Actin Inhibiting The Transcription And Expression Of Baculoviral Polyhedrin

Actin, as a component of the complex skeleton in the eucaryotic cell, takes part on many important cellular functional progresses. Recent researches have shown that lots of virus interacted with the actin during their infection. This illustrates the complicated interaction between viruses and cell, and an important role of actin in viral infection as well. Baculovirus is a kind of double-stranded circle large DNA virus, which has been developed as an environment sound pesticide and a powerful vector of foreign protein expression, as well as a potential vector of gene therapy. Revealing the molecular mechanism of viral replication, package, and transportation will lead the better understanding of baculovirus itself and enhance its application. As other viruses, in the progressing of baculovirus infection, they employ the actin in insect cells to promote nuclearcapsid transportation to nucleus, genome replication and expression, progeny nuclearcapsid morphogenesis, and prodution of polyhedra as well. In our research, we first analysed the recombinant virus AcMNPV-GFP-actin (ph"). We found that the expression of the fusion gene eGFP-actin didn't affect the viral infectivity on Sf9 cell. In the late phase of its infection, the actin transport from cytoplasm into nucleus, and then out of it. In order to further elucidate the function of actin in the late and very late infection stages, especially in the production of polyhedra, we constructed a recombinant virus vAc-ph/70GA using Bac-to-Bac system, in which eGFP-actin fusion gene under control of hsp70 promoter and polyhedrin gene with its' own promoter were inserted. The virus vAc-ph, which only contains ph, was also made as control. After vAc-ph/70GA infected Sf9 cells, actin was expressed persistently. However, there were no polyhedra, while the control virus vAc-ph did produce polyhedra. The polyhedra in the vAc-ph infected Sf9 cells indicated that ph was normal. Further analysis such as SDS-PAGE and RT-PCR did not detect the transcription and expression of polyhedrin when vAc-ph/70GA infected Sf9 cells, indicating that the expression of actin inhibited polyhedra formation. Expression of actin, however, did not change the viral infectivity. Thus, we can conclude that the persistent expression of actin during late stage of baculoviral infection inhibited the polyhedrin expression, and then the formation of polyhedra. Here, we show someresults from electro microscopy, SDS-PAGE and RT-PCR with vAc-ph/70GA. And we also constructed the recombinant virus, vAc-ph/PGA.It encompasses three parts. In the first one, we present a brief introduction of baculovirus, including the classification status, structure, replication, expression and regulation of genome, et al. We also summarize the interaction between actin and the baculovirus. And then we give the major aims of this study. In the second part, we describe the materials and methods used. The results and our opinion are presented in the last part.