title: A Cell-Free Assembly System for Generating Infectious Human Papillomavirus 16 Capsids Implicates a Size Discrimination Mechanism for Preferential Viral Genome Packaging.
abstract: We have established a cell-free in vitro system to study human papillomavirus type 16 (HPV16) assembly, a poorly understood process. L1/L2 capsomers, obtained from the disassembly of virus-like particles (VLPs), were incubated with nuclear extracts to provide access to the range of cellular proteins that would be available during assembly within the host cell. Incorporation of a reporter plasmid "pseudogenome" was dependent on the presence of both nuclear extract and ATP. Unexpectedly, L1/L2 VLPs that were not disassembled prior to incubation with a reassembly mixture containing nuclear extract also encapsidated a reporter plasmid. As with HPV pseudoviruses (PsV) generated intracellularly, infection by cell-free particles assembled in vitro required the presence of L2 and was susceptible to the same biochemical inhibitors, implying the cell-free assembled particles use the infectious pathway previously described for HPV16 produced in cell culture. Using biochemical and electron microscopy analyses, we observed that, in the presence of nuclear extract, intact VLPs partially disassemble, providing a mechanistic explanation to how the exogenous plasmid was packaged by these particles. Further, we provide evidence that capsids containing an <8-kb pseudogenome are resistant to the disassembly/reassembly reaction. Our results suggest a novel size discrimination mechanism for papillomavirus genome packaging in which particles undergo iterative rounds of disassembly/reassembly, seemingly sampling DNA until a suitably sized DNA is encountered, resulting in the formation of a stable virion structure.
  
  
  OBJECTIVE
  Little is known about papillomavirus assembly biology due to the difficulties in propagating virus in vitro. The cell-free assembly method established in this paper reveals a new mechanism for viral genome packaging and will provide a tractable system for further dissecting papillomavirus assembly. The knowledge gained will increase our understanding of virus-host interactions, help to identify new targets for antiviral therapy, and allow for the development of new gene delivery systems based on in vitro-generated papillomavirus vectors.
authors.affiliation: Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Carla
authors.initials: C
authors.lastname: Cerqueira
authors.affiliation: Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Yuk-Ying S
authors.initials: YY
authors.lastname: Pang
authors.affiliation: Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Patricia M
authors.initials: PM
authors.lastname: Day
authors.affiliation: Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Cynthia D
authors.initials: CD
authors.lastname: Thompson
authors.affiliation: Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Buck
authors.affiliation: Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Lowy
authors.affiliation: Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA schillej@mail.nih.gov.
authors.forename: John T
authors.initials: JT
authors.lastname: Schiller
mesh_terms: D036022:Capsid Proteins / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D017930:Genes, Reporter; D016679:Genome, Viral; D052162:Human papillomavirus 16 / Q000502:physiology*; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D010957:Plasmids; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral; C063316:L2 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; C063317:L1 protein, Human papillomavirus type 16