Qiaoli Wang

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

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 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. IMPORTANCE The actin cytoskeleton is involved in many crucial cellular processes and acts as an obstacle to pathogen entry into host cells. Because HSV-1 establishes lifelong latency in neurons and because neuronal cytoskeletal disruption is thought to be the main cause of HSV-1-induced neurodegeneration, understanding the F-actin remodeling pattern by HSV-1 infection and the molecular interactions that facilitate HSV-1 entry into neurons is important. In this study, we showed that HSV-1 infection induces the rearrangement of the cytoskeleton as well as the initial inactivation and subsequent activation of cofilin. Then, we determined that activation of the EGFR-PI3K-Erk1/2 signaling pathway inactivates cofilin and promotes F-actin polymerization. We postulate that by regulating actin cytoskeleton dynamics, cofilin biphasic activation could represent the specific cellular machinery usurped by pathogen infection, and these results will greatly contribute to the understanding of HSV-1-induced early and complex changes in host cells that are closely linked to HSV-1 pathogenesis. The actin cytoskeleton is involved in many crucial cellular processes and acts as an obstacle to pathogen entry into host cells. Because HSV-1 establishes lifelong latency in neurons and because neuronal cytoskeletal disruption is thought to be the main cause of HSV-1-induced neurodegeneration, understanding the F-actin remodeling pattern by HSV-1 infection and the molecular interactions that facilitate HSV-1 entry into neurons is important. In this study, we showed that HSV-1 infection induces the rearrangement of the cytoskeleton as well as the initial inactivation and subsequent activation of cofilin. Then, we determined that activation of the EGFR-PI3K-Erk1/2 signaling pathway inactivates cofilin and promotes F-actin polymerization. We postulate that by regulating actin cytoskeleton dynamics, cofilin biphasic activation could represent the specific cellular machinery usurped by pathogen infection, and these results will greatly contribute to the understanding of HSV-1-induced early and complex changes in host cells that are closely linked to HSV-1 pathogenesis.

Cofilin 1-Mediated Biphasic F-Actin Dynamics of Neuronal Cells Affect Herpes Simplex Virus 1 Infection and Replication

ABSTRACT Herpes simplex virus 1 (HSV-1) invades the nervous system and causes pathological changes. In this study, we defined the remodeling of F-actin and its possible mechanisms during HSV-1 infection of neuronal cells. HSV-1 infection enhanced the formation of F-actin-based structures in the early stage of infection, which was followed by a continuous decrease in F-actin during the later stages of infection. The disruption of F-actin dynamics by chemical inhibitors significantly reduced the efficiency of viral infection and intracellular HSV-1 replication. The active form of the actin-depolymerizing factor cofilin 1 was found to increase at an early stage of infection and then to continuously decrease in a manner that corresponded to the remodeling pattern of F-actin, suggesting that cofilin 1 may be involved in the biphasic F-actin dynamics induced by HSV-1 infection. Knockdown of cofilin 1 impaired HSV-1-induced F-actin assembly during early infection and inhibited viral entry; however, overexpression of cofilin 1 did not affect F-actin assembly or viral entry during early infection but decreased intracellular viral reproduction efficiently. Our results, for the first time, demonstrated the biphasic F-actin dynamics in HSV-1 neuronal infection and confirmed the association of F-actin with the changes in the expression and activity of cofilin 1. These results may provide insight into the mechanism by which HSV-1 productively infects neuronal cells and causes pathogenesis.

Proteomics analysis of autophagy-deficient Atg7−/− MEFs reveals a close relationship between F-actin and autophagy

Autophagy plays a crucial role in a wide array of physiological processes. To uncover the complex regulatory networks and mechanisms underlying basal autophagy, we performed a quantitative proteomics analysis of autophagy-deficient mouse embryonic fibroblast cells (MEFs) using iTRAQ labeling coupled with on-line 2D LC/MS/MS. We quantified a total of 1234 proteins and identified 114 proteins that were significantly altered (90% confidence interval), including 48 up-regulated proteins and 66 down-regulated proteins. We determined that F-actin was disassembled in autophagy-deficient Atg7(-/-) MEFs. Treatment of the WT MEFs with cytochalasin D (CD), which induces F-actin depolymerization, significantly induced autophagosome formation. However, treatment with cytochalasin D also increased the protein level of p62 under starvation conditions, suggesting that depolymerization of F-actin impaired autophagosome maturation and that the intact F-actin network is required for basal and starvation-induced autophagy. Our results demonstrate a close relationship between F-actin and autophagy and provide the basis for further investigation of their interactions.

Synthesis and in vitro anti-HSV-1 activity of a novel Hsp90 inhibitor BJ-B11

In this study, a novel Hsp90 inhibitor BJ-B11, was synthesized and evaluated for in vitro antiviral activity against several viruses. Possible anti-HSV-1 mechanisms were also investigated. BJ-B11 displayed no antiviral activity against coxsackievirus B(3) (CVB(3)), human respiratory syncytial virus (RSV) and influenza virus (H1N1), but exhibited potent anti-HSV-1 and HSV-2 activity with EC(50) values of 0.42±0.18 μM and 0.60±0.21 μM, respectively. Additionally, the inhibitory effects of BJ-B11 against HSV-1 were likely to be introduced at early stage of infection. Our results indicate that BJ-B11 with alternative mechanisms of action is potent as an anti-HSV clinical trial candidate.

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.

In vitro and in vivo anti-inflammatory effects of theaflavin-3,3′-digallate on lipopolysaccharide-induced inflammation

&NA; Inflammation is a defensive response against various harmful stimuli and stress conditions, such as tissue injury and one of the most common pathological processes occurring in human diseases. Theaflavin‐3,3′‐digallate, one of the theaflavins present in black tea, exhibits several bioactive properties, including the ability to lower the incidence of coronary heart disease, a positive effect on the bone mineral density, and the ability to prevent cancer. The aim of this study was to evaluate whether theaflavin‐3,3′‐digallate could reduce the production of pro‐inflammatory cytokines in vivo and in vitro and ameliorate acute lung injury (ALI) in a mouse model. In this study, we demonstrated that theaflavin‐3,3′‐digallate suppressed the lipopolysaccharide (LPS)‐induced phosphorylation of c‐Jun N‐terminal kinase and p38 mitogen‐activated protein kinase in RAW 264.7 macrophages. In addition, we also showed that theaflavin‐3,3′‐digallate inhibited the expression of tumor necrosis factor alpha, interleukin ‐1 beta, and interleukin 6 in phorbol myristate acetate ‐primed U937 and RAW 264.7 cells. Furthermore, theaflavin‐3,3′‐digallate treatment attenuated the severity of LPS‐induced ALI in mice. These results suggested that theaflavin‐3,3′‐digallate might be a potential therapeutic candidate for the treatment of inflammation and inflammatory diseases.

Silencing Herpes Simplex Virus Type 1 Capsid Protein Encoding Genes by siRNA: A Promising Antiviral Therapeutic Approach

Herpes simplex virus type 1 (HSV-1), a member of the herpesviridae, causes a variety of human viral diseases globally. Although a series of antiviral drugs are available for the treatment of infection and suppression of dissemination, HSV-1 remains highly prevalent worldwide. Therefore, the development of novel antiviral agents with different mechanisms of action is a matter of extreme urgency. During the proliferation of HSV-1, capsid assembly is essential for viral growth, and it is highly conserved in all HSV-1 strains. In this study, small interfering RNAs (siRNAs) against the HSV-1 capsid protein were screened to explore the influence of silencing capsid expression on the replication of HSV-1. We designed and chemically synthesized siRNAs for the capsid gene and assessed their inhibitory effects on the expression of target mRNA and the total intracellular viral genome loads by quantitative real-time PCR, as well as on the replication of HSV-1 via plaque reduction assays and electron microscopy. Our results showed that siRNA was an effective approach to inhibit the expression of capsid protein encoding genes including UL18, UL19, UL26, UL26.5, UL35 and UL38 in vitro. Interference of capsid proteins VP23 (UL18) and VP5 (UL19) individually or jointly greatly affected the replication of clinically isolated acyclovir-resistant HSV-1 as well as HSV-1/F and HSV-2/333. Plaque numbers and intracellular virions were significantly reduced by simultaneous knockdown of UL18 and UL19. The total intracellular viral genome loads were also significantly decreased in the UL18 and UL19 knockdown groups compared with the viral control. In conclusion, interfering with UL18 and UL19 gene expression could inhibit HSV-1 replication efficiently in vitro. Our research offers new targets for an RNA interference-based therapeutic strategy against HSV-1.

Long-Term Survival of AIDS Patients Treated with Only Traditional Chinese Medicine

Abstract Traditional Chinese herbal medicine (TCM) has been used in Chinese society for more than 5,000 years to treat diseases from inflammation to cancer. Here, we report the case of nine living AIDS patients in the age range of 51 to 67 who were treated with either a unique formula of TCM alone from 2001 to 2009 or the TCM from 2001 to 2006 and then switched to occasional antiretroviral therapy. Surprisingly, the viral loads of eight patients were at undetectable levels on June 28, 2016, while the remaining patient had a low viral load of 29 copies/ml. The CD4+ counts (170–592 cells/μl) and CD4+/CD8+ ratios (0.21–0.90) of the nine patients are excellent, contributing to their current good health. Thus, the case study suggests that the TCM has the potential to become a functional cure for HIV/AIDS.

Ubiquitin-proteasome-dependent slingshot 1 downregulation in neuronal cells inactivates cofilin to facilitate HSV-1 replication

Actin and its regulators are critical for neuronal function. Infection with herpes simplex virus 1 (HSV-1) remodels neuronal cell actin dynamics, which may relate virus-induced pathological processes in the nervous system. We previously demonstrated that cofilin is an actin regulator that participates in HSV-1-induced actin dynamics in neuronal cells, but how HSV-1 regulates cofilin has remained unclear. In the present study, we demonstrated the HSV-1-induced the inactivation of cofilin and the accumulation of phosphorylated cofilin in the nucleus, which together benefited viral replication. This consistent cofilin inactivation was achieved by the downregulation of slingshot 1 (SSH1). Notably, virus-induced SSH1 downregulation depended on the ubiquitin-proteasome system. Cofilin inactivation is therefore critical for HSV-1 replication during neuronal infection and is maintained by SSH1 downregulation. Moreover, these results provide new insight into the HSV-1-induced neurological pathogenesis and suggest potential new strategies to inhibit HSV-1 replication.

Calcium-signal facilitates herpes simplex virus type 1 nuclear transport through slingshot 1 and calpain-1 activation.

Herpes simplex virus type 1 (HSV-1) can establish its latency in neurons and is associated with virus-induced pathological neurodegeneration in the nervous system. Here we show that viral penetration-induced calcium release facilitated HSV-1 intracellular trafficking through activating slingshot 1 (SSH), a phosphatase regulating actin filament dynamics. More detailed studies revealed that phospholipase C gamma 1, and the inositol 1,4,5-trisphosphate receptor isoform 1 were required for SSH activation. Besides, calpain-1, a calcium-dependent cysteine protease, was involved in viral intracellular migration. These results may lead to new targets for antiviral therapy.