Studies On The Molecular Mechanism Of Interaction Between Baculovirus And Host Actin Cytoskeleton

Actin is a very important component of cytoskeleton, highly conserved and oneof the most abundant molecules in eucaryotic cells. Actin participates in cellmovement, endocytosis, cell division and many other functions important to cells. Itturns out that actin also has a fundamentally important role in viral infection.Cytoskeletal proteins have served in this regard as viral replication and/ortranscription factors and are involved in virus assembly and transportation. Takingbaculovirus as an example, release of baculoviral nucleocapsids into the cytoplasmafter entry induces the formation of thick actin cables. At the late stage of infection,F-actin is reorganized in the nucleus. However, our knowledge on the molecularmechanism of how to regulate cytoskeleton is still limited. This thesis takes HearNPV(Heliocoverpa armigera single nucleocapsid nucleopotyhedrovirus), a widely appliedbaculovirus, as the research object and reports the results of studies on functionalanalysis of HearNPV ORF2, a WASP homology protein, to explore the betterunderstanding of baculovirus transportation in host cell.Chapter one, an overview briefly introduces molecular kinetics of actincytoskeleton especially on the crucial components involved in actin filamentsassembly and outlines the actin-based motility of pathogen which rearrange and makeuse of the cellular actin cytoskeleton, following by the recently research progress on interaction between baculovirus and actin. At the end, the aims and significance ofthis thesis are presented.In Chapter two the open reading frame 2 (ha2) of the HearNPV, a conservedgene in most baculoviruses from lepidopteran insects was characterized in detail.Sequence analysis found that ha2 is the homologue of Autographa californica MNPV(AcMNPV) p78/83 and the member of WASP family. HearNPV ha2 transcription andtranslation results indicated that ha2 is a late expression gene. Western blot analysisshowed that the 50 kDa product of HA2 is a structural component of proteins of boththe budded virus (BV) and occlusion-derived virus (ODV) phenotypes. HA2-eGFPfusion protein showed that HA2 is localized primarily in the nucleus of HzAM1infected cells. The HA2 was found to co-localize with actin. These data indicated thatHA2 is a structural protein and interacts with the host cell actin.Actin polymerization is the main mechanism used in cell locomotion and is alsoutilized by pathogens for intracellular and intercellular movements. Baculovirus mayuse the general strategy similar to other pathogens such as Listeria, to regulate thedistribution of actin polymerization in cells. We first demonstrated, in Chapter three,that nucleocapsids of HearNPV were capable of nucleating actin polymerization invitro in a dose-dependent manner. Therefore, both Groupâ… (AcMNPV) and Groupâ…¡(HearNPV) NPVs are able to induce actin polymerization. Further work showed HA2could nucleate branched actin filaments in the presence of Arp2/3 complex in vitroand rearrange the host cytoskeleton in vivo. Co-localization of HA2 and Arp2/3complex in the nucleus of infected cells indicated that HA2 and Arp2/3 complex werealso involved in the nuclear F-actin dynamics. In conclusion HA2 and Arp2/3complex not only induce actin filament network formation in vitro and in vivo but alsoare involved in nuclear actin polymerization.Chapter four describes the function of each domain of HA2. In vitro analysisdemonstrated that the WCA domain of HA2 can accelerate Arp2/3-mediated actinassembly and is indispensable to the function of HA2. To further determine thebiological function of HA2 and each domain of HA2 in viral infection, the ha2knockout and ha2 repair bacmids were constructed. Transfection and infection analysis set out that the ha2 null bacmid was unable to produce infectious buddedvirus (BV) while the ha2 repair bacmid rescued the defect. We further repaired theha2 null recombinant with a series of truncated ha2, and found only the recombinantswith covered the WCA domain could yield infectious virions. Deletion of the 261-413aa of C terminal, a PtdIns 4-kinase homology, resulted in 1000-fold decrease of viraltiter, implying its functional importance. Subcellular localization analysis showed thatthe native HA2 and the fragments, which could rescue ha2 deletion recombinant,were distributed in the cytoplasm of the HzAM1 cells, and transported to the nucleusafter infected with HearNPV. On the contrary, the ha2 fragments, which could notrescue ha2 deletion recombinant, were localized in the whole cell with or withoutinfection of HearNPV. The motif from 170-193 aa played a pivotal role in thelocalization and transportation of HA2.HA2, the WASP homolog, is a baculovirus activator of actin nucleation andrequires the Arp2/3 complex to initiate an actin polymerization process similar toActA in Listeria, which represents a general strategy used to regulate the distributionof actin polymerization in cells. The assembly of a branched network of actinfilaments provides the mechanical propulsion that drives the transportation andassembly of baculovirus.