VHL negatively regulates SARS coronavirus replication by modulating nsp16 ubiquitination and stability
Xiao Yu,a,1 Shuliang Chen,b,1 Panpan Hou,a Min Wang,b Yu Chen,a and Deyin Guoa,b,∗
Biochem Biophys Res Commun. 2015 Apr 3; 459(2): 270–276.
Eukaryotic cellular and most viral RNAs carry a 5′-terminal cap structure, a 5′-5′ triphosphate linkage between the 5′ end of the RNA and a guanosine nucleotide (cap-0). SARS coronavirus (SARS-CoV) nonstructural protein nsp16 functions as a methyltransferase, to methylate mRNA cap-0 structure at the ribose 2′-O position of the first nucleotide to form cap-1 structures. However, whether there is interplay between nsp16 and host proteins was not yet clear. In this report, we identified several potential cellular nsp16-interacting proteins from a human thymus cDNA library by yeast two-hybrid screening. VHL, one of these proteins, was proven to interact with nsp16 both in vitro and in vivo. Further studies showed that VHL can inhibit SARS-CoV replication by regulating nsp16 ubiquitination and promoting its degradation. Our results have revealed the role of cellular VHL in the regulation of SARS-CoV replication.
Targeting Cullin–RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation
Emil Bulatov*†,1 and Alessio Ciulli*,2
Biochem J. 2015 May 1; 467(Pt 3): 365–386. Published online 2015 Apr 17. doi: 10.1042/BJ20141450
VIRAL HIJACKING OF CRLs
CRLs or their components can be hijacked by viral proteins to manipulate the host cellular processes, and to exploit the host UPS in order to promote viral replication [127,128]. Examples of CRL hijacking have been reported for several viral proteins. HIV-1 Vpu (viral protein U) protein binds the β-TrCP receptor of CRL1 to target T-cell surface glycoprotein CD4 for proteasomal degradation . HIV-1 Vpr was found to associate with CRL4AVPRBP and to trigger cell cycle arrest [130,131]. SV40 large T-antigen was found to bind F-box receptor Fbw7 of CRL1 and regulate the turnover of substrate cyclin E . HPV (high-risk human papillomavirus) E7 was shown to interact with the receptor Skp2 and be targeted for ubiquitination by CRL1Skp2 . Adenovirus proteins E4orf6 and E1B55K were shown to hijack the EloBC–Cul5–Rbx1 complex and promote ubiquitination of tumour-suppressor protein p53 .
Several types of paramyxoviruses, including HPIV-2 (human parainfluenza virus type 2) and SV5 are able to hijack the CRL4A machinery . These viruses produce a conserved V protein that recruits the DDB1 adaptor of CRL4A and promotes ubiquitination of substrate STAT1 and its targeted proteasomal degradation [136,137]. The crystal structure of DDB1 in complex with V protein revealed the structural basis of the interaction .
HIV-1 Vif (virion infectivity factor) protein suppresses the host antiviral activity by hijacking the CRL5 machinery. Specifically, Vif recruits the EloBC–Cul5–Rbx1 complex to form CRL5Vif and induce proteasomal degradation of the substrate APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G), a host protein that functions as an antiviral factor by inhibiting retrovirus replication . The recently solved crystal structure of Vif–CBFβ–Cul5–EloBC (CBFβ is core binding factor β) uncovered the details of the hijacking mechanism . The mechanism of viral hijacking was shown to involve extended interaction with cellular transcription factor CBFβ that promotes Vif-mediated degradation .
The area of viral CRL hijacking becomes more attractive for drug discovery as the number of solved crystal structures increases. Small-molecule inhibitors disrupting interaction of viral proteins with the CRL machinery or blocking the activity of the hijacked E3 ligase complex could lead to the development of novel antiviral therapies.
Cullin E3 Ligases and Their Rewiring by Viral Factors
Cathal Mahon,1 Nevan J. Krogan,2 Charles S. Craik,1 and Elah Pick3,*
Biomolecules. 2014 Dec; 4(4): 897–930.
The ability of viruses to subvert host pathways is central in disease pathogenesis. Over the past decade, a critical role for the Ubiquitin Proteasome System (UPS) in counteracting host immune factors during viral infection has emerged. This counteraction is commonly achieved by the expression of viral proteins capable of sequestering host ubiquitin E3 ligases and their regulators. In particular, many viruses hijack members of the Cullin-RING E3 Ligase (CRL) family. Viruses interact in many ways with CRLs in order to impact their ligase activity; one key recurring interaction involves re-directing CRL complexes to degrade host targets that are otherwise not degraded within host cells. Removal of host immune factors by this mechanism creates a more amenable cellular environment for viral propagation. To date, a small number of target host factors have been identified, many of which are degraded via a CRL-proteasome pathway. Substantial effort within the field is ongoing to uncover the identities of further host proteins targeted in this fashion and the underlying mechanisms driving their turnover by the UPS. Elucidation of these targets and mechanisms will provide appealing anti-viral therapeutic opportunities. This review is focused on the many methods used by viruses to perturb host CRLs, focusing on substrate sequestration and viral regulation of E3 activity.
DDB1 and Cul4A are required for human immunodeficiency virus type 1 Vpr-induced G2 arrest
Lindi Tan 1, Elana Ehrlich, Xiao-Fang Yu
J Virol. 2007 Oct;81(19):10822-30. doi: 10.1128/JVI.01380-07. Epub 2007 Jul 11.
Vpr-mediated induction of G2 cell cycle arrest has been postulated to be important for human immunodeficiency virus type 1 (HIV-1) replication, but the precise role of Vpr in this cell cycle arrest is unclear. In the present study, we have shown that HIV-1 Vpr interacts with damaged DNA binding protein 1 (DDB1) but not its partner DDB2. The interaction of Vpr with DDB1 was inhibited when DCAF1 (VprBP) expression was reduced by short interfering RNA (siRNA) treatment. The Vpr mutant (Q65R) that was defective for DCAF1 interaction also had a defect in DDB1 binding. However, Vpr binding to DDB1 was not sufficient to induce G2 arrest. A reduction in DDB1 or DDB2 expression in the absence of Vpr also did not induce G2 arrest. On the other hand, Vpr-induced G2 arrest was impaired when the intracellular level of DDB1 or Cullin 4A was reduced by siRNA treatment. Furthermore, Vpr-induced G2 arrest was largely abolished by a proteasome inhibitor. These data suggest that Vpr assembles with DDB1 through interaction with DCAF1 to form an E3 ubiquitin ligase that targets cellular substrates for proteasome-mediated degradation and G2 arrest.
HIV-1 Vpr mediates the depletion of the cellular repressor CTIP2 to counteract viral gene silencing
F Forouzanfar 1, S Ali 1 2, C Wallet 1, M De Rovere 1, C Ducloy 3, H El Mekdad 1, M El Maassarani 1, A Aït-Ammar 1 4, J Van Assche 1, E Boutant 5, F Daouad 1, F Margottin-Goguet 6 7 8, C Moog 3, C Van Lint 9, C Schwartz 10, O Rohr 11
Sci Rep. 2019 Sep 11;9(1):13154. doi: 10.1038/s41598-019-48689-x.
Mammals have evolved many antiviral factors impacting different steps of the viral life cycle. Associated with chromatin-modifying enzymes, the cellular cofactor CTIP2 contributes to HIV-1 gene silencing in latently infected reservoirs that constitute the major block toward an HIV cure. We report, for the first time, that the virus has developed a strategy to overcome this major transcriptional block. Productive HIV-1 infection results in a Vpr-mediated depletion of CTIP2 in microglial cells and CD4+ T cells, two of the major viral reservoirs. Associated to the Cul4A-DDB1-DCAF1 ubiquitin ligase complex, Vpr promotes CTIP2 degradation via the proteasome pathway in the nuclei of target cells and notably at the latent HIV-1 promoter. Importantly, Vpr targets CTIP2 associated with heterochromatin-promoting enzymes dedicated to HIV-1 gene silencing. Thereby, Vpr reactivates HIV-1 expression in a microglial model of HIV-1 latency. Altogether our results suggest that HIV-1 Vpr mediates the depletion of the cellular repressor CTIP2 to counteract viral gene silencing.
Hepatitis B virus regulatory HBx protein binding to DDB1 is required but is not sufficient for maximal HBV replication
Amanda J Hodgson 1, Joseph M Hyser, Victor V Keasler, Yong Cang, Betty L Slagle
Virology. 2012 Apr 25;426(1):73-82. doi: 10.1016/j.virol.2012.01.021. Epub 2012 Feb 17.
Robust hepatitis B virus (HBV) replication is stimulated by the regulatory HBx protein. HBx binds the cellular protein DDB1; however, the importance of this interaction for HBV replication remains unknown. We tested whether HBx binding to DDB1 was required for HBV replication using a plasmid based replication assay in HepG2 cells. Three DDB1 binding-deficient HBx point mutants (HBx(69), HBx(90/91), HBx(R96E)) failed to restore wildtype levels of replication from an HBx-deficient plasmid, which established the importance of the HBx-DDB1 interaction for maximal HBV replication. Analysis of overlapping HBx truncation mutants revealed that both the HBx-DDB1 binding domain and the carboxyl region are required for maximal HBV replication both in vitro and in vivo, suggesting the HBx-DDB1 interaction recruits regulatory functions critical for replication. Finally we demonstrate that HBx localizes to the Cul4A-DDB1 complex, and discuss the possible implications for models of HBV replication.
Small molecule degraders of the hepatitis C virus protease reduce susceptibility to resistance mutations
Mélissanne de Wispelaere 1, Guangyan Du 2 3, Katherine A Donovan 2 3, Tinghu Zhang 2 3, Nicholas A Eleuteri 3, Jingting C Yuan 3, Joann Kalabathula 3, Radosław P Nowak 2 3, Eric S Fischer 2 3, Nathanael S Gray 2 3, Priscilla L Yang 4
Nat Commun. 2019 Aug 1;10(1):3468. doi: 10.1038/s41467-019-11429-w.
Targeted protein degradation is a promising drug development paradigm. Here we leverage this strategy to develop a new class of small molecule antivirals that induce proteasomal degradation of viral proteins. Telaprevir, a reversible-covalent inhibitor that binds to the hepatitis C virus (HCV) protease active site is conjugated to ligands that recruit the CRL4CRBN ligase complex, yielding compounds that can both inhibit and induce the degradation of the HCV NS3/4A protease. An optimized degrader, DGY-08-097, potently inhibits HCV in a cellular infection model, and we demonstrate that protein degradation contributes to its antiviral activity. Finally, we show that this new class of antiviral agents can overcome viral variants that confer resistance to traditional enzymatic inhibitors such as telaprevir. Overall, our work provides proof-of-concept that targeted protein degradation may provide a new paradigm for the development of antivirals with superior resistance profiles.
BRD4 inhibition exerts anti-viral activity through DNA damage-dependent innate immune responses
Jiang Wang, Conceptualization, Data curation, Formal analysis, Methodology, Writing – original draft, Writing – review & editing,#1 Guo-Li Li, Investigation, Methodology,#1 Sheng-Li Ming, Investigation, Methodology,#1 Chun-Feng Wang, Investigation,#1 Li-Juan Shi, Investigation,1 Bing-Qian Su, Investigation,1 Hong-Tao Wu, Investigation,1 Lei Zeng, Investigation,1 Ying-Qian Han, Investigation,1 Zhong-Hu Liu, Conceptualization,1 Da-Wei Jiang, Funding acquisition, Investigation,1 Yong-Kun Du, Funding acquisition, Investigation,1 Xiang-Dong Li, Methodology,2 Gai-Ping Zhang, Conceptualization, Funding acquisition,1 Guo-Yu Yang, Conceptualization, Funding acquisition, Supervision,1,* and Bei-Bei Chu, Conceptualization, Formal analysis, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing1,*
PLoS Pathog. 2020 Mar; 16(3): e1008429. Published online 2020 Mar 24.
Chromatin dynamics regulated by epigenetic modification is crucial in genome stability and gene expression. Various epigenetic mechanisms have been identified in the pathogenesis of human diseases. Here, we examined the effects of ten epigenetic agents on pseudorabies virus (PRV) infection by using GFP-reporter assays. Inhibitors of bromodomain protein 4 (BRD4), which receives much more attention in cancer than viral infection, was found to exhibit substantial anti-viral activity against PRV as well as a range of DNA and RNA viruses. We further demonstrated that BRD4 inhibition boosted a robust innate immune response. BRD4 inhibition also de-compacted chromatin structure and induced the DNA damage response, thereby triggering the activation of cGAS-mediated innate immunity and increasing host resistance to viral infection both in vitro and in vivo. Mechanistically, the inhibitory effect of BRD4 inhibition on viral infection was mainly attributed to the attenuation of viral attachment. Our findings reveal a unique mechanism through which BRD4 inhibition restrains viral infection and points to its potent therapeutic value for viral infectious diseases.
Bromodomain Inhibitors as Therapeutics for Herpesvirus-Related Disease: All BETs Are Off?
Ian J. Groves*†, John H. Sinclair† and Mark R. Wills†
Front. Cell. Infect. Microbiol., 02 July 2020 | https://doi.org/10.3389/fcimb.2020.00329
Although the ubiquitous human herpesviruses (HHVs) are rarely associated with serious disease of the healthy host, primary infection and reactivation in immunocompromised individuals can lead to significant morbidity and, in some cases, mortality. Effective drugs are available for clinical treatment, however resistance is on the rise such that new anti-viral targets, as well as novel clinical treatment strategies, are required. A promising area of development and pre-clinical research is that of inhibitors of epigenetic modifying proteins that control both cellular functions and the viral life cycle. Here, we briefly outline the interaction of the host bromo- and extra-terminal domain (BET) proteins during different stages of the HHVs’ life cycles while giving a full overview of the published work using BET bromodomain inhibitors (BRDis) during HHV infections. Furthermore, we provide evidence that small molecule inhibitors targeting the host BET proteins, and BRD4 in particular, have the potential for therapeutic intervention of HHV-associated disease.
A proteomic model of SARS-COV2 infection by comparing the interactomes of BRD4 with BET-inhibition and SARS-COV2 viral proteins – implications for re-purposing approved drugs or ubiquitin-mediated degradation of select candidates
DOI: 10.21203/rs.3.rs-24573/v1. Download PDF
The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected 2,029,930 people worldwide and caused 136,320 deaths. Consequently, the hunt for drugs showing efficacy against this deadly disease, or vaccines for prevention, are being intensely investigated. Unfortunately, there is a scarcity of research data on the molecular mechanisms of SARS-CoV-2 infection for quickly finding effective therapies, or repurposing existing drugs approved by the US FDA. This report models existing knowledge of SARS-COV2 viral proteins and the cellular proteins they interact with by comparisons with BRD4 interacting proteins identified from B cells, with or without BET inhibition. The E protein of SARS-COV2 interacts with BRD4, and the Spike (S) protein with CANX. Extensive similarities were observed with published cellular interactants of 13 SARS-COV2 proteins resulting in 47 BRD4-interacting protein candidates, with or without BET inhibition. 61 cellular protein targets and 132 FDA approved drugs which use these proteins as targets are proposed, which can be investigated for efficacy against SARS-COV2 infections. The implications to SARS-COV2 disease diagnosis, therapy and vaccine creation are discussed.
Influences of cyclosporin A and non-immunosuppressive derivatives on cellular cyclophilins and viral nucleocapsid protein during human coronavirus 229E replication
Yue Ma-Lauer,a,b Yu Zheng,a,b Miroslav Malešević,c Brigitte von Brunn,a,b Gunter Fischer,d and Albrecht von Brunna,b,∗
Antiviral Res. 2020 Jan; 173: 104620.
The well-known immunosuppressive drug cyclosporin A inhibits replication of various viruses including coronaviruses by binding to cellular cyclophilins thus inactivating their cis-trans peptidyl-prolyl isomerase function. Viral nucleocapsid proteins are inevitable for genome encapsidation and replication. Here we demonstrate the interaction between the N protein of HCoV-229E and cyclophilin A, not cyclophilin B. Cyclophilin inhibitors abolish this interaction. Upon infection, cyclophilin A stays evenly distributed throughout the cell, whereas cyclophilin B concentrates at ER-bleb-like structures. We further show the inhibitory potential of non-immunosuppressive CsA derivatives Alisporivir, NIM811, compound 3 on HCoV-229E-GFP and -Luciferase replication in human Huh-7.5 hepatoma cells at 18 and 48 h time points post infection with EC50 s at low micromolar ranges. Thus, non-immunosuppressive CsA derivatives effectively inhibit HCoV-229E replication suggesting them as possible candidates for the treatment of HCoV infection. The interruption of interaction between CypA and N protein by CsA and its derivatives suggest a mechanism how CypA inhibitors suppress viral replication.
Replication of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E is inhibited by the drug FK506
Javier Carbajo-Lozoya,a Marcel A. Müller,b Stephan Kallies,b Volker Thiel,c,d Christian Drosten,b and Albrecht von Brunna,⁎
Virus Res. 2012 Apr; 165(1): 112–117. Published online 2012 Feb 10.
Recent research has shown that Coronavirus (CoV) replication depends on active immunophilin pathways. Here we demonstrate that the drug FK506 (Tacrolimus) inhibited strongly the growth of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E at low, non-cytotoxic concentrations in cell culture. As shown by plaque titration, qPCR, Luciferase- and green fluorescent protein (GFP) reporter gene expression, replication was diminished by several orders of magnitude. Knockdown of the cellular FK506-binding proteins FKBP1A and FKBP1B in CaCo2 cells prevented replication of HCoV-NL63, suggesting the requirement of these members of the immunophilin family for virus growth.
Cyclophilins and cyclophilin inhibitors in nidovirus replication
Adriaan H.de Wilde, Uyen Pham, Clara C.Posthuma, Eric J.Snijder
Virology. Volume 522, September 2018, Pages 46-55
Nidovirus replication is inhibited by cyclophilin inhibitors.
Arterivirus replication depends on cyclophilin A.
Cyclosporin A blocks arterivirus RNA synthesis.
Using cyclophilin inhibitors against nidoviruses in vivo needs more investigation.
Cyclophilins (Cyps) belong to the family of peptidyl-prolyl isomerases (PPIases). The PPIase activity of most Cyps is inhibited by the immunosuppressive drug cyclosporin A and several of its non-immunosuppressive analogs, which can also block the replication of nidoviruses (arteriviruses and coronaviruses). Cyclophilins have been reported to play an essential role in the replication of several other RNA viruses, including human immunodeficiency virus-1, hepatitis C virus, and influenza A virus. Likewise, the replication of various nidoviruses was reported to depend on Cyps or other PPIases. This review summarizes our current understanding of this class of nidovirus-host interactions, including the potential function of in particular CypA and the inhibitory effect of Cyp inhibitors. Also the involvement of the FK-506-binding proteins and parvulins is discussed. The nidovirus data are placed in a broader perspective by summarizing the most relevant data on Cyp interactions and Cyp inhibitors for other RNA viruses.
A SARS-CoV-2 protein interaction map reveals targets for drug repurposing
David E. Gordon, Gwendolyn M. Jang, […]Nevan J. Krogan
Nature volume 583, pages459–468(2020)Cite this article
A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein–protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.
Identification of key genes in SARS-CoV-2 patients on bioinformatics analysis
Gu H, Yuan G
Preprint from bioRxiv, 11 Aug 2020
Abstract The COVID-19 pandemic has infected millions of people and overwhelmed many health systems globally. Our study is to identify differentially expressed genes (DEGs) and associated biological processes of COVID-19 using a bioinformatics approach to elucidate their potential pathogenesis. The gene expression profiles of the GSE152075 datasets were originally produced by using the high-throughput Illumina NextSeq 500. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were performed to identify functional categories and biochemical pathways. GO and KEGG results suggested that several biological pathways such as “Fatty acid metabolism” and “Cilium morphogenesis” are mostly involved in the development of COVID-19. Moreover, several genes are critical for virus invasion and adhesion including FLOC, DYNLL1, FBXL3, and FBXW11 and show significant differences in COVID-19 patients. Thus, our study provides further insights into the underlying pathogenesis of COVID-19.
Identification of potential biomarkers and inhibitors for SARS-CoV-2 infection
Hanming Gu, View ORCID ProfileGongsheng Yuan
medRxiv. doi: https://doi.org/10.1101/2020.09.15.20195487. This article is a preprint.
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has overwhelmed many health systems globally. Here, we aim to identify biological markers and associated biological processes of COVID-19 using a bioinformatics approach to elucidate their potential pathogenesis. The gene expression profile of the GSE152418 dataset was originally produced by using the high-throughput Illumina NovaSeq 6000. Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) and Gene Ontology (GO) enrichment analyses were applied to identify functional categories and biochemical pathways. KEGG and GO results suggested that biological pathways such as Cancer pathways and Insulin pathways were mostly affected in the development of COVID-19. Moreover, we identified several genes including EP300, CREBBP, and POLR2A were involved in the virus activities in COVID-19 patients. We further predicted that some inhibitors may have the potential to block the SARS-CoV-2 infection based on the L1000FWD analysis. Therefore, our study provides further insights into the underlying pathogenesis of COVID-19.