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