Diverse Papillomavirus Types Induce Endosomal Tubulation

Abstract

Previous studies have shown that the endoplasmic reticulum (ER)-anchored protein VAP is strictly required by human papillomavirus type 16 (HPV-16) for successful infectious entry. Entry appeared to be mediated in part through the induction of endosomal tubulation and subsequent transport of the virion to the trans-Golgi network (TGN). In this study, we were interested in investigating whether this mechanism of infectious entry is conserved across multiple Papillomavirus types. To do this, we analyzed the role of VAP and endosomal tubulation following infection with Pseudovirions (PsVs) derived from the alpha, beta, delta, kappa, and pi papillomavirus genera, reflecting viruses that are important human and animal pathogens. We demonstrate that VAP is essential for infection with all PV types analyzed. Furthermore, we find that VAP and EGFR-dependent endosomal tubulation is also induced by all these different Papillomaviruses. These results indicate an evolutionarily conserved requirement for VAP-induced endocytic tubulation during Papillomavirus infectious entry.

Keywords: MICAL-L1; PV trafficking; endosomal tubulation; infectious entry; papillomavirus.

Figure 1

Infection by diverse papillomavirus types depends on the integral ER protein VAP. (A) List of different PVs analyzed in this study. (B) Western blot shows the efficacy of VAP-B knockdown. (C) WT HeLa (CTRL) and VAP DKO HeLa cells were infected with the indicated PVs for 48 h. Relative luminescence was measured and is plotted as a bar graph. The data shown here are the mean luciferase readings derived from 3 independent experiments, normalized against the respective infection in WT HeLa, where the bars indicate standard errors. Significance was determined using one way ANOVA (^****P < 0.00001).

Figure 2

Representative images of multiple PV types inducing VAP-dependent endosomal tubulation. WT HeLa (A) and VAP KDO HeLa (B) cells were infected with HPV-16 PsVs (150 vge/cell), and fixed at 2, 8, and 24 h post-infection. Reporter DNA encapsidated within the PsVs is detected by EdU labeling (red), whereas endogenous MICAL-L1 is stained with MICAL-L1 antibody (green) as a marker of an endosomal tubulation. Experiments are performed at least three times. Images were captured by confocal microscopy. The same analysis was performed with HPV-18 PsVs in WT HeLa (C) and in VAP KDO HeLa (D) cells; HPV-31 PsVs in WT HeLa (E) and in VAP KDO HeLa (F) cells; BPV-1 PsVs in WT HeLa (G) and in VAP KDO HeLa (H) cells; MmuPV-1 PsVs in WT HeLa (I) and in VAP KDO HeLa (J) cells. Scale bar: 20 μm. The right-hand column shows the zoomed images. Scale bar: 5 μm.

Figure 3

The number of tubules and EdU labeled viral DNA in WT HeLa and VAP KDO HeLa cells was quantified. At least 150 cells under each condition from three independent experiments were analyzed. (A) Number of endosomal tubules in WT HeLa cells were counted for UI, 2, 8, and 24 h post-infected cells and data was normalized to the uninfected cells. The data shown here is the mean number of tubules. An increase in tubulation was observed for all PV types analyzed: the increase observable as early as 2 h, becoming maximum at 8 h with a reduction at 24 h post-infection. Bars indicate standard errors. (B) Number of endosomal tubules in VAP-DKO HeLa cells were counted and data was normalized to the number of tubules counted in the WT HeLa for each respective PsV type (CTRL). Data shown here is the mean number of tubules. The dramatic decrease in tubulation in the absence of VAP is significant as found by Student's t-test comparing UI, 2, 8, 24 h WT with UI, 2, 8, 24 h VAP DKO HeLa cells for each PsVs, respectively (^****P < 0.00001). Bars indicate the standard error (C) EdU labeled viral DNA in VAP-DKO HeLa cells was counted for 2 h post-infection and data was normalized to the EdU counted in the WT HeLa for each PsV type (CTRL). The data shown here is the mean number of EdU particles. There is very slight difference in EdU in the absence of VAP. Bars indicate the standard error.

Figure 4

EGFR signaling is required for virus uptake and endocytic tubulation. (A) HeLa cells were treated with 300 nM of the EGFR-specific inhibitor (PD168393). DMSO-treated cells were used as a control. Cells were infected with HPV-16 PsVs (50 vge/cell) carrying a luciferase reporter plasmid and luciferase activity was measured at 48 h posttransduction. The data shown are the mean luciferase readings from three independent experiments, where bars indicate standard errors. Significance was determined using Student's t-test (^*P < 0.01). (B) Western blot for p-ERK1/2 following 10 min EGF (10 ng/ml) exposure in the presence or absence of PD168393 was performed in HeLa cells. (C) HeLa cells were treated with DMSO and infected with HPV-16 PsVs (150 vge/cell) for 8 and 24 h. Tubulation was detected with MICAL-L1 (green). (D) HeLa cells were treated with 300 nM PD168393 and infected with HPV-16 PsVs (150 vge/cell) for 8 and 24 h. (E) HeLa cells were infected with HPV-16 PsVs (150 vge/cell) and treated with 300 nM PD168393 6 h post- infection. Scale bar: 10 μm. The right-hand column shows the zoomed images. Scale bar 5 μm. (F) The number of tubules in HPV-16 PsVs infected HeLa cells (CTRL), EGFR inhibitor treated cells and 6 h post-infection EGFR inhibitor treated cells were quantified. At least 50 cells under each condition (UI, 8 and 24 h infection) from three independent experiments were analyzed and data was normalized to the uninfected cells. Data shown here is the mean number of tubules. The dramatic decrease in tubulation in the absence of EGFR signaling is significant as found by Student's t test comparing 8 h untreated to inhibitor treated cells (^**P < 0.001). Bars indicate the standard error.