cHuman papillomavirus type 16 (HPV16) is the primary etiologic agent for cervical cancer. The infectious entry of HPV16 into cells occurs via a so-far poorly characterized clathrin-and caveolin-independent endocytic pathway, which involves tetraspanin proteins and actin. In this study, we investigated the specific role of the tetraspanin CD151 in the early steps of HPV16 infection. We show that surface-bound HPV16 moves together with CD151 within the plane of the membrane before they cointernalize into endosomes. Depletion of endogenous CD151 did not affect binding of viral particles to cells but resulted in reduction of HPV16 endocytosis. HPV16 uptake is dependent on the C-terminal cytoplasmic region of CD151 but does not require its tyrosine-based sorting motif. Reexpression of the wild-type CD151 but not mutants affecting integrin functions restored virus internalization in CD151-depleted cells. Accordingly, short interfering RNA (siRNA) gene knockdown experiments confirmed that CD151-associated integrins (i.e., ␣3␤1 and ␣6␤1/4) are involved in HPV16 infection. Furthermore, palmitoylation-deficient CD151 did not support HPV16 cell entry. These data show that complex formation of CD151 with laminin-binding integrins and integration of the complex into tetraspanin-enriched microdomains are critical for HPV16 endocytosis.

The endoplasmic reticulum (ER) serves as the major intracellular Ca 2+ store and has a role in the synthesis and folding of proteins. BAX (BCL2-associated X protein) inhibitor-1 (BI-1) is a Ca 2+ leak channel also implicated in the response against protein misfolding, thereby connecting the Ca 2+ store and protein-folding functions of the ER. We found that BI-1-deficient mice suffer from leukopenia and erythrocytosis, have an increased number of splenic marginal zone B cells and higher abundance and nuclear translocation of NF-κB (nuclear factor-κ light-chain enhancer of activated B cells) proteins, correlating with increased cytosolic and ER Ca 2+ levels. When put into culture, purified knockout T cells and even more so B cells die spontaneously. This is preceded by increased activity of the mitochondrial initiator caspase-9 and correlated with a significant surge in mitochondrial Ca 2+ levels, suggesting an exhausted mitochondrial Ca 2+ buffer capacity as the underlying cause for cell death in vitro. In vivo, T-cell-dependent experimental autoimmune encephalomyelitis and B-cell-dependent antibody production are attenuated, corroborating the ex vivo results. These results suggest that BI-1 has a major role in the functioning of the adaptive immune system by regulating intracellular Ca 2+ homeostasis in lymphocytes. Cell Death and Differentiation (2016) 23, 358-368; doi:10.1038/cdd.2015; published online 16 October 2015The endoplasmic reticulum (ER) serves as the major intracellular calcium (Ca 2+ ) store, the release of which controls a vast array of cellular functions from short-term responses such as contraction and secretion to long-term regulation of cell growth and proliferation. 1 Dysregulated release of ER Ca 2+ , in contrast, initiates programmed cell death by several mechanisms including mitochondrial Ca 2+ overload, depolarization, ATP loss and cytochrome c release. 2 Besides this, the ER also has a key role in the synthesis, folding and sorting of proteins destined for the secretory pathway. The deleterious consequences of an increase in unfolded proteins is called ER stress and can be antagonized by the unfolded protein response (UPR), a mechanism that coordinates a simultaneous increase in the ER folding capacity and a decrease in folding load. In the case of insufficient adaptation to ER stress, cells undergo apoptosis. 3 BAX (BCL2-associated X protein) inhibitor-1 (BI-1) is an evolutionarily conserved protein that bridges both the Ca 2+ homeostasis and UPR functions of the ER. 4 BI-1 was first identified in a screen for human proteins capable of inhibiting BAX-mediated cell death in yeast. 5 In mammalian cells, BI-1's antiapoptotic function is most pronounced in paradigms of ER stress 6 and involves changes in the amount of Ca 2+ that can be released from intracellular stores. 6,7 BI-1 is a highly hydrophobic protein that forms a Ca 2+ pore responsible for its Ca 2+ leak properties 8 and is the founding member of a family of six proteins with similar properties. 9 The increase in the ER Ca 2+ lea...

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-...

Zusammenfassung Hintergrund Akut-symptomatische epileptische Anfälle treten in engem zeitlichem Zusammenhang mit einer akuten Störung der Hirnfunktion auf. Sie sind mit einem niedrigen Risiko späterer unprovozierter Anfallsrezidive assoziiert. Daher empfehlen aktuelle Leitlinien keine längerfristige medikamentöse Anfallsprophylaxe. Dennoch werden im klinischen Alltag oft langfristige sekundärprophylaktische Therapien begonnen. Die Anfallsprognose nach leitliniengerecht nicht oder nur kurzzeitig behandelten akut-symptomatischen Anfällen ist bisher unbekannt. Hypothese Nach einem akut-symptomatischen Erstanfall struktureller Ätiologie ist das 1‑Jahres-Risiko für unprovozierte Rezidivanfälle nicht höher als 25 %, auch wenn keine oder nur eine kurzfristige medikamentöse Anfallsprophylaxe eingesetzt wird. Methoden Das PROSA-Register ist eine einarmige, offene, prospektive, multizentrische Beobachtungsstudie. Eingeschlossen werden 115 volljährige Personen mit strukturell bedingtem akut-symptomatischem, epileptischem Erstanfall, sofern dieser kein Status epilepticus war. Der intrahospitale Verlauf wird der Krankenakte entnommen. Nach 3, 6 und 12 Monaten finden telefonische Nachbefragungen statt. Diskussion Das PROSA-Register wird als Beobachtungsstudie die derzeitige Behandlungspraxis nach akut-symptomatischem Anfall und die tatsächliche Anfallsprognose innerhalb eines Jahres beleuchten. Die Ergebnisse sollen die vorhandene Evidenz unterstreichen, dass eine medikamentöse Anfallsprophylaxe über die akute Phase der zugrunde liegenden Erkrankung hinaus nicht notwendig ist. Registrierung Die Studie wurde prospektiv im Deutschen Register Klinischer Studien unter der ID DRKS00017811 registriert.