Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment.
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.



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.
Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.
Rina
R
Barouch-Bentov
Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.
Gregory
G
Neveu
Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.
Fei
F
Xiao
Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.
Melanie
M
Beer
Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.
Elena
E
Bekerman
Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.
Stanford
S
Schor
Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.
Joseph
J
Campbell
Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.
Jim
J
Boonyaratanakornkit
Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut, USA.
Brett
B
Lindenbach
Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA.
Albert
A
Lu
Département de Virologie, Unité de Génétique Moléculaire des Virus ARN (GMVR), Institut Pasteur, Centre national de la recherche scientifique, and Université Paris Diderot, Paris, France.
Yves
Y
Jacob
Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA seinav@stanford.edu.
Shirit
S
Einav
D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D016174:Hepacivirus / Q000502:physiology*; D054884:Host-Pathogen Interactions; D025941:Protein Interaction Mapping; D054875:Ubiquitination; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D019065:Virus Assembly; D057074:Virus Release
D056827:Endosomal Sorting Complexes Required for Transport; D017361:Viral Nonstructural Proteins