To replicate, spread and persist in the host environment, viruses have evolved several immunological escape mechanisms via the action of specific viral proteins. The model “host shut off” adopted by virion host shut off (VHS) protein of Herpes simplex type 1 (HSV-1) represents an immune evasion mechanism which affects the best-characterized component of the innate immunological response, protein kinase R (PKR). However, up to now, the real mechanism employed by VHS to control PKR is still unknown. In this paper, we implement and extend our previous findings reporting that wild-type HSV-1 is able to control PKR, whereas a VHS mutant virus (R2621) clearly induces an accumulation of phosphorylated PKR in several cell types in a VHS-RNase activity-dependent manner. Furthermore, we demonstrate for the first time a new PKR-regulatory mechanism based on the involvement of Us3 and UL13 tegument viral proteins. The combined approach of transfection and infection assay was useful to discover the new role of both viral proteins in the immunological escape and demonstrate that Us3 and UL13 control the accumulation of the phosphorylated form (ph-PKR). Lastly, since protein kinases are tightly regulated by phosphorylation events and, at the same time, phosphorylate other proteins by inducing post-translational modifications, the interplay between Us3 and VHS during HSV-1 infection has been investigated. Interestingly, we found that VHS protein accumulates at higher molecular weight following Us3 transfection, suggesting an Us3-mediated phosphorylation of VHS. These findings reveal a new intriguing interplay between viral proteins during HSV-1 infection involved in the regulation of the PKR-mediated immune response.

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Depression has a high incidence and seriously endangers human health. Accumulated evidence indicates that targeting neuroinflammation is a potential avenue for neuroprotection and thus depression prevention. Herein, the effects of latroeggtoxin-VI (LETX-VI), a bioactive protein from the eggs of spider Latrodectus tredecimguttatus, on lipopolysaccharide (LPS)-induced inflammation and depression were systematically investigated using RAW264.7 macrophages and depression mouse model. Pretreatment with LETX-VI suppressed LPS-evoked NF-κB signaling pathway activation, inhibited LPS-induced over-production of NO, iNOS, IL-6 and TNF-α; at the same time LETX-VI mitigated the inhibitory effect of LPS on the expression of anti-inflammatory factors such as Arg-1, thereby suppressing oxidative stress and excessive inflammation. Culture of PC12 cells with the conditioned medium of RAW264.7 cells pretreated with LETX-VI demonstrated the neuroprotective effect of LETX-VI due to its anti-inflammation effect. In the LPS-induced depression mouse model, pretreatment with LETX-VI improved the LPS-induced depression-like behaviors, inhibited the activation of microglia and astrocytes, prevented the down-regulation of Nurr1 expression and alleviated the LPS-caused adverse changes in the brain tissues. Taken together, these in vitro and in vivo findings provide powerful insights into the anti-inflammation-based neuroprotective and antidepressant mechanisms of LETX-VI, which is helpful to deeply reveal the biological effects and potential applications of LETX-VI.

Latroeggtoxin‐VI (LETX‐VI) is a peptide neurotoxin discovered from Latrodectus tredecimguttatus eggs. In the current study, the action features of the neurotoxin on PC12 cells were systematically investigated. LETX‐VI could promote dopamine release from PC12 cells in the absence and presence of Ca2+, involving an even more complex action mechanism in the presence of Ca2+ and when the treatment time was longer. Although LETX‐VI enchanced the autophagy and secretion activity in PC 12 cells, it showed no remarkable influence on the proliferation, cell cycle, apoptosis and ultrastructure of the cells. Pulldown combined with CapLC‐MS/MS analysis suggested that LETX‐VI affected PC12 cells by interacting with multiple proteins involved in the metabolism, transport, and release of neurotransmitters, particularly dopamine. The low cytotoxicity and effective regulatory action of LETX‐VI on PC12 cells suggest the potential of the active peptide in the development of drugs for the treatment of some dopamine‐related psychotic diseases and cancers.

Latroeggtoxin-VI (LETX-VI) is a peptide neurotoxin newly found from the eggs of spider L. tredecimguttatus. To explore the mechanism of action of the LETX-VI on nerve cells, the effects of LETX-VI on PC12 cells, a commonly used neuron model, were analyzed using a pull-down assay-guided strategy. LETX-VI was shown to interact with 164 PC12 cell proteins that have diverse molecular functions such as binding, catalysis, regulation, structural activity, etc., thereby extensively affecting the biological processes in the PC12 cells, particularly protein metabolism, response to stimulus, substance transport, and nucleic acid metabolism, with 56.71%, 42.07%, 29.88% and 28.66% of the identified proteins being involved in these biological processes, respectively. By interacting with the relevant proteins, LETX-VI enhanced the synthesis of dopamine; positively regulated cell division and proliferation; and negatively regulated cell cycle arrest, cell death, and apoptotic processes, and therefore has limited cytotoxicity against the PC12 cells, which were further experimentally confirmed. In general, the effects of LETX-VI on PC12 cells are more regulatory than cytotoxic. These findings have deepened our understanding of the action mechanism of LETX-VI on nerve cells and provided valuable clues for further related researches including those on Parkinson’s disease.