Role For Cytoskeleton And MiRNA In HSV-1Infection And Antiviral Drugs Research

Background: Herpes simplex virus type1(HSV-1) infection is very commonworldwide and can cause significant health problems from periodic skin and corneallesions to encephalitis. HSV-1is neurotropic and can establish its latency infection incentral nerves system and reactivate when immune system is malfunction. Besides,infection of HSV-1is tightly correlated to the origination and development of severalneurodegenerative diseases such as Alzheimer disease and it also act as a pathogeniccofactor for HIV infection. Cellular cytoskeleton is such a skeleton network thatmodulates cell motivation, shape, and differentiation and signaling delivery ofneuronal cells, dysfunctional regulation of which may cause several neurologicdiseases. Our previous works had found that HSV-1interacted with cytoskeletonduring its life cycle. However, the mechanism by which HSV-1regulates hostcytoskeleton rearrangement and the role for cytoskeleton in virus entry into neuronalcells remain uncertain.Research contents and results: In this thesis, we for the first time investigatedthe regulatory pattern of cytoskeleton during the process of HSV-1entry into neuronalcells. It was found that HSV-1entry induces biphasic dynamics of F-actin (firstpolymerization then depolymerization), which is associated with the on-off switch ofcofilin activity (dephosphorylation and phosphorylation). Modulation of cofilinexpression and activity inhibited virus entry. More detailed studies revealed thatbinding of HSV-1to its membrane receptor in lipid rafts activated a signaling pathwayas EGFR-PI3K-Erk1/2-ROCK-LIMK to phosphorylate cofilin and led to F-actinpolymerization, which finally facilitated the membrane clustering of viral receptor sand efficient viral penetration. Besides, during HSV-1penetration, PLC and IP3R-1were activated through an unknown mechanism, led to the elevation of intracellularcalcium concentration. As a result, phosphatases Slingshot and Calpain-1were both activated to dephosphorylate cofilin and sever polymerizated F-actin, which therebyreleased viral particle from cell cortex and promoted its nuclear transport. It was alsofound that ubiquitin-proteasome-dependent Slingshot downregulation in neuronalcells inactivates cofilin to facilitate HSV-1replication.In addition, during HSV-1productive infection, several virus-encoded miRNAswere produced including miR-H1, expression of which is the largest with an unknownrole and unclarified target protein. In this thesis, by a combination of bioinformaticprediction and quantitative real-time PCR-based functional screening, ubiquitinprotein ligase E3component n-recognin1(UBR1) was found to be targeted bymiR-H1. Ubr1can recognize several viral protein and neurodegeneration associatedprotein fragments (e.g. β-amyloid), protein markers of Alzheimer disease, andmediate their degradation through proteasome pathway and its expression wasdownregulated by miR-H1during HSV-1infection. Those effects of miR-H1mayexplain the importance between HSV-1and neurodegeneration diseases. Moreover,antiviral effect of chloride channels inhibitors tamoxifen and NPPB was investigated.Both tamoxifen and NPPB showed a significant antiviral effect toward standardHSV-1or ACV-resistant HSV strains. Early infection of HSV-1increased theconcentration of intracellular chloride ion and tamoxifen and NPPB inhibited viralgene synthesis and protein expression. Several steps of viral life cycle, especially theentry step of HSV-1, were perturbed by the compounds. Tamoxifen and NPPBprevented viral binding, penetration and intracellular migration possibly throughreducing membrane lipid rafts location of virus and interrupting the calcium ion influxinduced by HSV-1penetration.Research Conclusion and significance: EGFR-PI3K signaling pathway andCalcium signals sequentially control cofilin activity to facilitate HSV-1entry intoneuronal cells through biphasic dynamic regulation of F-actin. Besides, HSV-1encoded miRNA miR-H1was largely expressed to downregulate Ubr1and therebyprotected viral proteins from degradation and promoted late gene expression. Inaddition, chloride channels inhibitors tamoxifen and NPPB exhibited a significant antiviral activity. Those results will help to understand the interaction betweenvirus and the host cells and lead to a greater understanding of viral entry andpathogenesis of HSV-1-induced neurological diseases or help the development ofmore efficient oncolytic virotherapy. Furthermore, targeting the cell host chloridechannel, as well as identifying approved drugs and probes with previouslyundocumented antiviral activity, is promising approaches in the development ofantiviral therapies to HSV infection.