Browsing by Author "Rossi, Katriina"

HIV-1 is a lentivirus causing a serious immunodeficiency disease called AIDS to almost 38 million people in the world. Like all retroviruses, HIV has an ability to insert a copy of its genome into the host cell DNA thus having a lifelong effect on the host cell. Two identical copies of single-stranded RNA are first reverse transcribed, and then a protein called integrase inserts the DNA into the genome of T-cells of the human immune system. Studies have shown that in some retroviruses the integrase protein has a unique integration pattern favoring certain areas of the host cell genome. For example, active transcription units or promoter units are found to attract integration activity. On the other hand, with nuclear localization signals in its structure and an ability to attach to the human genome, it raises a question if the integrase could have additional functions. Regulating certain gene expression levels could support viral replication and the survival of the virus. The aim of the study was to determine whether integration sites differ from integrase genomic contact sites, which could be an indication of integrase’s additional role. Study was started by method optimization. Two chromatin fragmentation methods, sonication and endo-exonuclease treatment, were tested in order to achieve optimal sized DNA fragments for ChIP-sequencing. Two cell lines were infected with HIV-1. Genomic DNA was collected for integration site sequencing. Integrase genomic contact sites were studied with ChIP-sequencing. Next generation sequencing for both, integration sites and integrase genomic contact sites, was carried out by an outsource biotechnology company. Sequences were processed with bioinformatics platforms, such as Galaxy and Homer. Study of the effects of transfected integrase was also initiated by transfection method optimization. Treatment with endo-exonuclease resulted in correctly sized DNA fragments when DNA was first purified. In a cell lysate, a correct ratio of MNase and cells was not found, although several cell lysis methods and buffers were tried. This is why actual integrase genomic contact site samples were fragmented by sonication. In MRC-5 cell line, integration site sequencing resulted in over 2 M reads with 289 aligned, unique integration sites. Integrase genomic contact site sequencing resulted in 1.8E+6 Bowtie mapped reads. A brief analysis of the sequences including sequence visualization, comparison of localization in the genome and GO terms, showed that integrase genomic contact sites have certain patterns that differ from integration sites. Therefore, it was confirmed that the study is repeatable, as the results were in line with the study conducted several years earlier. These results also suggest that method optimization had been successful. As bioinformatics methods were used with default parameters only, more attention should be payed when sequences are analyzed more deeply. This will offer a more thorough understanding of the functions of integrase.