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Polymyxin is the “last resort” of antibiotics. The self-induced resistance to polymyxin in Gram-negative bacteria could be mediated by lipopolysaccharide (LPS) modification, which is regulated by the two-component system, PhoP/PhoQ. Yersinia enterocolitica is a common foodborne pathogen. However, PhoP/PhoQ has not been thoroughly studied in Y. enterocolitica. In this study, the functions of PhoP/PhoQ in Y. enterocolitica intrinsic resistance were investigated. The resistance of Y. enterocolitica was found to decrease with the deletion of PhoP/PhoQ. Further, PhoP/PhoQ was found to play an important role in maintaining membrane permeability, intercellular metabolism, and reducing membrane depolarization. Based on subsequent studies, the binding ability of polymyxin to Y. enterocolitica was decreased by the modification of LPS with structures, such as L-Ara4N and palmitate. Analysis of the gene transcription levels revealed that the LPS modification genes, pagP and arn operon, were downregulated with the deletion of PhoP/PhoQ in Y. enterocolitica during exposure to polymyxin. In addition, pmrA, pmrB, and eptA were downregulated in the mutants compared with the wild-type strain. Such findings demonstrate that PhoP/PhoQ contributes to the intrinsic resistance of Y. enterocolitica toward polymyxins. LPS modification with L-Ara4N or palmitate is mainly responsible for the resistance of Y. enterocolitica to polymyxins. The transcription of genes related to LPS modification and PmrA/PmrB can be both affected by PhoP/PhoQ in Y. enterocolitica. This study adds to current knowledge regarding the role of PhoP/PhoQ in intrinsic resistance of Y. enterocolitica to polymyxin.

Enterovirus D68 (EV-D68) is a respiratory viral pathogen that causes severe respiratory diseases and neurologic manifestations. Since the 2014 outbreak, EV-D68 has been reported to cause severe complications worldwide. However, there are currently no approved antiviral agents or vaccines for EV-D68. In this study, we found that zinc ions exerted substantial antiviral activity against EV-D68 infection in vitro. Zinc salt treatment potently suppressed EV-D68 RNA replication, protein synthesis, and infectious virion production and inhibited cytopathic effects without producing significant cytotoxicity at virucidal concentrations (EC50=0.033mM). Zinc chloride (ZnCl2) treatment moderately inhibited EV-D68 attachment. Time-dose analysis of EV-D68 structural protein VP1 synthesis showed stronger suppression of VP1 in the culture medium than that in the cell lysates. Furthermore, a zinc ionophore, pyrrolidine dithiocarbamate, which can transport zinc ions into cells, also enhanced the anti-EV-D68 activity of ZnCl2 treatment. Taken together, our results demonstrated that the enhancement of zinc influx could serve as a powerful strategy for the therapeutic treatment of EV-D68 infections.

Long interspersed element 1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome that can serve as an endogenous upstream activator of cytoplasmic nucleic acid sensing pathways to elicit an antiviral immune response. In this study, we investigated the influence of enteroviral infection on L1 mobility. The results showed that infection with different enteroviruses, both EV-D68 and EV-A71, blocked L1 transposition. We screened diverse viral accessory proteins for L1 activity and identified EV-D68 2A, 3A, 3C, and EV-A71 ORF2p proteins as viral L1 inhibitors. EV-D68 2A suppressed L1 mobility by expression suppression of L1 proteins. Viral proteins 3A and 3C restricted ORF2p-mediated L1 reverse transcription in isolated L1 ribonucleoproteins. The newly identified enteroviral protein ORF2p inhibited the expression of L1 ORF1p. Altogether, our findings shed light on the strict modulation of L1 retrotransposons during enterovirus replication.

High-risk Human papillomaviruses (HPVs) types are associated with more than 90% of premalignant and malignant squamous lesions of the uterine cervix. The E6 oncoprotein of high-risk HPVs is a key determinant in cell transformation because it induces the degradation of the host pro-apoptotic tumor suppressor p53. E6 recruits the intracellular ubiquitin ligase E6AP and subsequently induces proteasome-dependent p53 degradation. Neither E6 nor E6AP alone interact with p53; however, the precise mechanism of the functional regulation of the E6/E6AP/p53 complex is unclear. Here, we showed that the high-risk HPV E6 proteins are ubiquitinated during E6/E6AP/p53 complex assembly and degraded by the proteasome system. Increasing p53 expression enhanced E6/E6AP/p53 assembly and facilitated E6 ubiquitination and degradation. The dominant negative mutant of p53 R175H, which does not efficiently bind E6, decreased E6 ubiquitination and increased stability. Furthermore, we showed that the ubiquitin ligase E6AP is essential for E6 ubiquitination, and downregulation of E6AP expression increased E6 stability. We also showed that p53 R175H inhibited E6-mediated p53 degradation. Consistently, the host deubiquitinating enzyme USP15 removed ubiquitin chains from E6 proteins and inhibited E6-mediated p53 degradation. Crucially, ectopic expression of either p53 R175H or USP15 promoted p53-triggered apoptosis in human cervical cancer cells. Considering the importance of ubiquitinated E6 on p53 degradation, the disruption of E6 ubiquitination represents an attractive pharmacological intervention against HPV-positive human cervical cancer.ImportanceVirtually 100% of cervical cancers are linked to HPV infection. Commercial HPV vaccines are estimated to prevent up to 90% of HPV-associated cancers, while they do not eliminate persistent HPV infections and have no effect on the progression to malignancy. Hence, the development of novel therapeutic interventions against HPV is urgently required. The HPV oncoprotein E6 binds to the intracellular E3 ubiquitin ligase E6AP and p53 resulting in the degradation of p53. In this study, we demonstrate that HPV E6 is ubiquitinated by E6AP in presence of p53. Crucially, ubiquitination of E6 is important for p53 degradation and blockage of E6 ubiquitination negatively interferes with E6-mediated p53 degradation and enhances the apoptotic effects of p53 and the cytotoxicity of DNA damage in HPV-positive cervical cancer cells. Importantly, our data suggest that the HPV oncogene E6 might be an optimal pharmacologic.

Enterovirus D68 (EV-D68) has emerged as a significant respiratory pathogen that can cause severe respiratory disease and acute neurologic disease. At present, there are no approved antiviral agents or vaccines for EV-D68. In this study, we demonstrate that andrographolide (ADO), an active component of Andrographis paniculata, exerts substantial antiviral activity against EV-D68 infection. ADO treatment dramatically inhibited EV-D68 RNA replication (EC50 = 3.45 μM) and protein synthesis without producing significant cytotoxicity at virucidal concentrations. ADO-treated cells did not show any changes in host immune activation, EV-D68 attachment, or viral 5′ UTR activity. Using a pH-sensitive fluorescent indicator system for endocytosis in living cells, we found that ADO prevented the acidification of endocytic vesicles after receptor-mediated endocytosis. Finally, we showed that ADO inhibited the viral replication of circulating isolated EV-D68 strains. In summary, our results demonstrate that ADO suppresses EV-D68 replication by targeting the maturation of virus-containing endosomes of EV-D68. This mechanism represents a promising strategy for drug development.