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 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 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

An autophagy-related protein Becn2 regulates cocaine reward behaviors in the dopaminergic system

Drug abuse is a foremost public health problem. Cocaine is a widely abused drug worldwide that produces various reward-related behaviors. The mechanisms that underlie cocaine-induced disorders are unresolved, and effective treatments are lacking. Here, we found that an autophagy-related protein Becn2 is a previously unidentified regulator of cocaine reward behaviors. Becn2 deletion protects mice from cocaine-stimulated locomotion and reward behaviors, as well as cocaine-induced dopamine accumulation and signaling, by increasing presynaptic dopamine receptor 2 (D2R) autoreceptors in dopamine neurons. Becn2 regulates D2R endolysosomal trafficking, degradation, and cocaine-induced behaviors via interacting with a D2R-bound adaptor GASP1. Inactivating Becn2 by upstream autophagy inhibitors stabilizes striatal presynaptic D2R, reduces dopamine release and signaling, and prevents cocaine reward in normal mice. Thus, the autophagy protein Becn2 is essential for cocaine psychomotor stimulation and reward through regulating dopamine neurotransmission, and targeting Becn2 by autophagy inhibitors is a potential strategy to prevent cocaine-induced behaviors

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

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