Host ESCRT factors are recruited during chikungunya virus infection and are required for the intracellular viral replication cycle

Chikungunya fever is a re-emerging zoonotic disease caused by chikungunya virus (CHIKV), a member of the Alphavirus genus in the Togaviridae family. Only a few studies have reported on the host factors required for intracellular CHIKV trafficking. Here, we conducted an imaging-based siRNA screen to identify human host factors for intracellular trafficking that are involved in CHIKV infection, examined their interactions with CHIKV proteins, and investigated the contributions of these proteins to CHIKV infection. The results of the siRNA screen revealed that host endosomal sorting complexes required for transport (ESCRT) proteins are recruited during CHIKV infection. Co-immunoprecipitation analyses revealed that both structural and nonstructural CHIKV proteins interact with hepatocyte growth factor–regulated tyrosine kinase substrate (HGS), a component of the ESCRT-0 complex. We also observed that HGS co-localizes with the E2 protein of CHIKV and with dsRNA, a marker of the replicated CHIKV genome. Results from gene knockdown analyses indicated that, along with other ESCRT factors, HGS facilitates both genome replication and post-translational steps during CHIKV infection. Moreover, we show that ESCRT factors are also required for infections with other alphaviruses. We conclude that during CHIKV infection, several ESCRT factors are recruited via HGS and are involved in viral genome replication and post-translational processing of viral proteins.

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ER-Shaping Atlastin Proteins Act as Central Hubs to Promote Flavivirus Replication and Virion Assembly

Proceedings ◽

10.3390/proceedings2020050031 ◽

2020 ◽

Vol 50 (1) ◽

pp. 31

Author(s):

Christopher J Neufeldt ◽

Mirko Cortese ◽

Pietro Scaturro ◽

Berati Cerikan ◽

Jeremy Wideman ◽

...

Keyword(s):

Viral Genome ◽

Viral Assembly ◽

Host Factors ◽

Genome Replication ◽

Membrane Structures ◽

Virion Assembly ◽

Virus Particles ◽

Membrane Invagination ◽

Viral Genome Replication ◽

Flavivirus Infection

Members of the Flavivirus genus rely extensively on the host cell endomembrane network to generate complex membranous replication organelles (ROs) that facilitate viral genome replication and the production of virus particles. For dengue virus and Zika virus, these ROs included vesicles which are formed through membrane invagination into the endoplasmic reticulum (ER) lumen, termed invaginated vesicles or vesicle packets (VPs), as well as large areas of bundled smooth ER, termed convoluted membranes. Though the morphology of these virus-induced membrane structures has been well characterized, the viral and host constituents that make up flaviviral ROs are still poorly understood. Here, we identified a subset of ER resident proteins (atlastins), normally required for maintaining ER tubule networks, as critical host factors for flavivirus infection. Specific changes in atlastin (ATL) levels had dichotomous effects on flaviviruses with ATL2 depletion, leading to replication organelle defects and ATL3 depletion to changes in viral assembly/release pathways. These different depletion phenotypes allowed us to exploit virus infection to characterize non-conserved functional domains between the three atlastin paralogues. Additionally, we established the ATL interactome and show how it is reprogrammed upon viral infection. Screening of specific ATL interactors confirmed non-redundant ATL functions and identified a role for ATL3 in vesicle trafficking. Our data demonstrate that ATLs are central host factors that coordinate the ER network and shape the ER during flavivirus infection.

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Telmisartan restricts Chikungunya virus infection in vitro and in vivo through the AT1/PPAR-γ/MAPKs pathways

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01489-21 ◽

2021 ◽

Author(s):

Saikat De ◽

Prabhudutta Mamidi ◽

Soumyajit Ghosh ◽

Supriya Suman Keshry ◽

Chandan Mahish ◽

...

Keyword(s):

Chikungunya Virus ◽

At1 Receptor ◽

Host Factors ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Global Public Health ◽

Chikv Infection ◽

Ppar Γ

Chikungunya virus (CHIKV) has re-emerged as a global public health threat. The inflammatory pathways of RAS and PPAR-γ are usually involved in viral infections. Thus, Telmisartan (TM) with known capacity to block AT1 receptor and activate PPAR-γ, was investigated against CHIKV. The anti-CHIKV effect of TM was investigated in vitro (Vero, RAW 264.7 cells and hPBMCs) and in vivo (C57BL/6 mice). TM was found to abrogate CHIKV infection efficiently (IC50 of 15.34-20.89μM in the Vero and RAW 264.7 cells respectively). Viral RNA and proteins were reduced remarkably. Additionally, TM interfered in the early and late stages of CHIKV life cycle with efficacy in both pre and post-treatment assay. Moreover, the agonist of AT1 receptor and antagonist of PPAR-γ increased CHIKV infection suggesting TM’s anti-viral potential by modulating host factors. Besides, reduced activation of all major MAPKs, NF-κB (p65) and cytokines by TM through the inflammatory axis supported the fact that the anti-CHIKV efficacy of TM is partly mediated through the AT1/PPAR-γ/MAPKs pathways. Interestingly, at the human equivalent dose, TM abrogated CHIKV infection and inflammation significantly leading to reduced clinical score and complete survival of C57BL/6 mice. Additionally, TM reduced infection in hPBMC derived monocyte-macrophage populations in vitro . Hence, TM was found to reduce CHIKV infection by targeting both viral and host factors. Considering its safety and in vivo efficacy, it can be a suitable candidate in future for repurposing against CHIKV.

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AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway

Molecular Biology of the Cell ◽

10.1091/mbc.e12-03-0251 ◽

2012 ◽

Vol 23 (18) ◽

pp. 3612-3623 ◽

Cited By ~ 35

Author(s):

Michael R. Dores ◽

May M. Paing ◽

Huilan Lin ◽

William A. Montagne ◽

Adriano Marchese ◽

...

Keyword(s):

Adaptor Protein ◽

Kinase Substrate ◽

Endosomal Sorting ◽

Escrt Machinery ◽

Early Endosomes ◽

Lysosomal Sorting ◽

Protease Activated Receptor 1 ◽

Signaling Receptors ◽

G Protein Coupled ◽

Hepatocyte Growth

The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor–regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein–coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail–localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting.

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NAP1L1 and NAP1L4 binding to Hypervariable Domain of Chikungunya Virus nsP3 Protein is bivalent and requires phosphorylation

10.1101/2021.05.19.444900 ◽

2021 ◽

Author(s):

Francisco Dominguez ◽

Nikita Shiliaev ◽

Tetyana Lukash ◽

Peter Agback ◽

Oksana Palchevska ◽

...

Keyword(s):

Viral Replication ◽

Chikungunya Virus ◽

Family Members ◽

Host Factors ◽

Nonstructural Proteins ◽

Host Proteins ◽

Chikv Infection ◽

Binding Motifs ◽

Specific Host ◽

Wide Range

Chikungunya virus (CHIKV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Within the last two decades, CHIKV has expanded its presence to both hemispheres and is currently circulating in both Old and New Worlds. Despite the severity and persistence of the arthritis it causes in humans, no approved vaccines or therapeutic means have been developed for CHIKV infection. Replication of alphaviruses, including CHIKV, is determined not only by their nonstructural proteins, but also by a wide range of host factors, which are indispensable components of viral replication complexes (vRCs). Alphavirus nsP3s contain hypervariable domains (HVDs), which encode multiple motifs that drive recruitment of cell- and virus-specific host proteins into vRCs. Our previous data suggested that NAP1 family members are a group of host factors that may interact with CHIKV nsP3 HVD. In this study, we performed a detailed investigation of the NAP1 function in CHIKV replication in vertebrate cells. Our data demonstrate that i) the NAP1-HVD interactions have strong stimulatory effects on CHIKV replication; ii) both NAP1L1 and NAP1L4 interact with the CHIKV HVD; iii) NAP1 family members interact with two motifs, which are located upstream and downstream of the G3BP-binding motifs of CHIKV HVD; iv) NAP1 proteins interact only with a phosphorylated form of CHIKV HVD and HVD phosphorylation is mediated by CK2 kinase; v) NAP1 and other families of host factors redundantly promote CHIKV replication and their bindings have additive stimulatory effects on viral replication.

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Structural and phenotypic analysis of Chikungunya Virus RNA structures during viral genome replication and translation

Access Microbiology ◽

10.1099/acmi.ac2019.po0346 ◽

2019 ◽

Vol 1 (1A) ◽

Author(s):

Catherine Kendall ◽

Henna Khalid ◽

Marietta Mueller ◽

Alain Kohl ◽

Andres Merits ◽

...

Keyword(s):

Viral Genome ◽

Chikungunya Virus ◽

Genome Replication ◽

Rna Structures ◽

Phenotypic Analysis ◽

Virus Rna ◽

Viral Genome Replication

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Flavivirus Replication Organelle Biogenesis in the Endoplasmic Reticulum: Comparison with Other Single-Stranded Positive-Sense RNA Viruses

International Journal of Molecular Sciences ◽

10.3390/ijms20092336 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2336 ◽

Cited By ~ 6

Author(s):

Masashi Arakawa ◽

Eiji Morita

Keyword(s):

Endoplasmic Reticulum ◽

Virus Replication ◽

Host Factors ◽

Genome Replication ◽

Organelle Biogenesis ◽

Membrane Structures ◽

Positive Sense ◽

Viral Genome Replication ◽

Amorphous Structures

Some single-stranded positive-sense RNA [ssRNA(+)] viruses, including Flavivirus, generate specific organelle-like structures in the host endoplasmic reticulum (ER). These structures are called virus replication organelles and consist of two distinct subdomains, the vesicle packets (VPs) and the convoluted membranes (CMs). The VPs are clusters of small vesicle compartments and are considered to be the site of viral genome replication. The CMs are electron-dense amorphous structures observed in proximity to the VPs, but the exact roles of CMs are mostly unknown. Several recent studies have revealed that flaviviruses recruit several host factors that are usually used for the biogenesis of other conventional organelles and usurp their function to generate virus replication organelles. In the current review, we summarize recent studies focusing on the role of host factors in the formation of virus replication organelles and discuss how these intricate membrane structures are organized.

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Arrestin-2 Interacts with the Endosomal Sorting Complex Required for Transport Machinery to Modulate Endosomal Sorting of CXCR4

Molecular Biology of the Cell ◽

10.1091/mbc.e10-02-0169 ◽

2010 ◽

Vol 21 (14) ◽

pp. 2529-2541 ◽

Cited By ~ 67

Author(s):

Rohit Malik ◽

Adriano Marchese

Keyword(s):

Molecular Mechanisms ◽

Lysosomal Degradation ◽

Kinase Substrate ◽

Endosomal Sorting ◽

Adaptor Molecule ◽

Escrt Machinery ◽

Early Endosomes ◽

G Protein Coupled ◽

Hepatocyte Growth ◽

Dependent Mechanism

The chemokine receptor CXCR4, a G protein-coupled receptor, is targeted for lysosomal degradation via a ubiquitin-dependent mechanism that involves the endosomal sorting complex required for transport (ESCRT) machinery. We have reported recently that arrestin-2 also targets CXCR4 for lysosomal degradation; however, the molecular mechanisms by which this occurs remain poorly understood. Here, we show that arrestin-2 interacts with ESCRT-0, a protein complex that recognizes and sorts ubiquitinated cargo into the degradative pathway. Signal-transducing adaptor molecule (STAM)-1, but not related STAM-2, interacts directly with arrestin-2 and colocalizes with CXCR4 on early endosomal antigen 1-positive early endosomes. Depletion of STAM-1 by RNA interference and disruption of the arrestin-2/STAM-1 interaction accelerates agonist promoted degradation of CXCR4, suggesting that STAM-1 via its interaction with arrestin-2 negatively regulates CXCR4 endosomal sorting. Interestingly, disruption of this interaction blocks agonist promoted ubiquitination of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) but not CXCR4 and STAM-1 ubiquitination. Our data suggest a mechanism whereby arrestin-2 via its interaction with STAM-1 modulates CXCR4 sorting by regulating the ubiquitination status of HRS.

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The ESCRT-0 subcomplex component Hrs/Hgs is a master regulator of myogenesis via modulation of signaling and degradation pathways

BMC Biology ◽

10.1186/s12915-021-01091-4 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

L. Coudert ◽

A. Osseni ◽

Y. G. Gangloff ◽

L. Schaeffer ◽

P. Leblanc

Keyword(s):

Muscle Development ◽

Signaling Cascades ◽

Degradation Pathways ◽

Master Regulator ◽

Kinase Substrate ◽

Endosomal Sorting ◽

Spatial Regulation ◽

First Time ◽

Inhibition Of Differentiation ◽

Hepatocyte Growth

Abstract Background Myogenesis is a highly regulated process ending with the formation of myotubes, the precursors of skeletal muscle fibers. Differentiation of myoblasts into myotubes is controlled by myogenic regulatory factors (MRFs) that act as terminal effectors of signaling cascades involved in the temporal and spatial regulation of muscle development. Such signaling cascades converge and are controlled at the level of intracellular trafficking, but the mechanisms by which myogenesis is regulated by the endosomal machinery and trafficking is largely unexplored. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery composed of four complexes ESCRT-0 to ESCRT-III regulates the biogenesis and trafficking of endosomes as well as the associated signaling and degradation pathways. Here, we investigate its role in regulating myogenesis. Results We uncovered a new function of the ESCRT-0 hepatocyte growth factor-regulated tyrosine kinase substrate Hrs/Hgs component in the regulation of myogenesis. Hrs depletion strongly impairs the differentiation of murine and human myoblasts. In the C2C12 murine myogenic cell line, inhibition of differentiation was attributed to impaired MRF in the early steps of differentiation. This alteration is associated with an upregulation of the MEK/ERK signaling pathway and a downregulation of the Akt2 signaling both leading to the inhibition of differentiation. The myogenic repressors FOXO1 as well as GSK3β were also found to be both activated when Hrs was absent. Inhibition of the MEK/ERK pathway or of GSK3β by the U0126 or azakenpaullone compounds respectively significantly restores the impaired differentiation observed in Hrs-depleted cells. In addition, functional autophagy that is required for myogenesis was also found to be strongly inhibited. Conclusions We show for the first time that Hrs/Hgs is a master regulator that modulates myogenesis at different levels through the control of trafficking, signaling, and degradation pathways.

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Telmisartan restricts Chikungunya virus infection in vitro and in vivo through the AT1/ PPAR-γ/MAPKs pathways

10.1101/2021.07.30.454559 ◽

2021 ◽

Author(s):

Saikat De ◽

Prabhudutta Mamidi ◽

Soumyajit Ghosh ◽

Supriya Suman Keshry ◽

Chandan Mahish ◽

...

Keyword(s):

Chikungunya Virus ◽

At1 Receptor ◽

Host Factors ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Global Public Health ◽

Chikv Infection ◽

Ppar Γ

Chikungunya virus (CHIKV) has re-emerged as a global public health threat. The inflammatory pathways of RAS and PPAR-γ are usually involved in viral infections. Thus, Telmisartan (TM) with known capacity to block AT1 receptor and activate PPAR-γ, was investigated against CHIKV. The anti-CHIKV effect of TM was investigated in vitro (Vero, RAW 264.7 cells and hPBMCs) and in vivo (C57BL/6 mice). TM was found to abrogate CHIKV infection efficiently (IC50 of 15.34-20.89µM in the Vero and RAW 264.7 cells respectively). Viral RNA and proteins were reduced remarkably with the TM driven modulation of host m-TOR signaling. Additionally, TM interfered in the early and late stages of CHIKV life cycle with efficacy in both pre and post-treatment assay. Moreover, the agonist of AT1 receptor and antagonist of PPAR-γ increased CHIKV infection suggesting TM’s anti-viral potential by modulating host factors. Besides, reduced activation of all major MAPKs, NF-κB (p65) and cytokines by TM through the inflammatory axis supported the fact that the anti-CHIKV efficacy of TM is partly mediated through the AT1/PPAR-γ/MAPKs pathways. Interestingly, at the human equivalent dose, TM abrogated CHIKV infection and inflammation significantly leading to reduced clinical score and complete survival of C57BL/6 mice. Additionally, TM reduced infection in hPBMC derived monocyte-macrophage populations in vitro. Hence, TM was found to reduce CHIKV infection by targeting both viral and host factors. Considering its safety and in vivo efficacy, it can be a suitable candidate in future for repurposing against CHIKV.

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ALIX binds a YPX3L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting

The Journal of Cell Biology ◽

10.1083/jcb.201110031 ◽

2012 ◽

Vol 197 (3) ◽

pp. 407-419 ◽

Cited By ~ 98

Author(s):

Michael R. Dores ◽

Buxin Chen ◽

Huilan Lin ◽

Unice J.K. Soh ◽

May M. Paing ◽

...

Keyword(s):

Mammalian Cells ◽

Interacting Protein ◽

Kinase Substrate ◽

Endosomal Sorting ◽

Ubiquitin Binding ◽

Lysosomal Sorting ◽

Protease Activated Receptor 1 ◽

Signaling Receptors ◽

G Protein Coupled ◽

Hepatocyte Growth

The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)–0, –I, –II, and –III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor–regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III–dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4–ESCRT-III interacting protein, bound to a YPX3L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPXnL motifs.

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