Matthew Woods (Western University, Canada: Barr Lab)
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Transcript of Matthew Woods (Western University, Canada: Barr Lab)
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
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
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.
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).
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
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
Cell
HIV
HERC5
HIV only HIV + HERC5
Does HERC5 restrict infectious HIV-1 production?
HERC5 inhibits infectious HIV-1 particle production
A B
Do biological levels of HERC5 reduce Gag production?
293T Cells Human MDM
What is causing the decrease of Gag within the cell pellet?
Is Gag degraded in the presence of HERC5?
Does HERC5 interfere with the nuclear export of Gag-containing mRNA?
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
HERC5 interferes with export of genomic RNA
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
HERC5 interferes with export of unspliced RNA
Viral mRNA nuclear export
Rev dependent Rev independent
HERC5 inhibits export of Rev/RRE-dependent RNA
Rev/RRE-dependent Rev/RRE-independent
HERC5 interferes with Rev localization
Which domain is required for inhibition?
RLD domain is required for the loss of Gag accumulation
E3 ligase-independent
Evolutionary Arms Race Between Host and Pathogen
VS
Neighbor-joining phylogenetic tree for progressive alignment of 13 HERC5 orthologs using COBALT
Strong positive selection is operating on the first 100 amino acids of the RLD
Strong positive selection is operating on the first 100 amino acids of the RLD
Conclusions
2. E3 ligase-dependent
ISG15
X
1. E3 ligase-independent
Xnucleus
cytoplasm
HERC5
3. HERC5 is evolving under positive selection
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