Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment
ABSTRACT Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-doma...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2016-12-01
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| Series: | mBio |
| Online Access: | https://journals.asm.org/doi/10.1128/mBio.01456-16 |
| _version_ | 1818822506163732480 |
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| author | Rina Barouch-Bentov Gregory Neveu Fei Xiao Melanie Beer Elena Bekerman Stanford Schor Joseph Campbell Jim Boonyaratanakornkit Brett Lindenbach Albert Lu Yves Jacob Shirit Einav |
| author_facet | Rina Barouch-Bentov Gregory Neveu Fei Xiao Melanie Beer Elena Bekerman Stanford Schor Joseph Campbell Jim Boonyaratanakornkit Brett Lindenbach Albert Lu Yves Jacob Shirit Einav |
| author_sort | Rina Barouch-Bentov |
| collection | DOAJ |
| description | ABSTRACT Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses. IMPORTANCE Viruses commonly bud at the plasma membrane by recruiting the host ESCRT machinery via conserved motifs termed late domains. The mechanism by which some viruses, such as HCV, bud intracellularly is, however, poorly characterized. Moreover, whether envelopment of HCV and other viruses lacking defined late domains is ESCRT mediated and, if so, what the entry points into the ESCRT pathway are remain unknown. Here, we report the interaction network of HCV with the ESCRT machinery and a critical role for HRS, an ESCRT-0 complex component, in HCV envelopment. Viral protein ubiquitination was discovered to be a signal for HRS binding and HCV assembly, thereby functionally compensating for the absence of late domains. These findings characterize how a virus lacking defined late domains co-opts ESCRT to bud intracellularly. Since the ESCRT machinery is essential for the life cycle of multiple viruses, better understanding of this virus-host interplay may yield targets for broad-spectrum antiviral therapies. |
| first_indexed | 2024-12-18T23:25:10Z |
| format | Article |
| id | doaj.art-7638b83bba084ee190baf218083848bc |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-18T23:25:10Z |
| publishDate | 2016-12-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-7638b83bba084ee190baf218083848bc2022-12-21T20:47:49ZengAmerican Society for MicrobiologymBio2150-75112016-12-017610.1128/mBio.01456-16Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral EnvelopmentRina Barouch-Bentov0Gregory Neveu1Fei Xiao2Melanie Beer3Elena Bekerman4Stanford Schor5Joseph Campbell6Jim Boonyaratanakornkit7Brett Lindenbach8Albert Lu9Yves Jacob10Shirit Einav11Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USADivision of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USADivision of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USADivision of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USADivision of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USADivision of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USADivision of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USADivision of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USADepartment of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut, USADepartment of Biochemistry, Stanford University School of Medicine, Stanford, California, USADépartement de Virologie, Unité de Génétique Moléculaire des Virus ARN (GMVR), Institut Pasteur, Centre national de la recherche scientifique, and Université Paris Diderot, Paris, FranceDivision of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USAABSTRACT Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses. IMPORTANCE Viruses commonly bud at the plasma membrane by recruiting the host ESCRT machinery via conserved motifs termed late domains. The mechanism by which some viruses, such as HCV, bud intracellularly is, however, poorly characterized. Moreover, whether envelopment of HCV and other viruses lacking defined late domains is ESCRT mediated and, if so, what the entry points into the ESCRT pathway are remain unknown. Here, we report the interaction network of HCV with the ESCRT machinery and a critical role for HRS, an ESCRT-0 complex component, in HCV envelopment. Viral protein ubiquitination was discovered to be a signal for HRS binding and HCV assembly, thereby functionally compensating for the absence of late domains. These findings characterize how a virus lacking defined late domains co-opts ESCRT to bud intracellularly. Since the ESCRT machinery is essential for the life cycle of multiple viruses, better understanding of this virus-host interplay may yield targets for broad-spectrum antiviral therapies.https://journals.asm.org/doi/10.1128/mBio.01456-16 |
| spellingShingle | Rina Barouch-Bentov Gregory Neveu Fei Xiao Melanie Beer Elena Bekerman Stanford Schor Joseph Campbell Jim Boonyaratanakornkit Brett Lindenbach Albert Lu Yves Jacob Shirit Einav Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment mBio |
| title | Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment |
| title_full | Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment |
| title_fullStr | Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment |
| title_full_unstemmed | Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment |
| title_short | Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment |
| title_sort | hepatitis c virus proteins interact with the endosomal sorting complex required for transport escrt machinery via ubiquitination to facilitate viral envelopment |
| url | https://journals.asm.org/doi/10.1128/mBio.01456-16 |
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