Journal of Tongji Medical University 19 (3): 170-174, 1999 170 I ~ ~ • Construction and Production of Fluorescent Papillomavirus- like Particles PENG Shiwen (j~'~), ZHOU Jian ( ~ fl~)" , Ian H Frazer 9 Department of Pediatrics, Xiehe Hospital, Tongji Medical University, Wuhan 430022 " Center for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Woolloongabba , Qld 4102, Australia Summary: The green fluorescent protein (GFP) from the jellyfish Aequorea victo- ria has attracted widespread interest since it was demonstrated to be fluorescent in vivo when expressed in other organisms. In order to investigate papillomavirus life cycle which harppered by the unavailability of conventional cell culture sys- tem, we constructed a chimeric bovine papillomavirus (BPV) type 1 virus-like particles (VLPs) containing GFP. It was found that fluorescent VLPs could be assembled from L2 protein in which GFP is inserted into the N-terminal region of L2 (aa 88). The fluorescent VLPs could also be assembled from a GFP/L2 fusion protein in which part of the L2 sequence had been deleted. In vitro, fluorescent VLPs could bind to CV-1 cells, and this VLP/cell interaction could be analyzed by FACS assay. These results demonstrated that GFP could incorporate into BPV1 VLPs without disruption of the VLP structure. Fluorescent VLPs might be a useful tool for study of papillomavirus virus/cell interaction. Key words : papillomavirus ; Study of the papillomaviruses (PVs) life cycle has been hampered by the unavail- ability of an in vitro cell culture system for propagation of the virus. It has been found that PV major eapsid protein L1, alone or with minor protein L2, could self-assemble into VLPs when expressed in eukaryotic systems by recombinant" viruses clJ. Evi- dence obtained using BPV 1 virions and HPV VLPs has demonstrated that PVs bind to a variety of cell lines, suggesting that the PV receptor is well conserved among vari- ous tissues, and this binding is Ll-mediated since binding of L1L2 VLP is not different from the binding of L1 VLP cz~. Although L2 is not necessary for in vitro capsid as- sembly, when co-expressed with L1, it is stably incorporated into capsid and enhances the yield and stability of VLPs E1"3~. Recent- ly, a sequence of BPV1L2 close to the N- terminus (aa 61 -- 123) has been identified as being exposed on the surface of viral par- ticles E4]. GFP of the jellyfish Aequorea victoria is a small molecule of 238 amino acids, whose natural function seems to be conversion of the blue chemiluminescence of the Ca z+- sensitive photoprotein aequorin into green emission F-s]. Heterologous expression of the PENG Shiwen, male, born in 1964, Lecturer virus-like particles; green fluorescent protein cloned GFP gene can generate strong green fluorescence without any additional Ae- quorea-specific enzymes Es-7]. Therefore, it has been widely used as a molecular re- porter to monitor gene expression, a tracer of cell lineage, and as a fusion tag to moni- tor protein localization and intracellular pro- tein trafficking within living cells Es'6~. In the present study, fluorescent BPV1L1L2/GFP VLPs were produced by fusing GFP gene to BPV1L2 sequence, in order to demonstrate that it might be a use- ful tool for study of papillomavirus virus/ cell interaction. 1 MATERIALS AND METHODS 1.1 Cell Lines Spodoptera frugiperda ( Sf-9 ) cells were maintained in Sf-900 1I medium, sup- plemented with Sf-900 I Supplement (Gib- co BRL, USA) and 10 ~ fetal bovine serum (FBS) (CSL, Australia), at 27 C. CV-1 cells were maintained in complete DMEM (CSL, Australia) plus 10 % FBS at 37 C in the presence of 5 % COz. 1.2 Generation of Chimeric ORF Express- ing BPV1L2-GFP Fusion Protein Genera 88 of a chimeric ORF express- ing BPV1L2-GFP fusion protein was achieved by firstly generating a BPV1L2 N-