Human Papillomavirus 16 Infection Induces VAP-Dependent Endosomal Tubulation

Abstract

Human papillomavirus (HPV) infection involves complex interactions with the endocytic transport machinery, which ultimately facilitates the entry of the incoming viral genomes into the -Golgi network (TGN) and their subsequent nuclear entry during mitosis. The endosomal pathway is a highly dynamic intracellular transport system, which consists of vesicular compartments and tubular extensions, although it is currently unclear whether incoming viruses specifically alter the endocytic machinery. In this study, using MICAL-L1 as a marker for tubulating endosomes, we show that incoming HPV-16 virions induce a profound alteration in global levels of endocytic tubulation. In addition, we also show a critical requirement for the endoplasmic reticulum (ER)-anchored protein VAP in this process. VAP plays an essential role in actin nucleation and endosome-to-Golgi transport. Indeed, the loss of VAP results in a dramatic decrease in the level of endosomal tubulation induced by incoming HPV-16 virions. This is also accompanied by a marked reduction in virus infectivity. In VAP knockdown cells, we see that the defect in virus trafficking occurs after capsid disassembly but prior to localization at the-Golgi network, with the incoming virion-transduced DNA accumulating in Vps29/TGN46-positive hybrid vesicles. Taken together, these studies demonstrate that infection with HPV-16 virions induces marked alterations of endocytic transport pathways, some of which are VAP dependent and required for the endosome-to-Golgi transport of the incoming viral L2/DNA complex. Human papillomavirus infectious entry involves multiple interactions with the endocytic transport machinery. In this study, we show that incoming HPV-16 virions induce a dramatic increase in endocytic tubulation. This tubulation requires ER-associated VAP, which plays a critical role in ensuring the delivery of cargoes from the endocytic compartments to the -Golgi network. Indeed, the loss of VAP blocks HPV infectious entry at a step after capsid uncoating but prior to localization at the-Golgi network. These results define a critical role for ER-associated VAP in endocytic tubulation and in HPV-16 infectious entry.

Diverse Papillomavirus Types Induce Endosomal Tubulation

“…HPV-16 PsVs were used as a control and, as can be seen from Figure 2, the maximum increase in endosomal tubulation was observed by 8 h post-infection in WT HeLa (Figure 2A). In contrast, endosomal tubulation was greatly reduced in VAP DKO HeLa cells (Figure 2B), which is consistent with previous studies (Siddiqa et al, 2018). We extended this analysis to other members of genus alpha (HPV-18, HPV-31), to genus delta (BPV-1), and to genus pi (MmuPV-1), to determine whether they also induce endosomal tubulation in a VAP-dependent manner.…”

“…After establishing that VAP is required for the efficient infectious entry of these diverse Papillomavirus types, we were interested to know whether VAP also has a role in inducing endosomal tubulation, as was observed previously for HPV-16 infection (Siddiqa et al, 2018). WT and VAP DKO HeLa cells were infected with diverse EdU-labeled PsVs.…”

“…Cells were fixed with 3.7% paraformaldehyde for 15 min at room temperature. Immunofluorescence for MICAL-L1 was performed as described before (Siddiqa et al, 2018). Images were obtained using a Zeiss Axiovert 100 M microscope, and analyzed by using an LSM image browser that supports the LSM 510 confocal unit.…”

“…The VAMP (vesicle-associated membrane protein)-associated protein (VAP) is thought to be important in making endosome-ER contact points, since depletion of VAP results in phosphatidylinositol 4-phosphate (PI4P) accumulation in the endosome, and dysfunctioning of the retromer and Wiskott-Aldrich Syndrome Protein and SCAR Homolog complex (WASH), leading to disruption of cargo trafficking from the endosome to the TGN (Dong et al, 2016). We have recently shown that VAP is important for HPV-16 PsV infectious entry and, moreover, HPV-16 PsV induces VAP-dependent endosomal tubulation, without which the incoming virus is unable to reach the TGN (Siddiqa et al, 2018). In this study we were therefore interested in investigating whether induction of tubulation was common to different Papillomavirus types, and, furthermore, whether the activity of VAP at endosomal-ER contact points was also required for infectious entry of multiple PV types.…”

Papillomaviruses and Endocytic Trafficking

“…The increase in tubulation is an early event, as it can be observed at 2 h post-infection, reaches a maximum at 8 h post-infection and is greatly reduced by 24 h post-infection. However, it is not yet clear how incoming virions affect endocytic tubulation, nor how HPV uses this to its advantage [ 82 ].…”