Caveolin-1-dependent infectious entry of human papillomavirus type 31 in human keratinocytes proceeds to the endosomal pathway for pH-dependent uncoating

Abstract

High-risk human papillomaviruses (HPVs) are small nonenveloped DNA viruses with a strict tropism for squamous epithelium. The viruses are causative agents of cervical cancer and some head and neck cancers, but their differentiation-dependent life cycles have made them difficult to study in simple cell culture. Thus, many aspects of early HPV infection remain mysterious. We recently showed the high-risk HPV type 31 (HPV31) enters its natural host cell type via caveola-dependent endocytosis, a distinct mechanism from that of the closely related HPV16 (Smith et al., J. Virol. 81:9922-9931, 2007). Here, we determined the downstream trafficking events after caveolar entry of HPV31 into human keratinocytes. After initial plasma membrane binding, HPV31 associates with caveolin-1 and transiently localizes to the caveosome before trafficking to the early endosome and proceeding through the endosomal pathway. Caveosome-to-endosome transport was found to be Rab5 GTPase dependent. Although HPV31 capsids were observed in the lysosome, Rab7 GTPase was dispensable for HPV31 infection, suggesting that viral genomes escape from the endosomal pathway prior to Rab7-mediated capsid transport. Consistent with this, the acidic pH encountered by HPV31 within the early endosomal pathway induces a conformational change in the capsid resulting in increased DNase susceptibility of the viral genome, which likely aids in uncoating and/or endosomal escape. The entry and trafficking route of HPV31 into human keratinocytes represents a unique viral pathway by which the virions use caveolar entry to eventually access a low-pH site that appears to facilitate endosomal escape of genomes.

FIG. 1.

HPV31 transiently localizes to caveolin-1 structures at the plasma membrane and intracellularly. HaCaT cells were exposed to AF594-labeled HPV31 virions at 10,000 vge/cell for 1 h at 4°C. Cells were washed and incubated at 37°C for 0 h (A), 30 min (B), or 16 h (C and D). Cells were fixed and stained for caveolin-1 (AF488; green), and nuclei were visualized with DAPI. The microscopy is representative, and the focal plane is mid-cell body/nucleus. Right panels are enlarged views of the boxed areas in the left panels.

FIG. 2.

HPV31 localizes to early endosomal pathway at late times postentry. HaCaT cells were exposed to AF594-labeled HPV31 virions at 10,000 vge/cell for 1 h at 4°C. Unbound virions were removed, and cells were incubated at 37°C for 16 h or 24 h. Cells were fixed and immunostained (AF488; green) for PDI (A), Golgin-97 (B), EEA1(C), or LAMP1 (D) as described in Materials and Methods. Nuclei were visualized with DAPI. The microscopy is representative, and the focal plane is mid-cell body/nucleus. Right panels are enlarged views of the boxed areas in the left panels.

FIG. 3.

HPV31 infection requires endosomal acidification and low pH induces conformational changes in the HPV31 virion. (A) HaCaT cells were pretreated for 30 min with bafilomycin A (75 or 100 nM; dotted line), NH₄Cl (15 or 20 mM; dashed line), or chloroquine (5 or 10 μM; solid line) and then infected with HPV31 at 100 vge/cell in the presence of inhibitors for 48 h. Cells were monitored for viability. Total RNA was analyzed for HPV31 E1^∧E4 by RT-qPCR. Values are expressed as the percentage of infection normalized to mock-treated cells. Error bars represent the SEM. (B) HPV31 virions (∼10⁸ vge) were treated in triplicate in vitro with 10 mM DTT at room temperature overnight or at the indicated pH for 2 h at 37°C. Samples were equally divided and mock or DNase I treated for 1 h at 37°C, blotted onto GeneScreen membrane, and probed for intact HPV31 genomes. (C) Quantification of panel B, where values are expressed as the percent DNase-resistant genome normalized to the corresponding mock-treated sample. Error bars represent the SEM. (D) A total of ∼10⁸ HPV31 virions were treated in vitro at indicated pH for 2 h at 37°C. Samples were bound to carbon-coated electron microscopy grids and stained with uranyl acetate before visualization by TEM.

FIG. 4.

Trafficking of HPV31 between the caveosome and endosomal pathway is Rab5 GTPase dependent and essential for the infection of human keratinocytes. (A) HaCaT cells were transfected with fluorescence-tagged Rab5 wild-type (Rab5wt), Rab5 S34N dominant-negative mutant (Rab11dn), Rab7wt, Rab7dn T22N, Rab11wt, or Rab11dn S25N constructs. At 48 h, AF594-HPV31 was exposed to cells at 10,000 vge/cell for 1 h at 4°C, washed, refed, and incubated at 37°C for 24 h. Cells were fixed and immunostained for caveolin-1 (cav-1; top row), EEA1 (middle row), or LAMP1 (bottom row) (AF680; pseudocolored green), and nuclei were visualized with DAPI. Images were captured on a Zeiss META confocal microscope with a ×63 objective and are representative. The focal plane was not limited to mid-cell body but was variable to assess colocalization. (B) Blinded quantification of HPV31 virions (red; AF594) colocalizing with caveolin-1-, EEA1-, or LAMP1-positive structures (pseudocolored green). Values are expressed as the percentage of HPV31 virions per cell colocalized with indicated marker compared to total virions. (C) Triplicate-transfected HaCaT cell cultures were infected with HPV31 at 100 vge/cell. Total RNA was harvested at 48 h postinfection and quantified in triplicate by RT-qPCR for spliced viral E1^∧E4 transcripts. Values are expressed as the percentage of infection compared to wild-type control transfected cells. Error bars represent the SEM of triplicate reactions. *, P > 0.01.

FIG. 5.

Model of HPV31 infectious entry in human keratinocytes. (A) HPV31 enters via caveola- and dynamin 2-mediated endocytosis and traffics to caveosomes. (B) Thereafter, virions are transported from the caveosome to the early endosome dependent on Rab5 GTPase. (C) As HPV31 proceeds through the endosomal pathway, the decreasing pH of the endosomal compartments causes a conformational change in the viral capsid, which results in DNase I sensitivity of genomes. We propose that this conformational change leads to endosomal escape of the viral genome or genome/L2 complex (D) before Rab7 GTPase-mediated capsids are transported to the lysosome (E). Empty/disassembled capsids can be visualized in the lysosome.