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| Intercellular distribution of hepatitis C virus F protein is associated with its mutation in phosphorylation sites |
| SHAO Sheng-wen; YU Jian-guo; WU Wen-bin; ZHAO Ping; DING Hui; QI Zhong-tian |
Department of Microbiology, PLA key Laboratory for Medical Microbiology, Second Military Medical University, Shanghai 200433, China |
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Abstract Objective To determine the relationship between phosphorylation site mutation and intercellular distribution of hepatitis C virus (HCV) F protein in HepG2 cells.Methods According to the phosphorylation sites of HCV F protein as predicted by NetPhos2.0 Server software, overlapping primers were designed and used to synthesize the HCV mutated f (mf) gene, in which the codons 13, 14, 114, 121 and 124 were mutated from TCT to GAT. Consequently, the amino acids shifted from serine(S) to aspartate (D). The recombinant pEGFP-mf and pEGFP-f were constructed by cloning the mf gene and f gene into pEGFP-N1, respectively. The resulting recombinant plasmids were transfected into HepG2 cells by liposome transfection method, and the distribution of mutant F protein or wild-type F protein in HepG2 cells was examined by laser confocal microscope.Results In HepG2 cells, the mutant F protein was located mainly in the nucleus (90%) and only 10% was in the cytoplasm. The mean fluorescence intensity of mutated F protein was significantly different between the nucleus and cytoplasm 〔63.70±3.20 and 7.06±0.34, respectively; (P<0.001,t=99.2)〕. On the contrary, the wild-type F protein was located mainly in the cytoplasm(94.9%) and only a small fraction was in the nucleus (5.1%). The mean fluorescence intensities for the wild-type F protein in the cytoplasm and nucleus were 83.34±4.07 and 4.48±0.22, respectively (P<0.001, t=106.5). Conclusions Phosphorylated and non-phosphorylated forms of the HCV F protein have different cellular distributions. The phosphorylated F protein lies mainly in cytoplasm and is involved in the regulation of HCV replication whereas the non-phosphorylated F protein exists mainly in the nucleus and may effect on gene transcription.
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Received: 01 January 1900
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Corresponding Authors:
QI Zhong-tian
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