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Progress in cell and molecular biology has furthered our understanding of eukaryotic gene transcriptional regulation and expanded the study of chromosomal and nuclear structure. Researchers of correlative gene transcriptional regulation need to grasp nuclear fine structure and formation in more detail. It is now appreciated that many DNA replication factors, transcription factors and splicing factors are not uniformly scattered in the nuclei but rather exist in condensed form aggregating into uneven "foci" within nuclear chromosome territories[4, 20]. The non-random distribution of these "foci" [7] and their functional role in cellular transcriptional regulation merit further investigation. Cell and molecular biology research has acknowledged the importance of understanding the formation of different functional domains although there is no consensus on the actual function of these nuclear "foci."
A number of studies have indicated that several important transcriptional regulatory factors in eukaryotes, such as E2F-1, GATA-1, SP1, and OCT1, could disperse or aggregate at specified regions in the nuclei [6, 10, 16, 18], and this location distinction might contribute to determining which phase a cell is in during the cell cycle. This deduction is mainly based on the observation that nuclear dispersed DNA replication factors only concentrate to about 150 replication focuses in S phase[1] and nuclear dissociation may also lead to disaggregation of the nuclear protein complex as parental cells divide into daughter cells. However, this deduction has not been fully confirmed by experiments. Therefore, research into viral transcriptional regulation factors expressed in host cells is warranted. Studies of the molecular biology of herpes simplex virus 1 (HSV-1) infection have identified many regulatory proteins generated after HSV-1 infected host cells could form typical punctiform structures in the nucleus[13, 14]. For example, as a multifunctional regulatory protein, HSV-1 infected-cell protein 22 (ICP22) not only regulates expression levels of viral and host genes but also affects mRNA splicing and changes the phosphorylated form of RNA polymerase LS[2, 8, 15, 17]. During the process of viral infection, the localization of ICP22 changes from aggregation to dispersion and back to aggregation[12]. ICP22 expressed alone is localized to small dense nuclear bodies and is paired with the SC-35 domain in the nucleus. Its biological characteristics are still unknown although these dense bodies have been found to correlate with its function by several experiments. Therefore, exploring the biological characteristics of this protein's cellular localization not only strengthens our knowledge of HSV-1 infection but also provides more information to understand cellular transcriptional regulation. Forthcoming experiments have confirmed that the structure of ICP22 provides two nuclear localization signals responsible for getting proteins into the nucleus. The N-terminus of ICP22 determines the formation of small dense nuclear bodies[5, 19]. Thus, a fusion protein (ICP22-EGFP), in which the C-terminus of ICP22 is combined with green fluorescence, was constructed to observe cellular localization of ICP22. Then we investigated ICP22's cellular biological characteristics by cell synchronization and a Tet-induced expression regulated system induced by vibramycin(Dox). Results indicated that cellular localization of ICP22 is not affected by cell cycle. However, it is worth noting that expression intensity of ICP22 correlated with its cellular localization. Results suggest that the cellular biological characteristics of proteins with transcriptional regulatory functions depend on the extent of protein self-expression. In addition, we developed a new non-protease expression reporter system, which could evaluate transcriptional control of internal ribosome entry sites (IRES), based on the observation that the localization of ICP22 reflects its own expression level.
Analysis of the Cellular Localization of Herpes Simplex Virus 1 Immediate-early Protein ICP22
- Received Date: 11 November 2009
- Accepted Date: 08 March 2010
Abstract: Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein, infected-cell protein 22 (ICP22), in the nucleus by observing the localization of ICP22-EGFP fusion protein. Results showed that, in high-level expression conditions, ICP22-EGFP gradually concentrates in the nucleus, persists throughout the cell cycle without disaggregation even in the cell division phase, and is finally distributed to daughter cells. We subsequently constructed a mammalian cell expression system, which had tetracycline- dependent transcriptional regulators. Consequently, the location of ICP22-EGFP in the nucleus changed with distinct induction conditions. This suggests that the cellular location of ICP22 is also influenced by promoter regulation, in addition to its own structure. Our findings provide new clues for the investigation of transcriptional regulation of viral genes. In addition, the non-protease reporter system we constructed could be utilized to evaluate the role of internal ribosome entry sites (IRES) on transcriptional regulation.