Kaposis sarcoma-associated herpesvirus induces the phosphatidylinositol 3-kinase-PKC-zeta-MEK-ERK signaling pathway in target cells early during contamination: implications for infectivity

Kaposis sarcoma-associated herpesvirus induces the phosphatidylinositol 3-kinase-PKC-zeta-MEK-ERK signaling pathway in target cells early during contamination: implications for infectivity. cofilin activity is usually important for computer virus access and cell ruffle production. (a) Changes in the mRNA levels of cofilin during early HSV-1 contamination. (b) Left: the knockdown of LIMK-1 inhibits HSV-1 access. Right: the efficacy of LIMK siRNA silencing effects on mRNA expression levels. (c) Colocalization between cofilin mutants and F-actin. The cells were transfected with p350 different cofilin mutant plasmids (2?g) and incubated for 24?h before being fixed and analyzed. Red, phalloidin staining; green, GFP fluorescence. The overexpression of Sal003 CFL/WT appeared in the cofilin-rod structure, which indicated the accumulation of Sal003 active cofilin; the overexpression of mutant CFL/S3E showed more F-actin accumulation and colocalization with cofilin compared with the overexpression of CFL/S3A, which showed lower F-actin levels and colocalization (arrows). (d) Effects of cofilin siRNA on cell ruffle production. Apparently, the knockdown of cofilin reduced the HSV-1-mediated production of filopodia and lamellipodia. (e) Active cofilin locates at the suggestions of filopodia (arrows). The cells were exposed to HSV-1 and stained with TRITC-phalloidin. Download Physique?S2, TIF file, 2.9 MB mbo001141716sf02.tif (2.8M) GUID:?1C86CF29-8845-4561-9A45-878F691A7721 Physique?S3: HSV-1 infection induces Lasp-1 translocation. (a) Subcellular localization of Lasp-1 during HSV-1 contamination. Lasp-1 migrates and colocalizes with F-actin. The cells were transfected with GFP-tagged Lasp-1 plasmid and incubated for 24?h before HSV-1 contamination. At different postinfection occasions, monolayer cells were fixed and stained with F-actin. (b) The knockdown or overexpression of Lasp-1 affects HSV-1 entry. The upper panel shows the efficacy of knockdown or overexpression, and the lower panel shows the effects on HSV-1 access. Download Physique?S3, TIF file, 4.6 MB mbo001141716sf03.tif (4.6M) GUID:?35F5E4FB-EF09-44F8-A7F2-C517B72824B7 Figure?S4: EGFR is activated and mediates the signaling transduction. (a) EGFR clustering upon HSV-1 contamination. (b) Percentage of HSV-1 access into serum-starved SKCNCSH cells in the presence of bFGF. (c) Experimental setup. The cells were pretreated either with or without AG-1478 for 1?h at 37C. After HSV-1 binding to cells for 1?h at 4C, during this time, HSV-1 binds to the cells but does Sal003 not efficiently enter; thus, the inoculum was removed, and the cells were incubated at 37C to allow for synchronous viral access. Dashed lines show the presence of an inhibitor. (d) Efficacy of siRNAs with respect to the mRNA expression level of EGFR. (e) HSV-1 contamination induces EGFR activation in different cell lines. MEF, Vero, and HeLa cells were exposed to HSV-1 for 10?min. Download Physique?S4, TIF file, 2.9 MB mbo001141716sf04.tif (2.9M) GUID:?20F5FC22-6EBF-4ABB-BA74-88FC942DA2DA Table?S1: List of all pharmacological inhibitors. Table?S1, DOCX file, 0 MB. mbo001141716st1.docx (12K) GUID:?D544118C-7B93-4749-BC16-CD5F3CE51FC1 Table?S2: List of antibodies. Table?S2, DOCX file, 0.1 MB. mbo001141716st2.docx (15K) GUID:?94C1A53B-1FAD-48A8-B20B-806C57400234 Table?S3: List of plasmids. Table?S3, DOCX file, 0.1 MB. mbo001141716st3.docx (12K) GUID:?F0421D2C-32A3-4B6F-80F8-8CBE8F90D63B Table?S4: List of siRNA sequences. Table?S4, DOCX file, 0.1 MB. mbo001141716st4.docx (12K) GUID:?030BAC77-3D76-4D99-80A0-2FC4296869A8 Table?S5: List of primer sequences that were used in quantitative real-time PCR. Table?S5, DOCX file, 0.1 MB. mbo001141716st5.docx (11K) GUID:?E898E8FC-5105-4529-AB48-1F7FEC7BCDB4 Table?S6: List of primer sequences that were utilized for plasmid construction and site-directed mutagenesis. Table?S6, DOCX file, 0.1 MB. mbo001141716st6.docx (12K) GUID:?8AABE388-1819-4080-99F1-D5C5AEACC763 ABSTRACT Herpes simplex virus type 1 (HSV-1) establishes latency in neurons and can cause severe disseminated infection with neurological impairment and high mortality. This neurodegeneration is usually thought to be tightly associated with virus-induced cytoskeleton disruption. Currently, the regulation pattern of the actin cytoskeleton and the involved molecular mechanisms during HSV-1 access into neurons remain unclear. Here, we demonstrate that this access of HSV-1 into neuronal cells induces biphasic remodeling of the actin cytoskeleton and an initial inactivation followed by the.