Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

Abstract

www.nature.com/scientificreports www.nature.com/scientificreports/ cluster increases at viral particle attachment sites. Our data suggests that viral particles use an entry platform containing integrin α6 for virus attachment and integrin α3 for internalisation. Resultsintegrin α3 is required for HPV16 infection of keratinocytes. To clarify the role of integrin α3 in the infection of keratinocytes, we infected integrin α3 depleted HaCaT cells by incubation with HPV16 pseudovirions (PsVs). For positive control, cells were depleted from integrin α6, which is required for infection of HeLa and KH-SV cells 5,21 . In Western blot analysis, knockdown reduced integrin α6 and integrin α3 protein levels by 87% and 97%, respectively ( Fig. S1). Intracellular processing of the L1 virus capsid protein was monitored by Western blot analysis and by microscopy. In the Western blots, we quantified the ~25 kDa cleavage product of L1, which is generated only after internalization and lysosomal degradation of the virus 22 . As seen in Fig. 1A, knockdown of integrin α3 and integrin α6 reduced the cleavage product by 73% and 44%, respectively. For microscopy, we employed antibody-detection of the L1-7 epitope, which only becomes detectable after intracellular capsid disassembly 4,23 . Knockdown of either one of the integrins reduced the number of L1-7 positive organelles to about 65% ( Fig. 1B,C). Moreover, organelles were slightly dimmer ( Fig. S2), suggesting that apart from less forming endocytic organelles the viral load per organelle diminishes.Next, we employed a luciferase-based infection assay to test whether less uptake and processing of the L1 protein would be associated with a lower infection rate. Infection is inhibited by 88% and 67% after integrin α3 and integrin α6 knockdown, respectively ( Fig. 1D). It should be noted that integrin knockdown has secondary effects ( Fig. S3). Still, after correcting for these secondary effects, knockdown of each integrin more than halves the infection rate ( Fig. S3). These experiments demonstrate that apart from integrin α6, integrin α3 plays a role in HPV infection of HaCaT cells as well. However, in Western blot analysis testing PsVs cell-surface primary binding only the knockdown of integrin α6 strongly inhibits binding by 40% ( Fig. 2), suggesting each of these integrins is differently involved during infection.PsVs associate with cluster crowds. Next, we asked whether PsV particles are closer to their potential binding partner integrin α6, and analysed the distances between PsVs and CD151/integrin maxima. On membrane sheets, PsVs are often close to or overlap with CD151 or integrin α6 maxima (Fig. 4A,B). Most distances are in the range of several 100 nm ( Fig. 4C), with no trend towards shorter distances to integrin α6 maxima. Also, no maxima preference is observed on PsVs/CD151/integrin α3 stainings of cells ( Fig. 4D-F), or PsVs/ CD151-GFP/integrin α3 stainings on membrane sheets (Fig. S11). Moreover, it should be noted that PsVs diminish the level of CD151 and integrins by 10-...

HPV caught in the tetraspanin web?

“…Cells were incubated with 4 × 10 7 viral genome equivalents (vge) (PsVs were prepared as previously described [10]) per well in culture medium without antibiotics for 3 h at 37 °C. Cells were washed twice in DPBS and either fixed directly or after membrane sheet generation.…”

“…Altogether, the data is consistent with the idea that PsVs associate with a tetraspanin web. In this web the tetraspanins would organize a platform [10] with different components such as OBSL1 for linking the virus to the actin cytoskeleton, or a receptor for virus attachment, e.g., integrin α6 that promotes papillomavirus binding [10,38,39]. It remains unclear whether the PsVs induce a special tetraspanin web or whether they associate to cluster crowds that are already present prior to virus contact.…”

“…Several reports document that TEMs modulate the cell entry for different types of viruses, including the human papillomavirus (HPV) [3], coronavirus [4], influenza A virus [5], hepatitis C virus [6,7] and human immunodeficiency virus [8,9]. In the case of HPV infection, TEMs organize viral entry platforms with components for virus binding and endocytosis [10]. However, tetraspanins may be as well binding receptors, as in the case of CD81 that directly binds to hepatitis C virus.…”

The mesoscale organization of syntaxin 1A and SNAP25 is determined by SNARE–SNARE interactions

“…To determine the point spread function at the specific laser power settings employed for STED microscopy, we used papillomavirus pseudovirions (PsVs, Finke et al, 2020 ) for calibration. PsVs (2 µl, which corresponds to ~4 × 10 7 viral genome equivalents) were diluted in 1 ml DMEM (see above) and vortexed vigorously.…”

HPV16 Entry into Epithelial Cells: Running a Gauntlet

“…This scenario is likely, as virions are found in complexes with GFs, their receptors, and soluble HSPGs [32]. Laminin-332 and lamininbinding integrins have been shown to bind HPV16 particles and are therefore suggested as additional components of the entry receptor complex [45][46][47][48][49][50]. The tetraspanin CD151 not only associates with EGFR but also regulates integrin activities by influencing their positioning within tetraspanin-enriched microdomains [34,[50][51][52][53][54][55] and is crucial for the process of HPV16 endocytosis into their host cells [56][57][58][59].…”