Human Herpesvirus-6 Lab Report

Viruses control the rate of cellular processes to support their own replication and manipulate already made machinery in cells to function in their benefit. Human herpesvirus-6 (HHV-6) can inhibit cellular processes by promoting cell-cycle arrest at certain points during cellular replication such as the G1/S phase and G2/M phase. HHV-6 typically infects young children and is characterized by a rash and high fever. Persisting in a dormant phase for many years, this virus can reactivate and cause many diseases and even death [1].
This virus also holds the information to produce a protein that is not found in cells without infection, U14, which is thought to interact with a common cellular protein, EDD, causing its nuclear localization. EDD normally …show more content…
Control cells were made in every experiment conducted by injecting an empty vector. Hemagglutinin (HA) tagging was used on the N-terminus of U14 in experiments involving transfecting the U14 gene; this was done because monoclonal antibodies are much cheaper and more reliable than polyclonal antibodies and allow for more precise detection [4]. The cells were lysed 72 hours after transfection and antibodies for HA and EDD were used to pull EDD and the HA-tagged U14 proteins out of a cell lysate into a pellet. The pellets and lysate of the infected and control cells were run through electrophoresis and Western blotting was performed using antibodies for EDD and U14 [3]. The second experiment was done the same way, except the antibodies used to pellet the proteins from the lysate were anti-U14 and anti-EDD …show more content…
Protein p53 is not usually activated in cells but can function with phosphorylation [5]. Connections between U14 and p53 could suggest that cell-cycle arrest is caused by the activation of p53 by U14. HA-tagged HHV-6 was transfected into 293T cells and the cells were lysed 72 hours after transfection. Antibodies for HA were used for immunoprecipitation to form a pellet which was separated using electrophoresis on a gel and then Western blotted with antibodies for HA, EDD, and p53 [3]. U14 was found to be present in all infected cells and EDD precipitated with only the unaltered U14 and mutant with intact C-terminus. P53 precipitated with the unaltered U14 and one of the U14 mutants lacking the C-terminus. Because p53 was not present in the precipitation of the intact C-terminus U14 mutant and EDD localized with that mutant without p53, EDD localization is not dependent on the presence of p53. The interaction of U14 with p53 and U14 with EDD are separate

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