Matthew Woods (Western University, Canada: Barr Lab)
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Transcript of Matthew Woods (Western University, Canada: Barr Lab)
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Interferon-induced HERC5 protein inhibits HIV-1 replication by two novel
mechanisms and is evolving under positive selection Matthew Woods (Western University, Canada: Barr Lab)
Session Title: Restriction Factors and HIV-1 Replication
21/07/2014
Interferon-induced HERC5 protein inhibits HIV-1 replication by two novel mechanisms and is evolving under positive selection
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Mature Virion
Binding
Fusion
Uncoating
ReverseTranscription
Nuclear Import
Integration
Transcription & Splicing
Nuclear Export
Translation
Assembly
Budding
Maturation
TRIM5α
Release
Tetherin
OAS1/RNaseL
PKRISG20
APOBEC
U
U
U U U U U U U U U
U
U
U
U
U
U UU
U U U U U U U U U
PKR
Trafficking TRIM22
T TT
T
T
T
T
T
Gag
Interferon Inhibits HIV-1 Replication
ViperinT
SAMHD1T
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Identifying new interferon induced anti-HIV-1 factors
• Our lab is interested in characterizing and identifying novel interferon-induced proteins capable of restricting HIV-1 particle production
• Surveyed IFN-induced genes for potential HIV-1 restriction factor candidates and identified HERC5.
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HERC5 is a novel interferon-induced antiviral protein
• HERC5 expression is up-regulated in response to IFN and both in vitro and in vivo virus infection.
• By virtue of its HECT domain, HERC5 is the main cellular E3 ligase that conjugates ISG15 to specific target proteins (Proc Natl Acad Sci U S A. 2006; J Biol Chem. 2006 Feb 17;281(7):4334-8)
• Tang and colleagues showed that HERC5 inhibits influenza A virus replication by catalyzing the conjugation of ISG15 to the viral NS1 protein (J Immunol. 2010 May 15;184(10):5777-90).
• Durfee and colleagues showed that HERC5 conjugates ISG15 to the human papillomavirus (HPV) L1 capsid protein, conferring a dominant-inhibitory effect on the infectivity of HPV16 pseudoviruses (Mol Cell. 2010 Jun 11;38(5):722-32).
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HERC5 is multifunctional
• We previously showed that HERC5 inhibits an early step of HIV-1 Gag assembly via the modification of Gag with ISG15 (Retrovirology. 2011 Nov 17;8:95)
1. E3 ligase-dependent
ISG15
X
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HERC5 is multifunctional• We previously showed that HERC5 inhibits an early step of HIV-1 Gag
assembly via the modification of Gag with ISG15 (Retrovirology. 2011 Nov 17;8:95)
1. E3 ligase-dependent
ISG15
X2. E3 ligase-independent
Xnucleus
cytoplasm
HERC5
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Cell
HIV
HERC5
HIV only HIV + HERC5
Does HERC5 restrict infectious HIV-1 production?
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HERC5 inhibits infectious HIV-1 particle production
A B
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Do biological levels of HERC5 reduce Gag production?
293T Cells Human MDM
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What is causing the decrease of Gag within the cell pellet?
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Is Gag degraded in the presence of HERC5?
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Does HERC5 interfere with the nuclear export of Gag-containing mRNA?
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GFP
MS2 Stem Loops
HIV-1 (Stem Loop) + MS2-GFPMS2-GFP
HIV-1 (Stem Loop) MS2-GFP
MS2 System To Localize HIV RNA Genomes Inside Cells
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HERC5 interferes with export of genomic RNA
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HIV-1 and REV
REV
REV
REV
REVREV
REV
Late Proteins (Ex. Gag, Env, and Genome) Early Proteins (Ex. Tat, Rev Nef)
Unspliced Fully spliced
Rev dependant Rev independent
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HERC5 interferes with export of unspliced RNA
Viral mRNA nuclear export
Rev dependent Rev independent
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HERC5 inhibits export of Rev/RRE-dependent RNA
Rev/RRE-dependent Rev/RRE-independent
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HERC5 interferes with Rev localization
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Which domain is required for inhibition?
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RLD domain is required for the loss of Gag accumulation
E3 ligase-independent
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Evolutionary Arms Race Between Host and Pathogen
VS
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Neighbor-joining phylogenetic tree for progressive alignment of 13 HERC5 orthologs using COBALT
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Strong positive selection is operating on the first 100 amino acids of the RLD
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Strong positive selection is operating on the first 100 amino acids of the RLD
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Conclusions
2. E3 ligase-dependent
ISG15
X
1. E3 ligase-independent
Xnucleus
cytoplasm
HERC5
3. HERC5 is evolving under positive selection
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Acknowledgements
SupervisorDr. Stephen Barr
Lab MembersJenna KellySean TomDivy KocharJohnny ZhangMike Ding
Past Lab MembersClayton HattlmannJess TongSherry XuMacon Coleman
Collaborators Dr. DikeakosCraig CavanaghDr. HaeryfarPeter Szabo
Funding Agencies