 2-LTR cycle DNA.
Highlights
The function of p53 in cancer biology has been studied extensively, but its role in anti-retrovirus infection has been elusive for many years
It was found that both late reverse transcription products and viral 2-Long terminal repeat sequence (LTR) cycle DNA were significantly increased in infected non-cycling HCT116 p53−/− cells
Our results suggested that p53 mediates the inhibition of retrovirus infection in non-cycling cells through it downstream gene Cyclin-dependent kinase inhibitor 1 (p21Cip1), and p53 functions to influence formation of 1-LTR cycle and 2-LTR cycle DNA
Summary
(Clontech, Mountain View, CA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) Flow cytometry Flow cytometry was used for both cell cycle analysis and quantification of infection. Cells were washed with PBS, fixed with ice-cold 70% ethanol, and stained with 0.1% (v/v) Triton X-100, 20 μg/ml propidium iodide (PI) (Sigma, St. Louis, MO, USA) and. 100 μg/ml DNase-free RNase (Life Technologies, Grand Island, NY, USA). Cells were disassociated by trypsin and washed with PBS. The infected GFP+ cells and uninfected cells were analyzed and quantified by a BD FACSVerseTM flow cytometer (BD Biosciences, San Jose, CA, USA). The FACSuite (BD Biosciences, San Jose, CA, USA) and the FlowJo (Ashland, OR) software were used for data analysis
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