Research Article: Translocation of the papillomavirus L2/vDNA complex across the limiting membrane requires the onset of mitosis

Date Published: May 2, 2017

Publisher: Public Library of Science

Author(s): Christine M. Calton, Matthew P. Bronnimann, Ariana R. Manson, Shuaizhi Li, Janice A. Chapman, Marcela Suarez-Berumen, Tatum R. Williamson, Sudheer K. Molugu, Ricardo A. Bernal, Samuel K. Campos, Craig Meyers.


The human papillomavirus type 16 (HPV16) L2 protein acts as a chaperone to ensure that the viral genome (vDNA) traffics from endosomes to the trans-Golgi network (TGN) and eventually the nucleus, where HPV replication occurs. En route to the nucleus, the L2/vDNA complex must translocate across limiting intracellular membranes. The details of this critical process remain poorly characterized. We have developed a system based on subcellular compartmentalization of the enzyme BirA and its cognate substrate to detect membrane translocation of L2-BirA from incoming virions. We find that L2 translocation requires transport to the TGN and is strictly dependent on entry into mitosis, coinciding with mitotic entry in synchronized cells. Cell cycle arrest causes retention of L2/vDNA at the TGN; only release and progression past G2/M enables translocation across the limiting membrane and subsequent infection. Microscopy of EdU-labeled vDNA reveals a rapid and dramatic shift in vDNA localization during early mitosis. At late G2/early prophase vDNA egresses from the TGN to a pericentriolar location, accumulating there through prometaphase where it begins to associate with condensed chromosomes. By metaphase and throughout anaphase the vDNA is seen bound to the mitotic chromosomes, ensuring distribution into both daughter nuclei. Mutations in a newly defined chromatin binding region of L2 potently blocked translocation, suggesting that translocation is dependent on chromatin binding during prometaphase. This represents the first time a virus has been shown to functionally couple the penetration of limiting membranes to cellular mitosis, explaining in part the tropism of HPV for mitotic basal keratinocytes.

Partial Text

Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that infect the basal keratinocytes of differentiating epidermal or mucosal epithelium. Most HPV infections are asymptomatic or cause benign lesions. However, several HPV types, termed high-risk HPVs, are associated with essentially all cases of cervical cancer, as well as a significant number of anogenital and oropharyngeal cancers [1]. HPV16 is the most common high-risk type and accounts for over 60% of cervical cancer cases. Combined, HPVs are responsible for approximately 5% of all human cancers [2].

A major barrier that all nonenveloped viruses must overcome is transport of their nucleic acid genomes across limiting membranes during infection of host cells. The papillomaviruses have evolved the L2 minor capsid protein, which complexes with the vDNA to ensure proper subcellular trafficking and subsequent penetration of limiting membranes. In this work we describe the development of the first direct assay for membrane translocation of L2, based on the bacterial biotin ligase BirA and its cognate BAP substrate. Our system involves the infection of GFP-BAP-expressing cells with virions encapsidating an L2-BirA fusion. Limiting membranes separate the lumenal virion-associated BirA from the cytosolic GFP-BAP substrate. Thus, biotinylation of GFP-BAP during infection is a direct consequence of L2 translocation across the limiting membrane. The enzymatic signal-amplifying nature of the system was intentional to favor the sensitive detection L2-BirA translocation. Similar BirA/BAP-based systems could be useful for trafficking and penetration studies of other viruses containing capsid proteins permissive to heterologous fusions, like adenovirus and the pIX capsid protein [54]. Likewise, we anticipate and are working towards extending the system to enable tracking of subcellular L2-BirA trafficking by engineering host cells to express BAP-tagged substrates in specific subcellular compartments.




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