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Interferon-induced HERC5 protein inhibits HIV-1 replication byistwo novel mechanisms and is evolving Interferon-induced HERC5 protein inhibits replication by two novel mechanisms and evolving under positiveHIV-1 selection under positive selection Matthew Woods (Western University, Canada: Barr Lab) Session Title: Restriction Factors and HIV-1 Replication 21/07/2014 Interferon Inhibits HIV-1 Replication Mature Virion Release Maturation Tetherin Budding Binding U U U Fusion Viperin Assembly Uncoating U APOBEC Gag TRIM22 U Trafficking Nuclear Import Translation U U Integration UU U U U U U U U UU U U U ISG20 U U U U PKR Nuclear Export U Transcription & Splicing U SAMHD1 U T T TRIM5α Reverse Transcription OAS1/RNaseL PKR 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 2. E3 ligase-independent X cytoplasm nucleus HERC5 X Does HERC5 restrict infectious HIV-1 production? HIV only Cell HIV HERC5 HIV + HERC5 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? MS2 System To Localize HIV RNA Genomes Inside Cells GFP MS2 Stem Loops HIV-1 (Stem Loop) MS2-GFP MS2-GFP HIV-1 (Stem Loop) + MS2-GFP HERC5 interferes with export of genomic RNA HIV-1 and REV Late Proteins (Ex. Gag, Env, and Genome) REV Early Proteins (Ex. Tat, Rev Nef) REV REV REV REV Unspliced Fully spliced REV Rev dependant Rev independent HERC5 interferes with export of unspliced RNA Rev dependent Rev independent Viral mRNA nuclear export 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 1. E3 ligase-independent cytoplasm nucleus HERC5 2. E3 ligase-dependent ISG15 X 3. HERC5 is evolving under positive selection X Acknowledgements Supervisor Past Lab Members Dr. Stephen Barr Clayton Hattlmann Jess Tong Lab Members Sherry Xu Jenna Kelly Macon Coleman Sean Tom Divy Kochar Collaborators Johnny Zhang Dr. Dikeakos Mike Ding Craig Cavanagh Dr. Haeryfar Peter Szabo Funding Agencies