TY - JOUR
T1 - Translocation of the papillomavirus L2/vDNA complex across the limiting membrane requires the onset of mitosis
AU - Calton, Christine M.
AU - Bronnimann, Matthew P.
AU - Manson, Ariana R.
AU - Li, Shuaizhi
AU - Chapman, Janice A.
AU - Suarez-Berumen, Marcela
AU - Williamson, Tatum R.
AU - Molugu, Sudheer K.
AU - Bernal, Ricardo A.
AU - Campos, Samuel K.
N1 - Funding Information:
SKC is supported by grant 1R01AI108751-01 from the National Institute for Allergy and Infectious Diseases. ARM and MSB were funded in part by a grant to UA from HHMI (52006942) that supports the Undergraduate Biology Research Program, UBRP. RAB is supported by grant NIH-NIGMS SC3GM113805 from the National Institute of General Medical Sciences, grant NSF-MRI-0923437 from the National Science Foundation, and grant AH-1649 from the Welch Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We gratefully acknowledge Dr. Martin M?ller for the K4L220-38monoclonal antibody, Dr. Patricia Day for the fur?1 furin-secreting CHO cells, and Dr. Steven Leppla for the furin deficient FD11 CHO cells. We thank Chris Buck for the 293TT cells. We thank Anne Cress for the HaCaT cells. We thank Felicia Goodrum, Kyle Roux, and Dan DiMaio for plasmid constructs. We thank Mike Kuhns for pericentrin antibody. We thank Dena Yoder of the UA BIO5 Media Facility, Patty Jansma of the UA ORD Imaging Core-Marley, and Paula Campbell and John Fitch of the UACC/ARL Cytometry Core Facility which is funded by a UA Cancer Center Support Grant (CCSG?CA 023074). We thank Greg Rogers, David Elliott, Jean Wilson, and the ?UA trafficking group? for critical feedback on various aspects of this work. We thank the Schelhaas group for critical reading of the manuscript.
PY - 2017/5
Y1 - 2017/5
N2 - 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.
AB - 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.
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U2 - 10.1371/journal.ppat.1006200
DO - 10.1371/journal.ppat.1006200
M3 - Article
C2 - 28463988
AN - SCOPUS:85020196790
VL - 13
JO - PLoS Pathogens
JF - PLoS Pathogens
SN - 1553-7366
IS - 5
M1 - e1006200
ER -