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Many of the biological studies on the Herpes Simplex Virus Type 1 (HSV-1) infectious process have elucidated the events of HSV-1 entry into host cells: HSV-1 releases its capsid and the tegument proteins into the cytosol of a host cell by fusing with the plasma membrane; the capsid is then transported by a specifically mediated mechanism to the nucleus where it binds to the cytosolic side of nuclear pore complexes, and the viral genome is rapidly released into the nucleus(6, 11). Although this process is very short, it is involved in complicated infectious dynamic actions generated between associated capsid protein complexes and the cytoskeleton (3). It has been reported that the HSV-1 UL31, UL34 etc. capsid proteins play an important role in the egress of virions from the nucleus into the cytoplasm (5, 12). Meanwhile, the functions and structures of several capsid proteins are most likely to offer necessary support for viral capsids and teguments to eventually complete the infection process (2). However, investigations regarding this aspect are insufficient, and the mechanistic details of several associated capsid proteins in this process remain unclear. The UL25 gene product encoding 1740 bp with a molecular weight of 62 KD is an essential capsid protein that is required for many crucial biological events that take place during viral infection. These include the facilitation of the route of transit, and proliferation and assembling etc. of virions in cells(1, 7, 8, 9, 10, 13, 15). These basic functions of the UL25 gene product suggest that this protein is most likely to interact with various molecules in cells. Based on this implication, we utilized biochemical assays for protein analysis to investigate the interaction of UL25 with other proteins, and its localization through fluorescence microscopy. Our results revealed that the UL25 protein plays an essential role in the transport of viral capsids to the nucleus.
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All of the plasmids used for co-localization in this study were constructed by PCR amplification, recombinant cloning, and verified by enzyme digestion and sequencing etc.
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By using a yeast two-hybrid assay, UL25 protein was found to interact with cellular microtubuleassociated protein. Interestingly, we also found interactions between UL25 and several cellular proteins with transcriptional and regulatory significance (Table 1). The interaction of UL25 with cellular microtubuleassociated protein was analyzed in detail using a β-D-galactosidase binding assay, and the results showed a specific interaction between them (Fig. 2).
Table 1. The genes coding proteins interacting with UL25 specifically from yeast trap
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In order to observe the localization of UL25 in cells, the UL25-EGFP fusion protein expression plasmid was transformed into Vero and KMB17 cells, respectively. The fluorescence examined at 24-48 posttransfection indicated that UL25 was scattered throughout the cell (Fig. 3).
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Based upon the yeast two-hybrid assay results and the observed localization of UL25 protein in cells, we further investigated the interaction of UL25 with microtubule-associated protein in cells using the UL25-EGFP fusion protein and an anti-cellular microtubule-associated immunofluorescence antibody. The results demonstrated that both the UL25-EFFP fusion protein and the microtubule-associated protein localized to the microtubule-organizing center (MT-OC), implying that UL25 is localized to the cytoskeleton via binding to cellular microtubule-associated protein (Fig. 4).
To further testify whether UL25 interacts with microtubule-associated protein in vivo, we inhibited the posttransfection cell with Colchicine as methods mentioned above. Colchicine can dissociate microtubules and inhibit cell growth. At this time, location of the UL25-EGFP fusion protein in microtubule dissociated Vero or KMB -17 cell changed, most of which accumulated at MTOC, but some were scattered in a manner consistent with tubulin (Fig. 5).
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In order to further biochemically characterize the interaction of UL25 with cellular microtubuleassociated protein, different deletion mutants of the UL25 gene were constructed (Fig. 1), recombined into the pEGFP-N2 vector and transformed into different cells, respectively. Interestingly, fluorescence microscopy showed that the C terminus (UL-C) of UL-25 was similar to its middle fragment (UL25-M), and the N-terminus domain (1-140AA) of UL25 localizes to the plasma membrane, as well as to nuclear pore complexes (Fig. 6). Along with our yeast two-hybrid data (Table 1), this suggests that UL25 also is involved in transcriptional and regulatory functions in nucleus. From these results, we conclude that UL25 protein is involved not only in its directed transfer in cells via interaction with cellular microtubule-associated protein, but also has unknown functions in the nucleus.