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As a DNA virus with complicated genomic structures (13), Herpes Simplex Virus1 (HSV-1) can interact specifically with host cells in different infectious phases via multifunctional viral proteins, resulting in modulation of the biological characteristics (17) of cells and establishment of latent and lytic infections (15). Although the mechanistic details of these two types of HSV-1 infection remain unclear, some data demonstrate that the monitoring of and switch from host cell to viral protein synthesis during HSV-1 infection are thought to be involved in the process (5). HSV-1 infection appears to functionally terminate translation and synthesis of many mRNAs and the linkage between degradation of an entire mRNA and protein synthesis (16). Of course, such common characteristics, which are required by many infectious viruses, have been extensively recognized. However, as far as HSV-1 infection is concerned, the mechanisms of redirecting infected cells to preferentially synthesize viral proteins during the lytic infectious phases are not completely clear. Previous studies have demonstrated that HSV-1 could redirect cellular viral protein synthesis via viral specific proteins, such as the ability of virion host shutoff protein to inhibit normal translation and mRNA synthesis (10). Further investigations of the molecular biology of HSV-1 will likely help elucidate the influences of HSV-1 or other viruses on host cellular functions in the context of multiple pathways and systems. Therefore, it seems wise to use more systematic approaches to study cellular protein translation and mRNA synthesis during HSV-1 infections.
Among investigations of the interaction between HSV-1 and host cells, an analysis based upon proteomics technology has been highlighted for a particular viral protein followed by investigation of other associated and interacting proteins (6, 19). The optimal pathway to systematically analyze mRNA synthesis and translation into proteins in HSV-1 infected cells should be based upon a comparative proteomic technology to analyze global variations in host cells with certain timing and spacing extensions, so as to facilitate phasic and integrated observations. Two-dimension electrophoresis (2-DE), which is currently well developed, is the basis for such a comparative technology. In our work, we performed preliminary analysis of the synthesis variation in HSV-1 infected normal human liver L-02 cells by using 2-DE. The synthesis variation associated with cellular mRNA decay and translation was identified in the process of HSV-1 infection of L-02 cells by 2-DE and matrix-assisted laser-desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) assays. The potential significance of this method is discussed.
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The Bradford Standard Curve was made by using a standard protein with known concentrations and their A595 values. The amount of protein sampling was calculated based upon the concentrations determined according to the A595 values, and the primary protein amount added to the gel strips was adjusted accordingly. As for the IPG gel strips with lengths of 17cm, pH3-pH10, 100 ~ 300 µg protein in 350 µL sampling volume was required for silver staining.
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Samples were collected from HSV-1 infected and control L-02 cells and loaded onto IEF and SDSPAGE gels respectively. Protein expression assays of HSV-1 infected and control L-02 cells at 24 h postinfection were obtained by silver staining (Fig. 1). The scanned images were processed by PDQuest professional software and 14 different protein spots were noted. The MALDI-TOF-MS assay indicated that among the HSV-1 infected L-02 cell at 24 h postinfection, the expression of 10 proteins were highly enhanced, 2 proteins were strikingly reduced, and 1 additional protein was identified as comparable to the controls (Table 1).
Figure 1. Two-dimensional gel electrophoresis analysis on L-02 cells infected by HSV-1. A: Uninfected L-02 cells. B: HSV-1 infected L-02 cells.
Table 1. Differential expression proteins in L-02 cells induced by HSVⅠinfection
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2-DE immunoblotting of protein extracted at 24 h post-infection revealed the expression levels of 2 proteins in HSV-1 infected L-02 cells were higher than those in control cells (Fig. 2A, 2B). The following MALDI identification and MASCOT retrieval demonstrated that these 2 proteins were acidic ribosomal phosphoproteins (P1) (Fig. 2C and 2D) and their position differences resulted from different modifications.
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By 2-DE immunoblotting of protein extracted from HSV-1 infected L-02 cells at 24 h post-infection, one protein was inhibited by HSV-1 infection (Fig. 3A and 3B). The following MALDI identification demonstrated that this protein was heterogeneous nuclear ribonucleoprotein H2 (hnRNP H2) (Fig. 3C).
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A KH-type splicing regulatory protein (KHSRP) identified by the MALDI assay was highly expressed in HSV-1 infected L-02 cells at different times (Fig. 4).