Featured interactome of homocysteine-inducible endoplasmic reticulum protein uncovers novel binding partners in response to ER stress.

Homocysteine-inducible endoplasmic reticulum protein (HERP) is an endoplasmic reticulum (ER)-resident protein and important for the adaptation of cellular protein homeostasis by ER-associated degradation (ERAD) system. HERP interactors are critical for cellular viability and the reaction to ER stress. To explore the exact mechanisms by which HERP performed the biological functions, we conducted an interaction analysis of HERP protein in HeLa cells by co-immunoprecipitation (Co-IP) and liquid chromatography-mass spectrometer (LC-MS)/MS coupled with label-free quantification (LFQ). Among the interactome results, 123 proteins significantly interacted with HERP, which leads to numerous biological processes including protein import into nucleus, ubiquitin-dependent ERAD pathway, negative regulation of apoptotic process, and protein transport from ER, along with multiple pathways including several diseases, protein processing in ER, fatty acid metabolism, and steroid biosynthesis. Furthermore, we selected several prey proteins from the interactome data and confirmed that HERP interacted with ancient ubiquitous protein 1 (AUP1), Fas-associated factor family member 2 (FAF2), tripartite motif containing 47 (TRIM47), acyl-CoA synthetase long-chain family member 3 (ACSL3), sequestosome 1 (SQSTM1), and poly(rC) binding protein 2 (PCBP2) by Co-IP and confocal microscopy experiments, respectively. Moreover, the expression and location of several interacted proteins were obviously altered in response to ER stress induced by Thapsigargin stimulation and Enterovirus 71 infection. In conclusion, our findings revealed that the vital proteins interacted with HERP to mediate signaling transduction, thus providing novel clues for the mechanisms of HERP associated with ERAD and metabolism in response to ER stress under physiological and pathological conditions.

Inhibition of PIKFYVE kinase interferes ESCRT pathway to suppress RNA virus replication.

Endosomal sorting complex required for transport (ESCRT) is essential in the functional operation of endosomal transport in envelopment and budding of enveloped RNA viruses. However, in nonenveloped RNA viruses such as enteroviruses of the Picornaviridae family, the precise function of ESCRT pathway in viral replication remains elusive. Here, we initially evaluated that the ESCRT pathway is important for viral replication upon enterovirus 71 (EV71) infection. Furthermore, we discovered that YM201636, a specific inhibitor of phosphoinositide kinase, FYVE finger containing (PIKFYVE) kinase, significantly suppressed EV71 replication and virus-induced inflammation in vitro and in vivo. Mechanistically, YM201636 inhibits PIKFYVE kinase to block the ESCRT pathway and endosomal transport, leading to the disruption of viral entry and replication complex in subcellular components and ultimately repression of intracellular RNA virus replication and virus-induced inflammatory responses. Further studies found that YM201636 broadly represses the replication of other RNA viruses, including coxsackievirus B3 (CVB3), poliovirus 1 (PV1), echovirus 11 (E11), Zika virus (ZIKV), and vesicular stomatitis virus (VSV), rather than DNA viruses, including adenovirus 3 (ADV3) and hepatitis B virus (HBV). Our findings shed light on the mechanism underlying PIKFYVE-modulated ESCRT pathway involved in RNA virus replication, and also provide a prospective antiviral therapy during RNA viruses infections.

Enterovirus 71 non-structural protein 3A hijacks vacuolar protein sorting 25 to boost exosome biogenesis to facilitate viral replication.

Human enterovirus 71 (EV71) is one of the major agents of the hand, foot, and mouth disease (HFMD), and occasionally causes severe neurological complications. There is clinical evidence that EV71 infection increases the exosomes in the serum of severe HFMD patients, suggesting a role of exosomes in EV71 pathogenesis. However, the relationship between exosomes and EV71 replication remains elusive. In this study, we initially found that EV71 infection elevated exosome biogenesis in the cultured cells. Among EV71 non-structural proteins, we identified EV71 3A, but not 3B, constitutively promoted exosome secretion. In detail, EV71 3A protein interacted with vacuolar protein sorting 25 (VPS25), while knock-down of VPS25 reduced EV71 3A protein- and EV71-induced exosome production. Further studies revealed VPS25 located on exosomes and its expression correlated to the exosome production. During EV71 infection, knock-down of VPS25 decreased exosome biogenesis to attenuate viral replication. Consistently, GW4869, an exosome inhibitor, exerted an obviously antiviral activity against EV71 replication companied with the decrease of exosome secretion or formation. These findings suggest the binding of EV71 3A and VPS25 benefited exosome biogenesis, thereby boosting viral replication. This study uncovers a novel mechanism underlying EV71-mediated exosomes in the regulation of viral replication, which provides potential anti-viral strategies against the EV71 infection and transmission in HFMD.

Advances in the development of therapeutic strategies against COVID-19 and perspectives in the drug design for emerging SARS-CoV-2 variants.

Since Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was identified in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has challenged public health around the world. Currently, there is an urgent need to explore antiviral therapeutic targets and effective clinical drugs. In this study, we systematically summarized two main therapeutic strategies against COVID-19, namely drugs targeting the SARS-CoV-2 life cycle and SARS-CoV-2-induced inflammation in host cells. The development of above two strategies is implemented by repurposing drugs and exploring potential targets. A comprehensive summary of promising drugs, especially cytokine inhibitors, and traditional Chinese medicine (TCM), provides recommendations for clinicians as evidence-based medicine in the actual clinical COVID-19 treatment. Considering the emerging SARS-CoV-2 variants greatly impact the effectiveness of drugs and vaccines, we reviewed the appearance and details of SARS-CoV-2 variants for further perspectives in drug design, which brings updating clues to develop therapeutical agents against the variants. Based on this, the development of broadly antiviral drugs, combined with immunomodulatory, or holistic therapy in the host, is prior to being considered for therapeutic interventions on mutant strains of SARS-CoV-2. Therefore, it is highly acclaimed the requirements of the concerted efforts from multi-disciplinary basic studies and clinical trials, which improves the accurate treatment of COVID-19 and optimizes the contingency measures to emerging SARS-CoV-2 variants.

Optimization of loop-mediated isothermal amplification (LAMP) assay for robust visualization in SARS-CoV-2 and emerging variants diagnosis.

Loop-mediated isothermal amplification (LAMP) is widely used in detection of pathogenic microorganisms including SARS-CoV-2. However, the performance of LAMP assay needs further exploration in the emerging SARS-CoV-2 variants test. Here, we design serials of primers and select an optimal set for LAMP-based on SARS-CoV-2 N gene for a robust and visual assay in SARS-CoV-2 diagnosis. The limit of detectable template reaches 10 copies of N gene per 25 µL reaction at isothermal 58℃ within 40 min. Importantly, the primers for LAMP assay locate at 12 to 213 nt of N gene, a highly conservative region, which serves as a compatible test in emerging SARS-CoV-2 variants. Comparison to a commercial qPCR assay, this LAMP assay exerts the high viability in diagnosis of 41 clinical samples. Our study optimizes an advantageous LAMP assay for colorimetric detection of SARS-CoV-2 and emerging variants, which is hopeful to be a promising test in COVID-19 surveillance.

Targeted Theranostics for Tuberculosis: A Rifampicin-Loaded Aggregation-Induced Emission Carrier for Granulomas Tracking and Anti-Infection.

Tuberculosis (TB) causes a global burden with its high rates of infection and death, especially the irrepressible threats of latent infection and drug resistance. Therefore, it is important to construct efficient theranostics for the prevention and control of TB. Herein, we created a targeted theranostic strategy for TB with a rifampicin-loaded aggregation-induced emission (AIE) carrier and performed testing in laboratory animals. The AIE carrier was constructed to localize in the granulomas and emit fluorescent signals at the early stage of infection, enabling the early diagnosis of TB. Subsequently, reactive oxygen species (ROS) were generated to eradicate infection, and the loaded rifampicin (RIF) was released for the synergistic treatment of persistent bacteria. Furthermore, targeted TB therapy was performed with the light-controlled release of ROS and accurate delivery of RIF, which realizes an anti-infection effect, providing an especially important treatment for drug-resistant TB. Thus, targeted theranostics for TB in laboratory animals possess the potential to become granulomas-tracking and anti-infection strategies for the diagnosis and treatment of TB.