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. IMPORTANCELittle 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 vitrogenerated papillomavirus vectors. P apillomaviruses (PVs) are a group of nonenveloped viruses with small circular double-stranded DNA genomes that infect the skin and mucosa. PV replication is tightly coupled to the differentiation of the epithelium (1). Human papillomavirus 16 (HPV16) is medically the most important HPV type, as it is responsible for about 50% of cervical cancers and for the vast majority of noncervical HPV-positive anogenital and oropharyngeal cancers (2). The expression of viral early genes occurs in the lower layers of the epithelium, while vegetative replication of the viral genome, late gene expression, and virus assembly occur in more differentiated suprabasal cells (1). The strict dependency of viral replication on epithelial differentiation has made it difficult to produce virus in reasonable amounts and quality in cultured cells, although organotypic epithelial cell "raft" culture s...

Bovine papillomavirus productive infection in cell cultures: First evidences

“…Similar results have been also described in humans, in which sequences of HPV were verified in blood and semen [54,55]. In addition, Cerqueira et al [47] currently demonstrated the cell-free assembly HPV-16 capsid. These results indicate the need to review the PVs natural history, as proposed by Munday [34].…”

“…This occur because, according to the BPV natural history, the viral replication is dependent of cell differentiation [45][46][47]. Considering the paradigm of PVs replication cycle, the expression of capsid proteins (L1 and L2) and viral assembly are only verified in most differentiated epithelium layers (hypergranulous) [48][49][50].…”

Brd4 Activates Early Viral Transcription upon Human Papillomavirus 18 Infection of Primary Keratinocytes

“…We noted that exogenous expression of E1 and E2 resulted in robust replication of HPV18 and hypothesized that this might increase packaging efficiency (though the resulting quasivirus could not be used to study early viral replication using the DpnI resistance assay, because the genomes would now be susceptible to DpnI digestion). The pMEP vectors used to express E1 and E2 are >10 kb in size and will not be packaged because they are above the 8-kb packaging limit (8, 46). Furthermore, despite the fact that E1 and E2 are expressed from the inducible metallothionein promoter, leaky expression ensures that they are expressed even in the absence of heavy metal induction (47).…”

Papillomavirus assembly: An overview and perspectives

“…A recent in vitro study suggested that papillomavirus genome incorporation may occur through a previously unrecognized size discrimination mechanism (Cerqueira et al, 2015). The viral capsid appears to undergo repeated rounds of assembly and disassembly in the cell nucleus sampling both cellular DNA loops and the viral genome.…”

“…To address this issue, we are in the process of developing strategies of producing the pseudovirions in cell-free systems in which high concentrations of purified L1/L2 protein and bacterially-derived peudogenome plasmids are used to drive the assembly reaction. In the first iteration, it was found that HPV16 L1/L2 proteins could generate high titers of peudovirions in the presence of purified bacterial plasmids, a nuclear extract and ATP (Cerqueira et al, 2015). In support of the size discrimination of genome packaging described above, assembly was efficient even when assembled VLPs were used in the reaction because the nuclear extracts were able to induce partial disassembly of the particles.…”