Assistant Professor, Albany College of Pharmacy and Health Sciences, NY
It is well known that retrovirus manipulates host cell machinery for its infection; while host cell has various defense systems against retrovirus infection. In mammalian cells, the tumor suppressor protein p53 has been described as "the guardian of the genome" because of its role in conserving genome stability by preventing mutations. In response to DNA damage, p53 arrests the cell cycle, induces the expression of DNA repair genes, and initiates apoptosis. Many p53 pathways are potentially highly relevant to retrovirus replication. In many steps of HIV reverse transcription and integration, the creation of linear and branched viral DNA can serve as DNA damage signals which induce the activation of p53. In addition, during retroviral infection, p53 expression gets up-regulated, and p53 protein is phosphorylated. Recent research has also identified that the p53 pathway belongs to the interferon-induced innate host cell defense system. The anti-retroviral roles of p53 have previously been suggested in the regulation of transcriptional elongation from the HIV LTR promoter, and in the induction of cell apoptosis. However, the influence of p53 on the early replication of retrovirus has not been studied in depth. We hypothesize that p53 plays a major role in blocking retrovirus early replication by activating the host cell defense system. p53 functions as a transcription factor, and regulates the expression of hundreds of downstream genes in p53 pathway. RNA-Seq next generation technology provides a powerful tool to study the function of p53 during retrovirus infection.
HIV-1 pNL4-3env-GFP+ and pVSV-G (vesicular stomatitis virus G protein) plasmids were co-transfected into HEK293 cells to produce pseudotyped HIV-1 GFP+ virus. The paired human cell lines HCT116 p53+/+ and its isogenic p53 knockout HCT116 p53-/- cell lines were infected in parallel by HIV-1 GFP+ viruses. Replicates were made for following tests: 1) Observe infected GFP+ cells under fluorescent microscope 24 hours post infection. 2) Analyze the infection percentages of HCT116 p53+/+ and HCT116 p53-/- cells 24 hours post infection by flow cytometer. 3) Quantify HIV-1 viral cDNA at multiple time points by real time PCR. 4) RNA-Seq experiment was performed by using total cellular RNA extracted from infected cells 20 hours post infection.
RNA-Seq data will be made available at a time to be determined, including two replicates each from infected HCT116 p53+/+ and HCT116 p53-/- cells at 20 hours post infection.
The preliminary result of this study suggested that HIV-1 reverse transcription is inhibited by a process mediated by p53. Since p53 is a transcription factor, the observed HIV-1 infection phenotype difference between HCT116 p53+/+ and HCT116 p53-/- cells could be the consequence of gene expressions
Genetics and Molecular Basis of Diseases is a core course for senior students in Department of Health Sciences. The course has four credits with lab sessions. Understanding gene expression and gene regulation are main course objectives. Students spend two weeks in both lecture and labs in this section of course. RNA-Seq next generation sequencing technology and Green Line sequence analysis in iPlant DNA Subway is used as an example of advanced technology to study gene expression and analyze gene regulation. The use of bioinformatics tools in the computer sequences analysis will also be introduced and emphasized. In the lab sessions DNA extraction, RNA extraction and real time PCR are also included.
Genetics is an elective course for second year students in our college. The course has three credits without lab sessions. RNA-Seq next generation sequencing and Green Line sequence analysis will be introduced in class as new technology to study gene expression and analyze gene regulation.
Independent Study is a three credits course to provide undergraduate students with extensive research opportunities. Students normally practice RNA extractions and real time PCR assays for gene expression quantification. Students will use the data set I obtained from RNA-Seq Workshop and use Green Line sequence analysis tools in iPlant DNA Subway to study next generation sequencing technology and bioinformatics tools. Data will be used for ongoing research in the understanding of the role of p53 in the inhibition of HIV-1 early replication.