Epidermal Growth Factor Receptor-PI3K Signaling Controls Cofilin Activity To Facilitate Herpes Simplex Virus 1 Entry into Neuronal Cells

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 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 entry into neurons remain unclear. Here, we demonstrate that the entry of HSV-1 into neuronal cells induces biphasic remodeling of the actin cytoskeleton and an initial inactivation followed by the subsequent activation of cofilin, a member of the actin depolymerizing factor family that is critical for actin reorganization. The disruption of F-actin dynamics or the modulation of cofilin activity by mutation, knockdown, or overexpression affects HSV-1 entry efficacy and virus-mediated cell ruffle formation. Binding of the HSV-1 envelope initiates the epidermal growth factor receptor (EGFR)-phosphatidylinositide 3-kinase (PI3K) signaling pathway, which leads to virus-induced early cofilin phosphorylation and F-actin polymerization. Moreover, the extracellular signal-regulated kinase (ERK) kinase and Rho-associated, coiled-coil-containing protein kinase 1 (ROCK) are recruited as downstream mediators of the HSV-1-induced cofilin inactivation pathway. Inhibitors specific for those kinases significantly reduce the virus infectivity without affecting virus binding to the target cells. Additionally, lipid rafts are clustered to promote EGFR-associated signaling cascade transduction. We propose that HSV-1 hijacks cofilin to initiate infection. These results could promote a better understanding of the pathogenesis of HSV-1-induced neurological diseases

Heat-Shock Protein 90 Promotes Nuclear Transport of Herpes Simplex Virus 1 Capsid Protein by Interacting with Acetylated Tubulin

Although it is known that inhibitors of heat shock protein 90 (Hsp90) can inhibit herpes simplex virus type 1 (HSV-1) infection, the role of Hsp90 in HSV-1 entry and the antiviral mechanisms of Hsp90 inhibitors remain unclear. In this study, we found that Hsp90 inhibitors have potent antiviral activity against standard or drug-resistant HSV-1 strains and viral gene and protein synthesis are inhibited in an early phase. More detailed studies demonstrated that Hsp90 is upregulated by virus entry and it interacts with virus. Hsp90 knockdown by siRNA or treatment with Hsp90 inhibitors significantly inhibited the nuclear transport of viral capsid protein (ICP5) at the early stage of HSV-1 infection. In contrast, overexpression of Hsp90 restored the nuclear transport that was prevented by the Hsp90 inhibitors, suggesting that Hsp90 is required for nuclear transport of viral capsid protein. Furthermore, HSV-1 infection enhanced acetylation of α-tubulin and Hsp90 interacted with the acetylated α-tubulin, which is suppressed by Hsp90 inhibition. These results demonstrate that Hsp90, by interacting with acetylated α-tubulin, plays a crucial role in viral capsid protein nuclear transport and may provide novel insight into the role of Hsp90 in HSV-1 infection and offer a promising strategy to overcome drug-resistance

Hsp90 plays a crucial role in ICP5 nuclear translocation.

<p>(A) Confocal images show capsid protein transport reduced by Hsp90 inhibition. MRC-5 cells exposed to HSV-1 (MOI = 10) for 4 h under the treatment of Hsp90 inhibitor (0.8 µM) were stained for ICP5 (red), total Hsp90 (green), and nuclei (blue). (B) Hsp90 is important for capsid protein nuclear transport. Cell monolayers infected with HSV-1 (MOI = 10) for different times were stained for ICP5 (red), total Hsp90 (green), and nuclei (blue). Five images per dish were acquired by LSM for counting of ICP5 docked in nuclear. The percentage of positive nuclei (nuclei with ICP5) was calculated. Each value represents the mean ± SD of three independent experiments (**, <i>P</i><0.01, compared with the viral control. ^##, <i>P</i><0.01, compared with the BJ-B11-treated group).</p

Hsp90 inhibitors suppress intracellular translocation of ACV-resistant virus capsid protein ICP5.

<p>(A) Effects of Hsp90 inhibitors on ICP5 transport. After 4h infection in the presence of BJ-B11 or 17-AAG, the cells were fixed and confocal images showed the inhibition of capsid transport ether in HSV-1 F strain or in ACV-resistant HSV-1. (B) Quantification of ICP5-positive nuclei. Each value represents the mean ± SD of three independent experiments (**<i>P</i><0.01, compared with the HSV-1 F strain control, or the ACV-resistant HSV-1 strain control, respectively).</p

Hsp90 inhibitors suppress viral RNA synthesis and protein expression.

<p>(<b>A</b>) Inhibition of viral RNA synthesis. MRC-5 cells were infected with HSV-1 (MOI = 10) in the presence of Hsp90 inhibitor (0.8 µM). RNA samples were extracted at 4, 6, and 9 h p.i. and reverse transcribed to cDNA, which was used for UL54 (immediate early gene), UL29 (early gene), and UL27 (late gene) detection, respectively. (<b>B</b>) Inhibition of viral protein expression. MRC-5 cells were infected with HSV-1 (MOI = 10) in the presence of Hsp90 inhibitor (0.8 µM). Protein samples were extracted at 4, 6, and 9 h p.i. and used for ICP27 (immediate early protein), ICP8 (early protein), and ICP5 (late protein) detection, respectively. The Western blotting results shown in the bar graph were normalized to GAPDH expression and were expressed as the fold increase relative to the cell control. (C, D) Time-dependent inhibition of viral RNA synthesis or protein expression. MRC-5 cells were infected with HSV-1 for indicated times in the presence of Hsp90 inhibitor (0.8 µM). Total RNA or protein was extracted and analyzed for UL29 (C) and ICP8 expression (D). The results were expressed as the fold increase relative to the cell control. Each value represents the mean ± SD of three independent experiments (*, <i>P</i><0.05; and **, <i>P</i><0.01, compared with the viral control).</p

Inhibition of Hsp90 reduces ICP5 expression.

<p>(A) MRC-5 cells infected with HSV-1 (MOI = 10) for 1, 2, 4, or 6 h in the presence of Hsp90 inhibitors (0.8 µM) were harvested, lysed, and analyzed by Western blotting for ICP5 and total Hsp90 expression. The Western blotting results shown in the bar graph were normalized to GAPDH expression and were expressed as the fold increase relative to the cell control. (B) Western blot analysis of the expression of ICP5 and Hsp90 in cells transfected with Hsp90 siRNA. The Western blotting results shown in the bar graph were normalized to GAPDH expression and were expressed as the fold increase relative to the cell control. (C) Overexpression of Hsp90 restores the expression level of ICP5. MRC-5 cells transfected with the pEGFP-Hsp90α or pEGFP-N1 plasmid were infected with HSV-1 (MOI = 10) and treated with BJ-B11 (0.8 µM). (**, <i>P</i><0.01, compared with the viral control. ^##, <i>P</i><0.01, compared with the BJ-B11-treated group).</p

HSV-1 infection induces Hsp90 upregulation and nuclear translocation.

<p>(A) MRC-5 cells were infected with HSV-1 (MOI = 10) for 0, 1, 2, 4, or 6 h. The cells were then harvested, lysed, and analyzed for total Hsp90 expression. The Western blotting results shown in the line graph were normalized to GAPDH expression and were expressed as the fold increase relative to the cell control. Each value represents the mean ± SD of three independent experiments. (B) MRC-5 cells infected with HSV-1 (MOI = 10) for 4 h were fixed, permeabilized and stained for ICP5 (red), total Hsp90 (green), and nuclei (blue). (C) Interaction between ICP5 and Hsp90. Cells transfected with or without pEGFP-Hsp90 were lysed, immunoprecipitated with anti-Hsp90 antibody and probed with indicated antibodies. Non-capsid protein ICP27 was used as a negative control.</p

HSV-1 infection facilitates α-tubulin acetylation which is inhibited by Hsp90 inhibition.

<p>(A) Western blotting shows HSV-1 infection enhanced acetylation of α<b>-</b>tubulin. (B) Colocalization between Hsp90 and acetylated α-tubulin is reduced by Hsp90 inhibition. MRC-5 cells were infected with HSV-1 (MOI = 10) for 4 h in the presence or absence of Hsp90 inhibitors. The cells were then fixed, stained for acetylated α-tubulin (green), total Hsp90 (red), and nuclei (blue). (C, D) Hsp90 inhibitors (0.8 µM) inhibit HSV-1-induced acetylation of α-tubulin (C) and the interaction between Hsp90 and acetylated α-tubulin (D). Co-IP experiment shows a reduced interaction between Hsp90 and α-tubulin. Each value represents the mean ± SD of three independent experiments (*, <i>P</i><0.05; and **, <i>P</i><0.01, compared with the viral control).</p

Cytotoxicity, anti-HSV activity, and therapeutic index of Hsp90 inhibitors.

<p>Note: The values are the mean ± SD of three independent experiments.</p>a<p>The cytotoxic effect was determined by the MTT assay. CC₅₀ was defined as the concentration reducing cell viability by 50%.</p>b<p>The antiviral activity was determined by the plaque reduction assay. IC₅₀ was the concentration that inhibited 50% of HSV replication.</p>c<p>The therapeutic index (TI) was defined as the ratio of CC₅₀ to IC₅₀.</p