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Analysis of key regions, roles, and interactions of the IE2 family of proteins throughout the human cytomegalovirus infection process

  • Author(s): Sanders, Rebecca L.
  • et al.
Abstract

Studies of the human cytomegalovirus (HCMV) major immediate early (MIE) protein, IE2 86 have yielded significant knowledge as to how this virus regulates many aspects of the viral infection. IE2 86 has been shown to be an essential regulatory factor during HCMV infection, and is thought to play many different roles throughout the different stages of the infection process. Two other proteins that are 3 prime co-terminal to IE2 86, IE2 60 and IE2 40, have also been found to be important at the later stages of the infection). Here, we have developed a system to overcome these limitations by using a translationally induced complementation system. Viruses expressing Cre and FLP recombinases have been constructed, and are used in conjunction with cell lines containing an inducible lenti-viral based vector to facilitate expression of IE2 86, IE2 60 or IE2 40. These cell lines are selected by a drug resistance marker placed upstream of IE2, and following a recombination event governed by Cre of FLP, the selectable marker is removed and allows expression of the 2nd open reading frame (ORF) encoding IE2 86, IE2 60 or IE2 40. These studies have also defined new roles of IE2 86, IE2 60, and IE2 40. We have found that expression of another early-late protein, UL84, is regulated post-transcriptionally by each of the IE2 proteins. This regulation can occur without any other viral proteins, but requires some cellular processes. We find that IE2 40 plays a major role in the expression of UL84 at late times post infection (p.i.), and that IE2 60 may also play a similar role, albeit to a lesser degree. IE2 86, IE2 60, and IE2 40 can interact with UL84 through the C-terminal region common to all three IE2 proteins. We have also identified that the first 105 aa of UL84 is responsible for an interaction with IE2 86 and IE2 40. Further, this domain is necessary and sufficient for the post-transcriptional regulation of UL84 governed by the IE2 proteins, and a protein-protein interaction is likely necessary for the regulation

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