| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
Upon viral entry into the host cell disassembly of the capsid and subsequent release of the viral genome is the essential steps for SV40 infection, though mechanism underlies of which is poorly defined. The viral disassembly, normally induced by cellular cue, was not properly regulated when the viral particles lack the minor capsid proteins, Vp2 and Vp3 (collectively Vp2/3) : the Vp2/3-less particles prematurely released the viral DNA upon cell entry (Nakanishi et al. J.Virol. 2007). In order to examine the contribution of Vp1 and Vp2/3 interaction on the regulation of viral disassembly, mutations were introduced to the residues involving Vp1 and Vp2/3 binding, and analyzed their impact on viral infectious cycle. The mutations are designed to (1) alter the ionic interaction interface between Vp1 and Vp2/3, Vp1 K116R and Vp3 Q174D, and to (2) form disulfide bond between Vp1 and Vp2/3, Vp1 V230C-Vp3 E159C and Vp1 E257C-Vp3 Q174C. In addition (3) a mutant combined with three distinct temp
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erature sensitive mutations (tsDs), each reported to exhibit 'uncoating defect', Vp3 P103S-M110I-Q113K, was also included in the analysis. Upon transfection of the mutant viral DNA, capsid protein expression and viral DNA replication takes place normally, and all the mutants form particles with similar composition with that of wild-type virion. However, the mutants, except Vp1 K116R, were severely defective in their ability to form plaques at 37C or at the restrictive temperature (Vp3 P103S-M110I-Q113K). The mutant particles are apparently be able to enter the cells, interact with importin alpha/beta heterodimer, though either defective or delayed in initiating viral early gene expression, indicating that the defect of the mutants lies after interaction with nuclear import machinery of the viral particles. The results imply that major conformational change of viral capsid involving shift of Vp1-Vp2/3 interaction occurs at the later phase of cell entry processes, including the post-nuclear entry events, during the infection. Less
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