Entry of Human Papillomavirus Type 16 by Actin-Dependent, Clathrin- and Lipid Raft-Independent Endocytosis

Abstract

Infectious endocytosis of incoming human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer, is poorly characterized in terms of cellular requirements and pathways. Conflicting reports attribute HPV-16 entry to clathrin-dependent and -independent mechanisms. To comprehensively describe the cell biological features of HPV-16 entry into human epithelial cells, we compared HPV-16 pseudovirion (PsV) infection in the context of cell perturbations (drug inhibition, siRNA silencing, overexpression of dominant mutants) to five other viruses (influenza A virus, Semliki Forest virus, simian virus 40, vesicular stomatitis virus, and vaccinia virus) with defined endocytic requirements. Our analysis included infection data, i.e. GFP expression after plasmid delivery by HPV-16 PsV, and endocytosis assays in combination with electron, immunofluorescence, and video microscopy. The results indicated that HPV-16 entry into HeLa and HaCaT cells was clathrin-, caveolin-, cholesterol- and dynamin-independent. The virus made use of a potentially novel ligand-induced endocytic pathway related to macropinocytosis. This pathway was distinct from classical macropinocytosis in regards to vesicle size, cholesterol-sensitivity, and GTPase requirements, but similar in respect to the need for tyrosine kinase signaling, actin dynamics, Na+/H+ exchangers, PAK-1 and PKC. After internalization the virus was transported to late endosomes and/or endolysosomes, and activated through exposure to low pH.

Incoming human papillomavirus type 16 genome resides in a vesicular compartment throughout mitosis

“…Following primary attachment and internalization, acidification of early endosomes triggers capsid disassembly (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Host cell cyclophilins release the majority of L1 from the L2 protein, which remains in complex with the viral genome (2,23,24).…”

Uncoating of non-enveloped viruses

“…It is worth noting that human papilloma virus 16 (HPV-16) also uses receptor binding to initiate its uncoating program. This critically involves exposure of the virion internal protein L2 and cleavage of its N-terminal sequence by furin [16], which enables the altered virus to use low pH in late endosomes to access the cytosol [17].…”

Molecular Signaling and Cellular Pathways for Virus Entry

“…Under different signaling regulations, the macropinosomes can fused with EE or recycled back to plasma membrane. To date, several viruses have been found to enter their host cells by macropinocytosis, including vaccinia virus, HIV-1, coxsackievirus B (CVB), herpes simplex virus type 1 (HSV-1), african swine fever virus (ASFV), human papillomavirus type 16 (HPV-16), and KSHV [11][12][13]69]. Recently, it was reported that vaccinia virus entry relies on type II membrane glycoprotein CD98 associated integrin 1 triggered PI3 K/Akt signaling and ERK, PKC, and PAK1 that are required for macropinosome closure [70,71].…”

“…Interestingly, Schelhaas and colleagues uncovered that HPV-16 utilizes a potentially novel ligand-induced endocytic pathway related to macropinocytosis. is pathway is different from classical macropinocytosis in regards to vesicle size, cholesterol-sensitivity, and GTPase requirements, but similar in the need for tyrosine kinase signaling, PAK-1, PKC, actin dynamics, and Na + /H + exchangers [13,69]. In the case of CVB, its entry and the macropinosomes trafficking are caveolin-depnendent but dynamin independent that require occludin, Ras-Rab5 pathway, and Rab34 activation [74].…”