Purification of Unanchored Polyubiquitin Chains from Influenza Virions

Yasu Miyake, Patrick Matthias, Yohei Yamauchi

Research output: Chapter in Book/Report/Conference proceedingChapter in a book

3 Citations (Scopus)
157 Downloads (Pure)


Influenza A virus (IAV) is an enveloped virus with a segmented single-stranded negative-strand RNA genome. In general, the role of virally encapsidated host cell proteins in the viral life cycle is unclear. The virion contains abundant ubiquitin molecules some of which have been identified as unanchored poly-
ubiquitin chains. These ubiquitin chains have been postulated to play a role in recruiting histone deacetylase 6 (HDAC6) to the cytosolic surface of late endosomes (LEs), promoting IAV uncoating via aggresome processing—a cellular machinery that disposes of protein waste. HDAC6, a class II HDAC, is unusual
because it resides mostly in the cytosol instead of the nucleus. It is a unique protein consisting of two catalytic domains (CDs) and a zinc-finger ubiquitin-binding domain (ZnF-UBP) close to its C-terminus. This ZnF-UBP recognizes the unconjugated ubiquitin C-terminus (di-Gly motif) with very high affinity.
Biochemical analyses showed that free di-Gly motifs are present in the form of unanchored ubiquitin inside IAV virions. These motifs are exposed following low pH-triggered viral fusion at the LEs and attract HDAC6 transiently to the cytosolic surface of vesicles. The binding of the two components promotes
viral uncoating via HDAC6 interaction with cellular motor proteins dynein and myosin II and the viral M1 capsid. The cellular mechanism involved is related to aggresome processing, a pathway that promotes degradation of misfolded protein aggregates. K63-linked ubiquitin chains are thought to be the trigger for
aggresome processing, though it is still not clear whether such types of chains are prevalent within the IAV capsid. Here, we present methods using purified ZnF-UBP domain of HDAC6 to immunoprecipitate viral unanchored ubiquitin chains, which can then be used for further biochemical analyses of ubiquitin chain linkage.
Original languageEnglish
Title of host publicationInfluenza Virus
Subtitle of host publicationMethods and Protocols
PublisherHumana Press
Number of pages14
ISBN (Electronic)9781493986781
ISBN (Print)9781493986774
Publication statusPublished - 28 Aug 2018

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745


  • Influenza virus
  • Ubiquitin
  • Unanchored ubiquitin
  • Di-glycine motif
  • HDAC6
  • Aggresome processing


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