Farnesyltransferase inhibitor lonafarnib suppresses respiratory syncytial virus infection by blocking conformational change of fusion glycoprotein.

Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in young children and the elderly. There are currently no approved RSV-specific therapeutic small molecules available. Using high-throughput antiviral screening, we identified an oral drug, the prenylation inhibitor lonafarnib, which showed potent inhibition of the RSV fusion process. Lonafarnib exhibited antiviral activity against both the RSV A and B genotypes and showed low cytotoxicity in HEp-2 and human primary bronchial epithelial cells (HBEC). Time-of-addition and pseudovirus assays demonstrated that lonafarnib inhibits RSV entry, but has farnesyltransferase-independent antiviral efficacy. Cryo-electron microscopy revealed that lonafarnib binds to a triple-symmetric pocket within the central cavity of the RSV F metastable pre-fusion conformation. Mutants at the RSV F sites interacting with lonafarnib showed resistance to lonafarnib but remained fully sensitive to the neutralizing monoclonal antibody palivizumab. Furthermore, lonafarnib dose-dependently reduced the replication of RSV in BALB/c mice. Collectively, lonafarnib could be a potential fusion inhibitor for RSV infection.

Enterohemorrhagic Escherichia coli effector EspF triggers oxidative DNA lesions in intestinal epithelial cells.

Attaching/effacing (A/E) pathogens induce DNA damage and colorectal cancer by injecting effector proteins into host cells via the type III secretion system (T3SS). EspF is one of the T3SS-dependent effector proteins exclusive to A/E pathogens, which include enterohemorrhagic Escherichia coli. The role of EspF in the induction of double-strand breaks (DSBs) and the phosphorylation of the repair protein SMC1 has been demonstrated previously. However, the process of damage accumulation and DSB formation has remained enigmatic, and the damage response is not well understood. Here, we first showed a compensatory increase in the mismatch repair proteins MutS homolog 2 (MSH2) and MSH6, as well as poly(ADP-ribose) polymerase 1, followed by a dramatic decrease, threatening cell survival in the presence of EspF. Flow cytometry revealed that EspF arrested the cell cycle at the G2/M phase to facilitate DNA repair. Subsequently, 8-oxoguanine (8-oxoG) lesions, a marker of oxidative damage, were assayed by ELISA and immunofluorescence, which revealed the accumulation of 8-oxoG from the cytosol to the nucleus. Furthermore, the status of single-stranded DNA (ssDNA) and DSBs was confirmed. We observed that EspF accelerated the course of DNA lesions, including 8-oxoG and unrepaired ssDNA, which were converted into DSBs; this was accompanied by the phosphorylation of replication protein A 32 in repair-defective cells. Collectively, these findings reveal that EspF triggers various types of oxidative DNA lesions with impairment of the DNA damage response and may result in genomic instability and cell death, offering novel insight into the tumorigenic potential of EspF.IMPORTANCEOxidative DNA lesions play causative roles in colitis-associated colon cancer. Accumulating evidence shows strong links between attaching/effacing (A/E) pathogens and colorectal cancer (CRC). EspF is one of many effector proteins exclusive to A/E pathogens with defined roles in the induction of oxidative stress, double-strand breaks (DSBs), and repair dysregulation. Here, we found that EspF promotes reactive oxygen species generation and 8-oxoguanine (8-oxoG) lesions when the repair system is activated, contributing to sustained cell survival. However, infected cells exposed to EspF presented 8-oxoG, which results in DSBs and ssDNA accumulation when the cell cycle is arrested at the G2/M phase and the repair system is defective or saturated by DNA lesions. In addition, we found that EspF could intensify the accumulation of nuclear DNA lesions through oxidative and replication stress. Overall, our work highlights the involvement of EspF in DNA lesions and DNA damage response, providing a novel avenue by which A/E pathogens may contribute to CRC.

Development of a novel virus-like particle-based vaccine for preventing tick-borne encephalitis virus infection.

Tick-borne encephalitis virus (TBEV) is an important tick-borne pathogen that poses as a serious public health concern. The coverage and immunogenicity of the currently available vaccines against TBEV are relatively low; therefore, it is crucial to develop novel and effective vaccines against TBEV. The present study describes a novel strategy for the assembly of virus-like particles (VLPs) by co-expressing the structural (core/prM/E) and non-structural (NS2B/NS3Pro) proteins of TBEV. The efficacy of the VLPs was subsequently evaluated in C57BL/6 mice, and the resultant IgG serum could neutralize both Far-Eastern and European subtypes of TBEV. These findings indicated that the VLP-based vaccine elicited the production of cross-subtype reactive antibodies. The VLPs provided protection to mice lacking the type I interferon receptor (IFNAR-/-) against lethal TBEV challenge, with undetectable viral load in brain and intestinal tissues. Furthermore, the group that received the VLP vaccine did not exhibit significant pathological changes and the inflammatory factors were significantly suppressed compared to the control group. Immunization with the VLP vaccine induced the production of multiple-cytokine-producing antiviral CD4+ T cells in vivo, including TNF-α+, IL-2+, and IFN-γ+ T cells. Altogether, the findings suggest that noninfectious VLPs can serve as a potentially safe and effective vaccine candidate against diverse subtypes of TBEV.

Sterile 20-like kinase 3 promotes tick-borne encephalitis virus assembly by interacting with NS2A and prM and enhancing the NS2A-NS4A association.

Tick-borne encephalitis virus (TBEV) is the causative agent of a potentially fatal neurological infection in humans. Investigating virus-host interaction is important for understanding the pathogenesis of TBEV and developing effective antiviral drugs against this virus. Here, we report that mammalian ste20-like kinase 3 (MST3) is involved in the regulation of TBEV infection. The knockdown or knockout of MST3, but not other mammalian ste20-like kinase family members, inhibited TBEV replication. The knockdown of MST3 also significantly reduced TBEV replication in mouse primary astrocytes. Life cycle analysis indicated that MST3 remarkably impaired virion assembly efficiency and specific infectivity by respectively 59% and 95% in MST3-knockout cells. We further found that MST3 interacts with the viral proteins NS2A and prM; and MST3 enhances the interaction of NS2A-NS4A. Thus, MST3-NS2A complex plays a major role in recruiting prM-E heterodimers and NS4A and mediates the virion assembly. Additionally, we found that MST3 was biotinylated and combined with other proteins (e.g., ATG5, Sec24A, and SNX4) that are associated with the cellular membrane required for TBEV infection. Overall, our study revealed a novel function for MST3 in TBEV infection and identified as a novel host factor supporting TBEV assembly.

SARS-CoV-2 infection activates CREB/CBP in cellular cyclic AMP-dependent pathways.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global coronavirus disease 2019 (COVID-19) pandemic that has affected the lives of billions of individuals. However, the host-virus interactions still need further investigation to reveal the underling mechanism of SARS-CoV-2 pathogenesis. Here, transcriptomics analysis of SARS-CoV-2 infection highlighted possible correlation between host-associated signaling pathway and virus. In detail, cAMP-protein kinase (PKA) pathway has an essential role in SARS-CoV-2 infection, followed by the interaction between cyclic AMP response element binding protein (CREB) and CREB-binding protein (CBP) could be induced and leading to the enhancement of CREB/CBP transcriptional activity. The replication of Delta and Omicron BA.5 were inhibited by about 49.4% and 44.7% after knockdown of CREB and CBP with small interfering RNAs, respectively. Furthermore, a small organic molecule naphthol AS-E (nAS-E), which targets on the interaction between CREB and CBP, potently inhibited SARS-CoV-2 wild-type (WT) infection with comparable the half-maximal effective concentration (EC50 ) 1.04 µM to Remdesivir 0.57 µM. Compared with WT virus, EC50 in Calu-3 cells against Delta, Omicron BA.2, and Omicron BA.5 were, on average, 1.5-fold, 1.1-fold, and 1.5-fold higher, respectively, nAS-E had a satisfied antiviral effect against Omicron variants. Taken together, our study demonstrated the importance of CREB/CBP induced by cAMP-PKA pathway during SARS-CoV-2 infection, and further provided a novel CREB/CBP interaction therapeutic drug targets for COVID-19.

EspF of Enterohemorrhagic Escherichia coli Enhances Apoptosis via Endoplasmic Reticulum Stress in Intestinal Epithelial Cells: An Isobaric Tags for Relative and Absolute Quantitation-Based Comparative Proteomic Analysis.

There have been large foodborne outbreaks related to Enterohemorrhagic Escherichia coli (EHEC) around the world. Among its virulence proteins, the EspF encoded by locus of enterocyte effacement is one of the most known functional effector proteins. In this research, we infected the HT-29 cells with the EHEC wild type strain and EspF-deficient EHEC strain. Via the emerging technique isobaric tags for relative and absolute quantitation (iTRAQ), we explored the pathogenic characteristics of EspF within host cells. Our data showed that the differences regarding cellular responses mainly contained immune regulation, protein synthesis, signal transduction, cellular assembly and organization, endoplasmic reticulum (ER) stress, and apoptosis. Notably, compared with the EspF-deficient strain, the protein processing in the ER and ribosome were upregulated during wild type (WT) infection. Our findings proved that the EspF of Enterohemorrhagic Escherichia coli induced ER stress in intestinal epithelial cells; the ER stress-dependent apoptosis pathway was also activated within the host cells. This study provides insight into the virulence mechanism of protein EspF, which will deepen our general understanding of A/E pathogens and their interaction with host proteins.

An Escherichia coli Effector Protein EspF May Induce Host DNA Damage via Interaction With SMC1.

Enterohemorrhagic Escherichia coli (EHEC) O157: H7 is an important foodborne pathogen that causes human diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. EspF is one of the most important effector proteins injected by the Type III Secretion System. It can target mitochondria and nucleoli, stimulate host cells to produce ROS, and promote host cell apoptosis. However, the mechanism of the host-pathogen interaction leading to host oxidative stress and cell cytotoxic effects such as DNA damage remains to be elucidated. Here, we used Cell Counting Kit-8 (CCK-8) assays and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG) ELISA to study cell viability and DNA oxidative damage level after exposure to EspF. Western blot and immunofluorescence were also used to determine the level of the DNA damage target protein p-H2AX and cell morphology changes after EspF infection. Moreover, we verified the toxicity in intestinal epithelial cells mediated by EspF infection in vivo. In addition, we screened the host proteins that interact with EspF using CoIP-MS. We found that EspF may more depend on its C-terminus to interact with SMC1, and EspF could activate SMC1 phosphorylation and migrate it to the cytoplasm. In summary, this study revealed that EspF might mediate host cell DNA damage and found a new interaction between EspF and the DNA damage repair protein SMC1. Thus, EspF may mediate DNA damage by regulating the subcellular localization and phosphorylation of SMC1.

Prevalence of sexually transmitted infections among foreigners living in Guangzhou, China: a cross-sectional study (2010-2017).

BACKGROUND: The prevalence of HIV/HCV/HBV/ Treponema pallidum is an essential health issue in China. However, there are few studies focused on foreigners living in China. This study aimed to assess the prevalence and socio-demographic distribution of HIV, HBV, HCV, and T. pallidum among foreigners in Guangzhou in the period of 2010-2017. METHODS: A cross-sectional study was conducted to screen serological samples of 40,935 foreigners from 2010 to 2017 at the Guangdong International Travel Health Care Center in Guangzhou. Samples were tested for hepatitis B surface antigen (HBsAg), anti-HCV, syphilis antibody (anti-TPPA) and anti-HIV 1 and 2. We collected secondary data from laboratory records and used multiple logistic regression analyses to verify the association between different factors and the seroprevalence of HIV/HBV/HCV/ T. pallidum. RESULTS: The prevalence of HBV/HCV/HIV/ T. pallidum was 2.30, 0.42, 0.02, and 0.60%, respectively, and fluctuated slightly for 7 years. The results of multiple logistic regression showed that males were less susceptible to HBV than females (odds ratio [OR] = 0.77, 95% CI: 0.67-0.89). Participants under the age of 20 had a lower risk of HBV (OR = 0.25, 95% CI: 0.18-0.35), HCV (OR = 0.06, 95% CI: 0.02-0.18), and T. pallidum (OR = 0. 10, 95% CI: 0.05-0.20) than participants over the age of 50. Participants with an education level below high school were more likely to have HBV (OR = 2.98, 95% CI: 1.89-4.70) than others, and businessmen (OR = 3.02, 95% CI: 2.03-4.49), and designers (OR = 3.83, 95% CI: 2.49-5.90) had a higher risk of T. pallidum than others. Co-infection involved 58 (4.20%) total cases, and the highest co-infection rate was observed for HBV and T. pallidum (2.60%). CONCLUSION: The prevalence of HBV/HCV/HIV/ T. pallidum was low among foreigners in Guangzhou. Region, gender, age, educational level, and occupation were risk factors for positive infection.

Enterohemorrhagic Escherichia coli Effector Protein EspF Interacts With Host Protein ANXA6 and Triggers Myosin Light Chain Kinase (MLCK)-Dependent Tight Junction Dysregulation.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen that can cause bloody diarrhea and hemolytic uremic syndrome (HUS) in humans. EspF is one of the best-characterized effector proteins secreted from the type three secretion system to hijack host cell functions. However, the crucial pathogen-host interactions and the basis for the intestinal barrier disruption during infections remain elusive. Our previous study screened and verified the interaction between host protein ANXA6 and EspF protein. Here, by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (CO-IP), we verified that EspF interacts with ANXA6 through its C-terminal domain. Furthermore, we found that both the constitutive expression of EspF or ANXA6 and the co-expression of EspF-ANXA6 could decrease the levels of tight junction (TJ) proteins ZO-1 and occludin, and disrupt the distribution of ZO-1. Moreover, we showed that EspF-ANXA6 activated myosin light chain kinase (MLCK), induced the phosphorylation of myosin light chain (MLC) and PKCα, and down-regulated the expression level of Calmodulin protein. Collectively, this study revealed a novel interaction between the host protein (ANXA6) and EspF. The binding of EspF to ANXA6 may perturb TJs in an MLCK-MLC-dependent manner, and thus may be involved in EHEC pathogenic function.

The EspF N-Terminal of Enterohemorrhagic Escherichia coli O157:H7 EDL933w Imparts Stronger Toxicity Effects on HT-29 Cells than the C-Terminal.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 EspF is an important multifunctional protein that destroys the tight junctions of intestinal epithelial cells and promotes host cell apoptosis. However, its molecular mechanism remains elusive. We knocked out the espF sequence (747 bp, ΔespF), N-terminal sequence (219 bp, ΔespFN ), and C-terminal sequence (528 bp, ΔespFC ) separately using the pKD46-mediated λ Red homologous recombination system. Then, we built the corresponding complementation strains, namely, ΔespF/pespF, ΔespFN/pespFN , and ΔespFC/pespFC by overlap PCR, which were used in infecting HT-29 cells and BALB/C mice. The level of reactive oxygen species, cell apoptosis, mitochondrial trans-membrane potential, inflammatory factors, transepithelial electrical resistance (TER), and animal mortality were evaluated by DCFH-DA, double staining of Annexin V-FITC/PI, JC-1 staining, ELISA kit, and a mouse assay. The wild-type (WT), ΔespF, ΔespF/pespF, ΔespFC , ΔespFC/pespFC , ΔespFN , and ΔespFN/pespFN groups exhibited apoptotic rates of 68.3, 27.9, 64.9, 65.7, 73.4, 41.3, and 35.3% respectively, and mean TNF-α expression levels of 428 pg/mL, 342, 466, 446, 381, 383, and 374 pg/mL, respectively. In addition, the apoptotic rates and TNF-α levels of the WT, ΔespF/pespF, and ΔespFC were significantly higher than that of ΔespF, ΔespFN , ΔespFC/pespFC , and ΔespFN/pespFN group (p < 0.05). The N-terminal of EspF resulted in an increase in the number of apoptotic cells, TNF-α secretion, ROS generation, mitochondria apoptosis, and pathogenicity in BalB/c mice. In conclusion, the N-terminal domain of the Enterohemorrhagic E. coli O157:H7 EspF more strongly promotes apoptosis and inflammation than the C-terminal domain.