Prediction of protein assemblies, the next frontier: The CASP14-CAPRI experiment.

We present the results for CAPRI Round 50, the fourth joint CASP-CAPRI protein assembly prediction challenge. The Round comprised a total of twelve targets, including six dimers, three trimers, and three higher-order oligomers. Four of these were easy targets, for which good structural templates were available either for the full assembly, or for the main interfaces (of the higher-order oligomers). Eight were difficult targets for which only distantly related templates were found for the individual subunits. Twenty-five CAPRI groups including eight automatic servers submitted ~1250 models per target. Twenty groups including six servers participated in the CAPRI scoring challenge submitted ~190 models per target. The accuracy of the predicted models was evaluated using the classical CAPRI criteria. The prediction performance was measured by a weighted scoring scheme that takes into account the number of models of acceptable quality or higher submitted by each group as part of their five top-ranking models. Compared to the previous CASP-CAPRI challenge, top performing groups submitted such models for a larger fraction (70-75%) of the targets in this Round, but fewer of these models were of high accuracy. Scorer groups achieved stronger performance with more groups submitting correct models for 70-80% of the targets or achieving high accuracy predictions. Servers performed less well in general, except for the MDOCKPP and LZERD servers, who performed on par with human groups. In addition to these results, major advances in methodology are discussed, providing an informative overview of where the prediction of protein assemblies currently stands.

COVID-19 Docking Server: a meta server for docking small molecules, peptides and antibodies against potential targets of COVID-19.

MOTIVATION: The coronavirus disease 2019 (COVID-19) caused by a new type of coronavirus has been emerging from China and led to thousands of death globally since December 2019. Despite many groups have engaged in studying the newly emerged virus and searching for the treatment of COVID-19, the understanding of the COVID-19 target-ligand interactions represents a key challenge. Herein, we introduce COVID-19 Docking Server, a web server that predicts the binding modes between COVID-19 targets and the ligands including small molecules, peptides and antibodies. RESULTS: Structures of proteins involved in the virus life cycle were collected or constructed based on the homologs of coronavirus, and prepared ready for docking. The meta-platform provides a free and interactive tool for the prediction of COVID-19 target-ligand interactions and following drug discovery for COVID-19. AVAILABILITY AND IMPLEMENTATION: http://ncov.schanglab.org.cn. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Hepatitis C virus NS5A hijacks ARFGAP1 to maintain a phosphatidylinositol 4-phosphate-enriched microenvironment.

UNLABELLED: Phosphatidylinositol 4-phosphate (PI4P) is well known to be upregulated during hepatitis C virus (HCV) replication. The role of PI4 kinases in HCV has been extensively investigated. Whether the PI4P phosphatase Sac1 is altered by HCV remains unclear. Here, we identified ARFGAP1 to be a novel host factor for HCV replication. We further show that Sac1 interacts with ARFGAP1 and inhibits HCV replication. The elevation of PI4P induced by HCV NS5A is abrogated when the coatomer protein I (COPI) pathway is inhibited. We also found an interaction between NS5A and ARFGAP1. Furthermore, we identified a conserved cluster of positively charged amino acids in NS5A critical for interaction between NS5A and ARFGAP1, induction of PI4P, and HCV replication. Our data demonstrate that ARFGAP1 is a host factor for HCV RNA replication. ARFGAP1 is hijacked by HCV NS5A to remove COPI cargo Sac1 from the site of HCV replication to maintain high levels of PI4P. Our findings provide an additional mechanism by which HCV enhances formation of a PI4P-rich environment. IMPORTANCE: PI4P is enriched in the replication area of HCV; however, whether PI4P phosphatase Sac1 is subverted by HCV is not established. The detailed mechanism of how COPI contributes to viral replication remains unknown, though COPI components were hijacked by HCV. We demonstrate that ARFGAP1 is hijacked by HCV NS5A to remove COPI cargo Sac1 from the HCV replication area to maintain high-level PI4P generated by NS5A. Furthermore, we identify a conserved cluster of positively charged amino acids in NS5A, which are critical for interaction between NS5A and ARFGAP1, induction of PI4P, and HCV replication. This study will shed mechanistic insight on how other RNA viruses hijack COPI and Sac1.

Hepatitis C virus NS5A drives a PTEN-PI3K/Akt feedback loop to support cell survival.

BACKGROUND & AIMS: Decreased levels of phosphatase and tensin homologue (PTEN) are associated with hepatocellular carcinoma (HCC) pathogenesis and poor prognosis in hepatitis C virus (HCV)-infected HCC patients. The molecular processes governing the reduction in PTEN and outcome of PTEN dysfunction in hepatocytes are poorly understood. METHODS: The levels of proteins and mRNA were assessed by real time PCR and immunoblot. PTEN promoter activity was measured by reporter assay. Signalling pathways were perturbed using siRNAs or pharmacological inhibitors. RESULTS: Here, we report that HCV down-regulates PTEN expression at the transcriptional level by decreasing its promoter activity, mRNA transcription, and protein levels. We further identify NS5A protein as a key determinant of PTEN reduction among HCV proteins. NS5A-mediated down-regulation of PTEN occurs through a cooperation of reactive oxygen species (ROS)-dependent Nuclear Factor- kappa B (NF-κB) and ROS-independent phosphoinositol-3-kinase (PI3K) pathways. Moreover, NS5A protects cells against apoptosis. In addition, we found that down-regulation of PTEN relieves its inhibitory effect on PI3K-Akt pathway and triggers cumulative activation of Akt. This PTEN-PI3K/Akt feedback network mediates the suppression of cell apoptosis caused by NS5A. CONCLUSIONS: These data demonstrate that HCV NS5A down-regulates PTEN expression through a cooperation of ROS-dependent and -independent pathways that subsequently drives a PTEN-PI3K/Akt feedback loop to support cell survival. Our findings provide new insights suggesting that NS5A contributes to HCV-related hepatocarcinogenesis.

Docking guided phase display to develop fusion protein with novel scFv and alkaline phosphatase for one-step ELISA salbutamol detection.

Introduction: Salbutamol (SAL) is a ß2 adrenergic receptor agonist which has potential hazardous effects for human health. It is very important to establish a sensitive and convenient method to monitor SAL. Methods: Here we introduce a method to combine the information from docking and site specific phage display, with the aim to obtain scFv with high affinity to SAL. First, single chain variable fragment (scFv) antibodies against SAL were generated through phage display. By using molecular docking approach, the complex structure of SAL with antibody was predicted and indicated that H3 and L3 contribute to the binding. Then new libraries were created by randomization specific residues located on H3 and L3 according to the docking results. Results and discussion: Anti-SAL scFv antibodies with high efficiency were finally identified. In addition, the selected scFv was fused with alkaline phosphatase and expressed in E coli to develop a rapid and low-cost one step ELISA to detect SAL.

Hepatitis C virus NS5A competes with PI4KB for binding to ACBD3 in a genotype-dependent manner.

Although genotype-dependency of PI4KB involved in HCV replication has been reported, the mechanism underlying that is unknown. In this study, we found that NS5A and PI4KB competed for association of acyl-coenzyme A binding domain containing protein 3 (ACBD3), which inhibited HCV replication. ACBD3 bind to GT1b NS5A with a higher affinity than to GT2a NS5A, which was consistent with higher co-localization between PI4KB and phosphatidylinositol 4-phosphate (PI4P) in GT1b HCV-infected cells than that in GT2a HCV-infected cells. These results suggested that NS5A could rob the preexisting ACBD3/PI4KB complex to form NS5A/ACBD3 complex and PI4KB could relocate to the viral RNA replication sites to facilitate HCV replication. Our findings not only revealed the anti-HCV function of ACBD3, but also shed mechanistic light on how ACBD3 was manipulated by NS5A from different GT of HCV.

[Roles of COPI related proteins during virus replication].

COPI is a protein complex that transports vesicles from the Golgi complex back to endoplasmic reticulum. Many viruses such as RNA viruses, DNA viruses and retroviruses, hijack or adapt COPI related proteins including coatomer, ARF1 and GBF1 for their own benefits. Here, we summarize the current progress of the roles of COPI related proteins in virus replication.