IFIT1 IFIT2 IFIT3 IFIT5 Mxa Interferon … Stimulated Hundreds of Genes Other Genes

Lecture 3- Vignette 1 Discovery of a novel overprinting gene in polyomaviruses

Matt Daugherty, former postdoc now Assistant Professor, UC San Diego WHAT IS OVERPRINTING? HOW DOES IT EVOLVE?

DNA CCTATTAATGGAACAGCCA Frame 1 P I N G T A Frame 2 L Y M E Q P

hiv.lanl.gov UNUSUAL EVOLUTIONARY CONSERVATION

Virus A CCTATTAATGGAACAGCCA Frame 1 P I N G T A Frame 2 L L M E Q P

Virus B CCTATAAATGGAACAGCCA Frame 1 P I N G T A Synonymous change Frame 2 L * M E Q P Nonsynonymous change

Observe depression of ‘synonymous’ mutations in overprinting regions A NOVEL GENE IN MCPYV

Polyomaviruses (PyV’s) are small DNA viruses

9 distinct human viruses, infect >50% of people

SV40: Causes cancer in monkeys (also has a very useful promoter) MousePyV: Causes cancer in rodents Merkel cell polyomavirus (MCPyV): Causes cancer in humans

YGS/T LxCxE motif motif JCPyV LargeT DNAj OBD ATPase/Helicase 1 688

MCPyV LargeT 1 817 ALTO (+1 frame) *

Start Hydrophobic motif YGS/T LxCxE LTERNATE motifANTIGENmotif A JCPyV LargeT DNAj T ORFOBD (ALTO)ATPase/Helicase 1 688

MCPyV LargeT 1 817 ALTO (+1 frame) *

Start Hydrophobic motif 248 amino acid protein No recognizable primary or secondary structure except a hydrophobic C-terminus No similarity to any other viral protein except 3 closely related viruses

D 1 Region of decreased dS

0.5

MCPyV-GorillaPyV1

Synonymous MCPyV-ChimpPyV1a

site divergence (dS) MCPyV-ChimpPyV2a 0 0-100 100-200 200-300 300-400 400-500 500-600 600-700 700-800 Amino acid window

MCPyV LT j D ATPase/HelicaseAT

ALTO

How did ALTO come to exist? A WHOLE CLADE OF OVERPRINTED LT GENES

Are there other PyVs with a large uninterrupted ORF overprinting on LT?

Overlap with LT OBD domain WUPyV * A 20 amino acids KIPyV BKPyV * * JCPyV SV40 HPyV10 * * HPyV6 HPyV7 HPyV9 * LPV * and LT ancestor

TSPyV * Avipolyomaviruses Exon 2 of MPyV MT OrangutanPyVBo MPyV * * HamsterPyV BatPyVB1130 RacoonPyV BatPyVR104 * Birth of ChimpPyV1a *alternate ORF GorillaPyV1 * * ChimpPyV2a *

Almipolyomaviruses MCPyV * ALTO protein of MCPyV Conserved stop codon

Includes another human virus (TSPyV) and oncogenic MousePyV The overprinting MousePyV gene (called MiddleT) is sufﬁcient to cause cancer Deﬁnes a clade of Almi(ALTO or MiddleT containing)polyomaviruses ALTO WAS BORN IN ONE VIRAL LINEAGE

RaccoonPyV ChimpPyV2a GgPyV1 MCPyV

OtomopsPyV2 ChaerephonPyV ChimpPyV1a

BatPyVR104 BatPyVB1130 RaccoonPyV BatPyVR104 EidolonPyV OtomopsPyV1 OrangutanPyVBo Almipolyomaviruses CardiodermaPyV VervetPyV1 TSPyV

84 OrangutanPyVPi MPyV 99 84 ChimpPyV1aMCPyVHamsterPyV OrangutanPyVPi ChimpPyV2a 93 100 91 GorillaPyVBatPyVB1130 100 72 92 MPyV ChimpPyVBob 84 HamsterPyV 100 HPyV9 100 LPV 16 76 AGMPyV

53 100 HPyV6 TSPyV

86 BirthH ofPy V7 100 alternateHP ORFyV10 OrangutanPyVBo BKPyV JCPyV 100 65 100 SV40 Non-Almipolyomaviruses EquinePyV BatPyVA504 100 100 100 MastomysPyV 100 100 VervetPyV3 100

Murinepneum100 otropicPyV LPV 84 BaboonPyV2 HPyV9 VervetPyV2 SquirrelmonkeyPyV SV40 BKPyV BaboonPyV1 JCPyV WUPyV KIPyV

WUPyV BudgerigarFledglinHPyV10 gPyV KIPyV FinchPyV CrowPyV Avipolyomaviruses GoosePyV

CanaryPyV HPyV7

Papillomavirus E1 Papillomaviruses HPyV6 A STEPWISE MODEL FOR OVERPRINTING BIRTH

Ancestral LT gene LT frame Potential for +1 frame +1 frame *** JCPyV, KiPyV start codon and hydrophobic motif Expansion of LT protein already present

* * * LPV, HPyV9

Innovation of ALTO/MT Removal of stop codons

* Ancestral Almipolyomavirus Subfunctionalization Create MT through splicing of ALTO/MT Expand LT/ALTO Expand LT/MT

* MCPyV * MPyV HOW WAS THIS NEW GENE BORN? YGS/T LxCxE motif motif JCPyV LargeT DNAj OBD ATPase/Helicase What was in place1 before ALTO was born? 688

MCPyV LargeT 1 817 ALTO (+1 frame) *

Start Hydrophobic motif LT YGS/T motif

DNA WUPyV LT frame +1 frame JCPyV

SV40

HPyV6

LPV

TSPyV

MPyV

RacoonPyV

BatPyVR104

GorillaPyV1

MCPyV

ALTO start codon HOW WAS THIS NEW GENE BORN? YGS/T LxCxE motif motif JCPyV LargeT DNAj OBD ATPase/Helicase What was in place1 before ALTO was born? 688

MCPyV LargeT 1 817 ALTO (+1 frame) *

Start Hydrophobic motif Hydrophobicity

TSPyV MPyV HamsterPyV BatPyVB1130 RacoonPyV BatPyVR104 ChimpPyV1a MCPyV Start of LT OBD domain

Hydrophobicity

HPyV9 LPV HPyV10 HPyV6 HPyV7 Start of LT OBD domain HOW WAS THIS NEW GENE BORN? The start codon and hydrophobic motif were in place before ALTO was born

Where did all of the extra sequence come from?

YGS/T LxCxE motif motif JCPyV LargeT DNAj OBD ATPase/Helicase 1 688

MCPyV LargeT 1 817 ALTO (+1 frame) * B YGS/T motif Start Hydrophobic motif WUPyV KIPyV BKPyV JCPyV SV40 HPyV10 HPyV6 HPyV7 HPyV9 LPV TSPyV OrangutanPyVBo MPyV HamsterPyV BatPyVB1130 RacoonPyV BatPyVR104 Birth of ChimpPyV1a GorillaPyV1 ALTO/MT ChimpPyV2a MCPyV 0 50 100 150 200 Start of exon 2 to OBD domain (residues) A STEPWISE MODEL FOR OVERPRINTING BIRTH

Ancestral LT gene LT frame Potential for +1 frame +1 frame *** JCPyV, KiPyV start codon and hydrophobic motif Expansion of LT protein already present

* * * LPV, HPyV9

Innovation of ALTO/MT Removal of stop codons

* Ancestral Almipolyomavirus Subfunctionalization Create MT through splicing of ALTO/MT Expand LT/ALTO Expand LT/MT

* MCPyV * MPyV WHAT DOES ALTO DO?

The short answer: we don’t know! WHAT DOES ALTO DO?

ChimpPyV2a GgPyV1 MCPyV ALTO ChimpPyV1a Function unknown BatPyVR104 RaccoonPyV

OrangutanPyVBo Almipolyomaviruses TSPyV OrangutanPyVPi MiddleT MPyV HamsterPyV Oncoprotein BatPyVB1130 ChimpPyVBob

HPyV9 LPV AGMPyV

HPyV6 HPyV7 EvolutionaryHPyV1 0connection to MousePyV allows BKPyV usJC PtoyV use MiddleT to understand ALTO SV40 Non-Almipolyomaviruses EquinePyV BatPyVA504 MiddleTMastomysPyV transforms cells using short linear MurinepneumotropicPyV motifsSquirrelmon ktoeyPy Vdisrupt cell cycle control WUPyV KIPyV BudgerigarFledglinSeveralgPyV of these motifs have phosphorylated Y’s FinchPyV CrowPyV Avipolyomaviruses GoosePyV CanaryPyV Does ALTO have conserved Y motifs? Do these motifs activate signaling cascades? Papillomavirus E1 Papillomaviruses Viral Other mammalian polyomaviruses genome (including 8 other human polyomaviruses)

Birth of a novel viral protein Human polyomavirus (TSPyV) Mouse polyomavirus Hamster polyomavirus Raccoon polyomavirus ALTO Bat polyomavirus Chimpanzee polyomavirus Merkel cell polyomavirus Lecture 3- Vignette 2 Deconvolution of two ancient mammalian antiviral families

Matt Daugherty, former postdoc now Assistant Professor, UC San Diego Antiviral protein Viral protein Heterozygosity or gene duplications provides host genomes with an advantage against viruses

Host Virus Host Virus IFIT genes: a case-study in diversity

Interferon IFIT1 IFIT2 IFIT3 IFIT5 MxA Interferon … stimulated Hundreds of genes other genes

IFIT: Interferon-induced with tetratricopeptide (TPR) repeats

Human 22 33 22 3 1B1B 21c 3c 131 5251c51b 3c1B1B 31 1c11b1 3c 515 1b 1 Chromosome 10

Mouse 2 3 1c2 3c 3 1c1b 3c 1 1b 1 Chromosome 19

What are the functional consequences of IFIT evolution? IFIT1 distinguishes ‘self’ from ‘non-self’

Me Me PPP PPP GPPPNN NN G NN Me pre-mRNA Cap0 Cap1 Nucleus Cytoplasm

Me Me PPP PPP GPPPNN NN G NN X Me Viral Cap0 Cap1 transcript

Mouse IFIT1

Dafﬁs et al, Nature, 2010 A discrepancy in IFIT1 function

Me Me GPPPNN PPPNN GPPPNN Me Viral Cap0 Cap1 transcript

Mouse IFIT1 Human IFIT1 Pichlmair, 2011 Andrejeva, 2013 IFIT phylogeny violates mammalian phylogeny

PRIMATES 2 3 1B 1 5

RODENTS 2 3 1c 3c 1b 1

CARNIVORES 2 3 1B 1 5

RODENT IFIT1 Whole gene phylogeny of IFIT1 and IFIT1B

PRIMATE IFIT1 Suggests many independent duplications (into the same chromosomal location)

PRIMATE IFIT1B Is there another possible explanation for this phylogeny?

DOG IFIT1 & 1B

CAT IFIT1 & 1B

PANDA IFIT1 & 1B

FERRET IFIT1 & 1B IFIT5 Recurrent recombination among IFIT1 parallels in mammals

Pairwise comparison of ferret IFIT1 & IFIT1B (black indicates divergence) RODENT IFIT1

PRIMATE IFIT1 Recombining 5’ end Non-recombining 3’ end Pairwise comparison of ferret IFIT1 & Panda IFIT1 PRIMATE IFIT1B

DOG IFIT1 & 1B

CAT IFIT1 & 1B RODENTS PANDA IFIT1 & 1B

FERRET IFIT1 & 1B PRIMATES IFIT5 DOG

CAT PANDA

FERRET

Identical breakpoint in all 4 carnivores! TWO ancient families of IFIT1 genes

Human Chromosome 2 10 3 2 3 1B1B 1c2 3c 131 521c51b 3c 1B 31 1c11b 3c 15 1b 1 Mouse Chromosome 19 2 3 1c2 3c 3 1B 1c1b 3c 1 1B 1b 1B 1 X 1B 1 IFIT1 Primates (Human IFIT1) Missing IFIT1: X Rodents Mice, rats, hamsters, guinea pigs Carnivores IFIT1B Humans (Human IFIT1B) Gibbons Missing IFIT1B: Old World monkeys Chimpanzees, New World monkeys, X New World monkeys Rodents (Mouse IFIT1,1b,1c) pigs, cows Carnivores IFIT2 IFIT3 IFIT5 IFIT1 and IFIT1B have been diverging for >100 million years! IFIT1 But many speciesPrimates only (Human have IFIT1) one or the other! X Rodents Carnivores IFIT1B X Humans (Human IFIT1B) Gibbons Old World monkeys X New World monkeys Rodents (Mouse IFIT1,1b,1c) Carnivores IFIT2 IFIT3 IFIT5 FUNCTIONAL DIVERGENCE OF IFIT1 PROTEINS?

IFIT1 Primates (Human IFIT1) Me Me Rodents GPPPNN GPPPNN X Me Carnivores Cap0 Cap1 IFIT1B Humans (Human IFIT1B) Gibbons Old World monkeys X New World monkeys Rodents (Mouse IFIT1,1b,1c) IFIT1B IFIT1 Carnivores IFIT2 IFIT3 IFIT5

IFIT1 Primates (Human IFIT1) Want toX test RodentsIFIT activity independent of rest of innate immune system Carnivores IFIT1B X Humans (Human IFIT1B) Gibbons Old World monkeys X New World monkeys Rodents (Mouse IFIT1,1b,1c) Carnivores IFIT2 IFIT3 IFIT5 A NOVEL YEAST ASSAY FOR IFIT FUNCTION

Me Me GPPPNN GPPPNN Human FTSJD2 Me Cap0 Cap1

IFIT1B Budding yeast IFIT off IFIT on Empty Mouse IFIT1B Mouse IFIT1B + FTSJD2

Yeast accurately recapitulatesEmpty IFIT1B molecular speciﬁcity

Human IFIT1

Human IFIT1 + FTSJD2

IFIT1 recognizes a different molecular pattern to arrest yeast growth Empty Empty Empty 7 Mouse IFIT1B 7 Mouse IFIT1B 8 Mouse IFIT1B 6 Human IFIT1 6 Human IFIT1 7 Human IFIT1 5 5 6 5 4 4 4 3 3 3 2 2

Log viral titer (PFU/ml) Log viral titer (PFU/ml) Log viral titer (PFU/ml) 2 1 1 1 0 24 48 0 24 48 0 12 18 24 Hours post-infection Hours post-infection Hours post-infection

Empty Empty Empty 7 Mouse IFIT1B 7 Mouse IFIT1B 8 Mouse IFIT1B Empty Empty Empty Human IFIT1 Human IFIT1 7 Human IFIT1 76 Mouse IFIT-IFIT1B MEDIATED7 6ANTIVIRAL RESTRICTION8 Mouse IFIT1B 6 65 Human IFIT1 65 7 5 Me 6 54 Me 54 PPP G4PPPNN G NN X 5 Me 43 43 Vaccinia J3 3 Cap0 4 Cap1 2 2 methyltransferase

Log viral titer (PFU/ml) Log viral titer (PFU/ml) Log viral titer (PFU/ml) 2 3 3 3 21 21 1 Log viral titer (PFU/ml) 0 24Vaccinia 48 Log viral titer (PFU/ml) IFIT1B0 24 48 Log viral titer (PFU/ml) 2 0 12 18 24 1 Hours post-infectionvirus 1 Hours post-infection 1 Hours post-infection 0 24 48 0 24 48 0 12 18 24 Hours post-infection Hours post-infection Hours post-infection Mutant vaccinia virusWildtype (Cap0) vaccinia virus (Cap1) Empty Empty Empty Empty Empty Empty Empty Empty 7 Mouse IFIT1B 7 Mouse IFIT1B 7 Mouse IFIT1B88 Mouse IFIT1B 7 Mouse IFIT1B 8 Mouse IFIT1B 7 Mouse IFIT1B Human IFIT1 Human IFIT1 Human IFIT17 Human IFIT1 Human IFIT1 Human IFIT1 6 Human IFIT1 6 6 77 6 7 6 6 5 5 5 6 5 6 5 55 5 4 4 4 4 4 44 4 3 3 3 3 3 33 3 2 2 2 2 Log viral titer (PFU/ml) Log viral titer (PFU/ml) Log viral titer (PFU/ml) Log viral titer (PFU/ml) Log viral titer (PFU/ml) Log viral titer (PFU/ml) Log viral titer (PFU/ml) Log viral titer (PFU/ml) 2 Log viral titer (PFU/ml) Log viral titer (PFU/ml) 2 2 Log viral titer (PFU/ml) 2 Log viral titer (PFU/ml) 1 1 1 11 1 1 1 0 24 48 0 24 0 48 240 12 48 18 0 24 24 48 0 12 18 24 0 24 48 0 12 18 24 Hours post-infection Hours post-infection Hours post-infectionHoursHours post-infection post-infection Hours post-infection Hours post-infection Hours post-infection

Empty Empty Empty 7 Mouse IFIT1B 8 7 EmptyMouse IFIT1B Empty Empty Empty Empty Empty 6 7 67 MouseHuman IFIT1BIFIT1 7 7 Mouse IFIT1B8 Mouse IFIT1B 7 8 Mouse IFIT1B Human IFIT1 5 Human IFIT167 7 56 6 6 6 4 56 6 45 5 5 5 45 5 4 43 4 4 3 34 4 2 3 Log viral titer (PFU/ml) 3 3 3 2 Log viral titer (PFU/ml) 23 3 Log viral titer (PFU/ml) 2 21 2 1 2 Log viral titer (PFU/ml) Log viral titer (PFU/ml) Log viral titer (PFU/ml) 1 0 24 Log viral titer (PFU/ml) 48 2 Log viral titer (PFU/ml) Log viral titer (PFU/ml) 2 0 24 48 0 12 18 24 1 1 Hours post-infection1 1 Hours post-infection1 1 0 Hours post-infection24 48 0 24 0 48 240 12 48 18 0 24 24 48 0 12 18 24 Hours post-infection Hours post-infection Hours post-infectionHours post-infection Hours post-infection Hours post-infection

Empty Empty Empty Empty Empty Empty 7 Mouse IFIT1B 8 7 Mouse IFIT1B 8 7 Mouse IFIT1B 7 Mouse IFIT1B Human IFIT1 6 7 6 7 6 6 Human IFIT1 5 6 5 6 5 5 5 4 4 5 4 4 4 4 3 3 3 3 3 3 2 2 Log viral titer (PFU/ml) Log viral titer (PFU/ml) Log viral titer (PFU/ml) 2 2 2 Log viral titer (PFU/ml) 2 Log viral titer (PFU/ml) 1 Log viral titer (PFU/ml) 1 1 1 1 0 24 1 48 0 12 18 0 24 24 48 0 24 48 0 24 48 0 12 18 24 Hours post-infection Hours post-infection Hours post-infection Hours post-infection Hours post-infection Hours post-infection IFIT-MEDIATED ANTIVIRAL RESTRICTION

Me Me GPPPNN X GPPPNN Vaccinia J3 Me Cap0 methyltransferase Cap1

Vaccinia IFIT1B virus

100 100 100

10 10 10 Methyltransferase-mutant Methyltransferase-mutant Methyltransferase-mutant 1 1

Vaccinia virus replication (%) Vaccinia 1 virus replication (%) Vaccinia Vaccinia virus replication (%) Vaccinia

Flag-IFITFlag-IFIT Flag-IFIT Vector Vector Vector Patas1B Patas1B Patas1A Patas1A Patas1B Patas1A Mouse1B Mouse1B Mouse1B Langur1B Langur1B Langur1A Langur1A Langur1B Langur1A Human1B Human1B Human1A Human1A Gibbon1B Gibbon1B Gibbon1A Gibbon1A Human1B Human1A Gibbon1B Gibbon1A Anti-FlagAnti-Flag Anti-Flag CONSEQUENCES OF IFIT1 EVOLUTION

Antiviral Molecular speciﬁcity recognition

IFIT1 Primates (Human IFIT1) X Rodents Carnivores ? ? IFIT1B Humans (HumanX IFIT1B) Gibbons Old World monkeys Speciﬁc Cap0 New World monkeys X recognition Rodents (Mouse IFIT1,1b,1c) Cap0 viruses Carnivores IFIT2 IFIT3 IFIT5

What is the function of IFIT1? Does it have antiviral activity? IFIT1 Primates (Human IFIT1) X Rodents Carnivores IFIT1B X Humans (Human IFIT1B) Gibbons Old World monkeys X New World monkeys Rodents (Mouse IFIT1,1b,1c) Carnivores IFIT2 IFIT3 IFIT5 Empty Empty Empty 7 Mouse IFIT1B 7 Mouse IFIT1B 8 Mouse IFIT1B 6 Human IFIT1 6 Human IFIT1 7 Human IFIT1 5 5 6 5 4 4 4 3 3 3 2 2

Log viral titer (PFU/ml) Log viral titer (PFU/ml) Log viral titer (PFU/ml) 2 1 1 1 0 24 48 0 24 48 0 12 18 24 Hours post-infection Hours post-infection Hours post-infection

Empty Empty Empty 7 Mouse IFIT1B 7 Mouse IFIT1B 8 Mouse IFIT1B Empty Empty Empty Human IFIT1 Human IFIT1 Human IFIT1 67 Mouse IFIT1B 67 78 Mouse IFIT1B 6 56 Human IFIT1 56 7 5 45 45 IFIT1 INHIBITS C6AP1 VIRUS REPLICATION 45 34 34 3 4 Me 23 23 PPP Log viral titer (PFU/ml) Log viral titer (PFU/ml) Log viral titer (PFU/ml) 2 G NN 3 Me 12 12 1 Log viral titer (PFU/ml) 0 24 48 Log viral titer (PFU/ml) 0 24 48 Log viral titer (PFU/ml) 2 0 12Cap1 18 24 1 Hours post-infection 1 Hours post-infection 1 Hours post-infection 0 24 48 0 24 48 0 12 18 24 Hours post-infection Hours post-infectionVesicular HoursIFIT1 post-infection stomatitis Empty EmptyEmpty virus (VSV) Empty EmptyEmpty Empty Empty 7 Mouse IFIT1B 77 Mouse IFIT1B 8 Mouse IFIT1B 7 MouseMouse IFIT1BIFIT1B Mouse IFIT1B Mouse IFIT1B 6 Human IFIT1 66 Human IFIT1 7 Human IFIT1 66 Human IFIT1 6 5 55 6 5 5 4 44 4 4 3 33 33 3 3 2

2 Log viral titer (PFU/ml) 2 Log viral titer (PFU/ml) Log viral titer (PFU/ml) Log viral titer (PFU/ml) Log viral titer (PFU/ml) Log viral titer (PFU/ml) 2 Log viral titer (PFU/ml) Log viral titer (PFU/ml) 2 Log viral titer (PFU/ml) 1 11 1 1 0 24 48 00 2424 4848 0 12 18 24 00 2424 4848 0 24 48 0 12 18 24 Hours post-infection HoursHours post-infection post-infection Hours post-infection HoursHours post-infectionpost-infection Hours post-infection Hours post-infection

Empty IFIT1 is a potent antiviral, but with speciﬁcity that differs from IFIT1B Empty Empty 7 Mouse IFIT1B 8 7 EmptyMouse IFIT1B Empty Empty 6 7 67 MouseHuman IFIT1BIFIT1 7 8 Mouse IFIT1B Human IFIT1 5 67 56 6 4 56 45 5 45 4 43 3 34 2 3 Log viral titer (PFU/ml) 3 2 Log viral titer (PFU/ml) 23 Log viral titer (PFU/ml) 2 21 1 Log viral titer (PFU/ml) 1 Log viral titer (PFU/ml) 0 24 48 Log viral titer (PFU/ml) 2 0 24 48 0 12 18 24 1 1 Hours post-infection 1 Hours post-infection 0 Hours post-infection24 48 0 24 48 0 12 18 24 Hours post-infection Hours post-infection Hours post-infection

Empty Empty Empty 7 Mouse IFIT1B 8 7 Mouse IFIT1B 6 7 6 Human IFIT1 5 6 5 4 5 4 4 3 3 3 2 Log viral titer (PFU/ml) 2 Log viral titer (PFU/ml) 2 Log viral titer (PFU/ml) 1 1 1 0 24 48 0 12 18 24 0 24 48 Hours post-infection Hours post-infection Hours post-infection IFIT1 EVOLUTION ALTERS ANTIVIRAL DEFENSES

Antiviral Molecular speciﬁcity recognition

IFIT1 Primates (Human IFIT1) X Rodents Carnivores ? VSV, others? IFIT1B Humans (Human IFIT1B) Gibbons Old World monkeys Speciﬁc Cap0 New World monkeys X recognition Rodents (Mouse IFIT1,1b,1c) Cap0 viruses Carnivores IFIT2 IFIT3 IFIT5

IFIT1 and IFIT1B are distinct gene families that diverged millions of years ago IFIT1 IFIT1 and IFIT1B havePrimates evolved (Human different IFIT1) mechanisms and antiviral speciﬁcities X Rodents Carnivores IFIT1B X Humans (Human IFIT1B) Gibbons Old World monkeys X New World monkeys Rodents (Mouse IFIT1,1b,1c) Carnivores IFIT2 IFIT3 IFIT5 Recombination may also help explain MxA/MxB evolution

Mitchell et al. PLOS Pathogens 2015 Recombination may also help explain MxA/MxB evolution

GARD segment A

Mitchell et al. PLOS Pathogens 2015 Lecture 3- Vignette 3 An unusual origin for a host immune gene

Seemay Chou Matt Daugherty, former postdoc former postdoc, Mougous lab, UW now Assistant Professor, UC San Diego now Assistant Professor, UC San Francisco ‘Stealing’ immunity genes

Host Pathogen

Horizontal gene transfer Interbacterial warfare

Bacteria Bacteria Bacterial genes in eukaryotic genomes

Anabaena

Cyanothece

Acidovorax

ChondromycesChondromyces

Salmonella Salmonella Cronobacter Burkholderia Elioraea Ticks and mites Escherichia Skermanella Beijerinckia Janthinobacterium Burkholderia Duganella Acidovorax

Burkholderia BurkholderiaBurkholderia Rhodanobacter Sagittula Ixodes scapularis Acidovorax Daphnia pulex

Azospirillum Metaseiulus occidentalis

0.2

Magnetospirillum Janthinobacterium

OxalobacteraceaeOxalobacteraceae Phaeospirillum

Daphnia Chou*, Daugherty*,…Malik, Mougous Nature 2015 Antibacterial effector genes were ‘stolen’ at least 6 times

Ticks and mites Oxytricha Mollusks

Daphnia Naegleria Sea anemone, Lancelet and others

Surrounded by other eukaryotic genes

Contain introns

Chou*, Daugherty*,…Malik, Mougous Nature 2015 GENES WERE ‘STOLEN’ AT LEAST 6 TIMES

A Heterolobosea (Naegleria, dae3) Jakobida tae3 Excavata Oxymonads and diplomonads

tae3 Archeplastida (plants) Ciliates (Oxytricha, dae3) tae2 Dinoflagellates Chromalveolata Diatoms Crustaceans (Daphnia, dae2) Insects tae2 Arachnids (Mites & ticks, dae2) tae4 Rhizaria Lophotrochozoa (Capitella & mollusks, dae4)

Eukaryotic ancestor Mammals Cephalochordate (Lancelet, dae4) Amoebozoa Hemichordate (Acorn worm, dae4) Cnidaria (Sea anemone, dae4) Choanoflagellates (Monosiga, dae4) Opisthokonta tae4 Fungi

Splice junctions B 0.97 C Burkholderia I. scapularis: * * Daphnia magna Renamed* DomesticatedDaphnia pulex amidaseM. occidentalis effector: (Dae) genes 0.93 Daphnia pulex Daphnia pulex * 0.96 Cyanobacteria D Amblyomma cajennense *Amblyomma triste Metastriate ticks Amblyomma parvum (Amblyomma) * Amblyomma maculatum * Rhipicephalus pulchellus Metastriate ticks Rhipicephalus sanguineus Chou*,(Rhipicephalus) Daugherty*,…Malik, Mougous Nature 2015 tae2 * 0.97 Ixodes scapularis Prostriate ticks (Ixodes) * Metaseiulus occidentalis Parasitiform mites Dermanyssus gallinae (Metaseiulus & Dermanyssus) ther Acariform mites O

Dermatophagoides pteronyssinus arthropods (Dermatophagoides) * Burkholderia * Beijerinckia indica 0 200 400 Divergence time (MYA) * Rhodospirillales

Other bacteria * Chondromyces apiculatus * Chronobacter spp * Rhodanobacter spathiphylli Burkholderia sp 0.2

C Conserved splice junction I. scapularis: RWVRGQH VKSNCGSIP Chou et al, Figure 1 M. occidentalis: SWRKGAK VRGICNSIP * :* : *:. *.*** Conservation of catalytic residues … and a new way to get out of cells

A CatalyticCatalytic CatalyticCatalytic Catalytic Catalytic Catalytic Catalytic A N-terminusA Acysteine histidine N-terminus ConservedcysteineN-terminus catalytichistidine residues NewN-terminus cysteinesecretion signalhistidine cysteine histidine Tae2Tae2 Tae2 Bacterial Tae2 BacterialBacterial consensusconsensus Bacterial consensusconsensus Bacterial consensus A. cajennense MKGFVICAALLVLGMAVGSSQAI KECVALVK HAAIFES A. cajennense MKGFVICAALLVLGMAVGSSQAA. cajennenseI MKGFVICAALLVLGMAVGSSQAKECVALVK A.H cajennenseAAIFES IMKGFVICAALLVLGMAVGSSQAKECVALVK HAAIFEI S KECVALVK HAAIFES A. triste MNGFLLCAALLVLGMAVGSQAI KECVALVK HAAIFES A. triste MNGFLLCAALLVLGMAVGSQAIA. triste MNGFLLCAALLVLGMAVGSQAKECVALVK HAAIFEA. tristeS I MNGFLLCAALLVLGMAVGSQAKECVALVK HAAIFEI S KECVALVK HAAIFES A. parvum MKCFVTCTALLVLGMAVGSQAI KECVALVK HAAIFES A. parvum MKCFVTCTALLVLGMAVGSQAA. Iparvum MKCFVTCTALLVLGMAVGSQAKECVALVK HAAIFEA. parvumS I MKCFVTCTALLVLGMAVGSQAKECVALVK HAAIFEI S KECVALVK HAAIFES A. maculatum MNGFILSAALLVLGMAVGSQAI KECVALVK HAAIFES A. maculatum MNGFILSAALLVLGMAVGSQAA. maculatumI MNGFILSAALLVLGMAVGSQAKECVALVK A.H maculatumAAIFES I MNGFILSAALLVLGMAVGSQAKECVALVK HAAIFEI S KECVALVK HAAIFES R. pulchellus>400 millionMKGFVVSVGLLLLGMAIVIQA years I RECVALVK HAAIFES R. pulchellus MKGFVVSVGLLLLGMAIVIQAR. pulchellusI MKGFVVSVGLLLLGMAIVIQARECVALVK R.H AAIFEpulchellusS I MKGFVVSVGLLLLGMAIVIQARECVALVK HAAIFEI S RECVALVK HAAIFES R. sanguineus MKGFVISVGLLLLGMTVVTQAI KECVALVK HAAIFES R. sanguineusof tickMKGFVISVGLLLLGMTVVTQA and mite R. sanguineusI MKGFVISVGLLLLGMTVVTQAKECVALVK R.H sanguineusAAIFES I MKGFVISVGLLLLGMTVVTQAKECVALVK HAAIFEI S KECVALVK HAAIFES I. scapularis MKMGVTPLVLVVSIAFSLNCEVDAI KECVALVK HAAIFES I. scapularis evolutionMKMGVTPLVLVVSIAFSLNCEVDAI. scapularisI MKMGVTPLVLVVSIAFSLNCEVDAKECVALVK HI. AAIFEscapularisS MKMGVTPLVLVVSIAFSLNCEVDAI KECVALVK HAAIFEIS KECVALVK HAAIFES M. occidentalis MKLFLISAALVVLGLAAVSDAM GSAAALIK HAGIFVR M. occidentalis MKLFLISAALVVLGLAAVSDAM. occidentalisM MKLFLISAALVVLGLAAVSDAGSAAALIK M. HoccidentalisAGIFVR M MKLFLISAALVVLGLAAVSDAGSAAALIK HAGIFVM R GSAAALIK HAGIFVR D. gallinae MSPSTVTGALLALLAFTVGVSGM GSAAGLVK HAAILVR D. gallinae MSPSTVTGALLALLAFTVGVSGMD. gallinae MSPSTVTGALLALLAFTVGVSGMGSAAGLVK HD.AAILV gallinaeR MSPSTVTGALLALLAFTVGVSGMGSAAGLVK HAAILVR GSAAGLVK HAAILVR D. pteronyssinus MYSFKYCLAAAILLLVGNVCQAF QECAALVQ HTAIFVR D. pteronyssinus MYSFKYCLAAAILLLVGNVCQAD. pteronyssinusF MYSFKYCLAAAILLLVGNVCQAQECAALVQ D. pteronyssinusHTAIFVR FMYSFKYCLAAAILLLVGNVCQAQECAALVQ HTAIFVF R QECAALVQ HTAIFVR

D. magna MKFFIVTVFVITFIDQSMGG GECVSLYK HVAIYVG D. magna MKFFIVTVFVITFIDQSMGGD. magna MKFFIVTVFVITFIDQSMGGGECVSLYK HVAIYVGD. magna MKFFIVTVFVITFIDQSMGGGECVSLYK HVAIYVG GECVSLYK HVAIYVG D. pulex MGSSMKLFTFTFVLLALIELGFGG GECVSLYK HVAIYVG D. pulex MGSSMKLFTFTFVLLALIELGFGGD. pulex MGSSMKLFTFTFVLLALIELGFGGGECVSLYK HVAIYVGD. pulex MGSSMKLFTFTFVLLALIELGFGGGECVSLYK HVAIYVG GECVSLYK HVAIYVG D. pulex MAKLLLLLFLAATLKSSFGA GECVSFYK HTAIYVG D. pulex MAKLLLLLFLAATLKSSFGA D. pulex MAKLLLLLFLAATLKSSFGAGECVSFYK HTAIYVGD. pulex MAKLLLLLFLAATLKSSFGAGECVSFYK HTAIYVG GECVSFYK HTAIYVG D. pulex MPLCEPSKHVGTLLADPWGGQ GECVSFVK HAAVYMG D. pulex MPLCEPSKHVGTLLADPWGGQD. pulex MPLCEPSKHVGTLLADPWGGQGECVSFVK HAAVYMGD. pulex MPLCEPSKHVGTLLADPWGGQGECVSFVK HAAVYMG GECVSFVK HAAVYMG BB B B Tae3Tae3 Tae3 Tae3 Chou*, Daugherty*,…Malik, Mougous Nature 2015 BacterialBacterial consensusconsensus Bacterial consensus Bacterial consensus N. gruberi MSQVKTVILLLLIALVSAV GKCAKYTA HPDGHMQM N. gruberi MSQVKTVILLLLIALVSAV N. gruberi MSQVKTVILLLLIALVSAVGKCAKYTA HPDGN. HgruberiMQM MSQVKTVILLLLIALVSAVGKCAKYTA HPDGHMQM GKCAKYTA HPDGHMQM N. gruberi MHSSKLIIAVLLMVIASCVVVSSR GHCARAVR HPHGHIQI N. gruberi MHSSKLIIAVLLMVIASCVVVSSRN. gruberi MHSSKLIIAVLLMVIASCVVVSSRGHCARAVR HPHGN. HgruberiIQI MHSSKLIIAVLLMVIASCVVVSSRGHCARAVR HPHGHIQI GHCARAVR HPHGHIQI N. gruberi MHKKTLILGVALLLFALFAVVARAA GYCARAVR HPHGHMQV N. gruberi MHKKTLILGVALLLFALFAVVARAAN. gruberi MHKKTLILGVALLLFALFAVVARAAGYCARAVR HPHGN. HgruberiMQV MHKKTLILGVALLLFALFAVVARAAGYCARAVR HPHGHMQV GYCARAVR HPHGHMQV

O. trifallax MLSKALAFGALALTVSAD GLCAKYVR HIHGHITV O. trifallax MLSKALAFGALALTVSAD O. trifallax MLSKALAFGALALTVSADGLCAKYVR HIHGO. HtrifallaxITV MLSKALAFGALALTVSADGLCAKYVR HIHGHITV GLCAKYVR HIHGHITV CC C C Tae4Tae4 Tae4 Tae4 BacterialBacterial consensusconsensus Bacterial consensus Bacterial consensus B. floridae MKATVWLVVVLFACVWNESSAW NTCAMRVS TGHVDLWD B. floridae MKATVWLVVVLFACVWNESSAWB. floridae MKATVWLVVVLFACVWNESSAWNTCAMRVS TGHB.VDLWD floridae MKATVWLVVVLFACVWNESSAWNTCAMRVS TGHVDLWD NTCAMRVS TGHVDLWD B. floridae MLKTTVWFAAVLFACVWHGSSAW NTCTMRVS TGHVDLWD B. floridae MLKTTVWFAAVLFACVWHGSSAWB. floridae MLKTTVWFAAVLFACVWHGSSAWNTCTMRVS TGHB.VDLWD floridae MLKTTVWFAAVLFACVWHGSSAWNTCTMRVS TGHVDLWD NTCTMRVS TGHVDLWD B. floridae MRPRPGWPKFSELKSNYPSY NTCAMRVS TGHVDLWD B. floridae MRPRPGWPKFSELKSNYPSYB. floridae MRPRPGWPKFSELKSNYPSYNTCAMRVS TGHB.VDLWD floridae MRPRPGWPKFSELKSNYPSYNTCAMRVS TGHVDLWD NTCAMRVS TGHVDLWD B. floridae MEKKLLLCLVLLAIPVATAQ NTCAMRVS TGHVDLWD B. floridae MEKKLLLCLVLLAIPVATAQB. floridae MEKKLLLCLVLLAIPVATAQNTCAMRVS TGHB.VDLWD floridae MEKKLLLCLVLLAIPVATAQNTCAMRVS TGHVDLWD NTCAMRVS TGHVDLWD S. kowalevskii MSTWPSFEELWENYPNYRDW NTCAIRLS TGHVDLWD S. kowalevskii MSTWPSFEELWENYPNYRDWS. kowalevskii MSTWPSFEELWENYPNYRDWNTCAIRLS S.TG kowalevskiiHVDLWD MSTWPSFEELWENYPNYRDWNTCAIRLS TGHVDLWD NTCAIRLS TGHVDLWD N. vectensis MNCIIQLLELFCVIGHISHT NTYAIRLS TGHVDLYD N. vectensis MNCIIQLLELFCVIGHISHTN. vectensis MNCIIQLLELFCVIGHISHTNTYAIRLS TGN.H vectensisVDLYD MNCIIQLLELFCVIGHISHTNTYAIRLS TGHVDLYD NTYAIRLS TGHVDLYD N. vectensis MDLVLSLHLELSVYEVNFAI NTCAIRLS TGHVDLYD N. vectensis MDLVLSLHLELSVYEVNFAIN. vectensis MDLVLSLHLELSVYEVNFAINTCAIRLS TGN.H vectensisVDLYD MDLVLSLHLELSVYEVNFAINTCAIRLS TGHVDLYD NTCAIRLS TGHVDLYD M. brevicollis MALNFDKMWQDPVTAMDHAS NYCAIRLS TGHVTLWN M. brevicollis MALNFDKMWQDPVTAMDHASM. brevicollis MALNFDKMWQDPVTAMDHASNYCAIRLS TGM.H brevicollisVTLWN MALNFDKMWQDPVTAMDHASNYCAIRLS TGHVTLWN NYCAIRLS TGHVTLWN

V. lienosa MNHAFMRRWTATLLLLAATPAKSE DTSALRLS KGHVALWD V. lienosa MNHAFMRRWTATLLLLAATPAKSEV. lienosa MNHAFMRRWTATLLLLAATPAKSEDTSALRLS KGHV.VALWD lienosa MNHAFMRRWTATLLLLAATPAKSEDTSALRLS KGHVALWD DTSALRLS KGHVALWD E. complanata MNHAFMRRWTATLLLLAATPAKSE DTSALRLS KGHVALWD E. complanata MNHAFMRRWTATLLLLAATPAKSEE. complanata MNHAFMRRWTATLLLLAATPAKSEDTSALRLS E.KG complanataHVALWD MNHAFMRRWTATLLLLAATPAKSEDTSALRLS KGHVALWD DTSALRLS KGHVALWD L. gigantea MIFKVLLVVLYCTLSVIAD DTSALRLS KGHVALWD L. gigantea MIFKVLLVVLYCTLSVIADL. gigantea MIFKVLLVVLYCTLSVIADDTSALRLS KGL.H VALWDgigantea MIFKVLLVVLYCTLSVIADDTSALRLS KGHVALWD DTSALRLS KGHVALWD L. stagnalis MKVLVTLCVILTKCAVTRGE DTSALRLS KGHVALWD L. stagnalis MKVLVTLCVILTKCAVTRGEL. stagnalis MKVLVTLCVILTKCAVTRGEDTSALRLS KGL.H VALWDstagnalis MKVLVTLCVILTKCAVTRGEDTSALRLS KGHVALWD DTSALRLS KGHVALWD A. californica MKQGLLLFVLLTGVLTPTKAY DTSALRLS KGHVALWD A. californica MKQGLLLFVLLTGVLTPTKAYA. californica MKQGLLLFVLLTGVLTPTKAYDTSALRLS KGA. HcalifornicaVALWD MKQGLLLFVLLTGVLTPTKAYDTSALRLS KGHVALWD DTSALRLS KGHVALWD S. constricta MAAEQGLTSVALRDALTLRQ DTSALRLS KGHVALWD S. constricta MAAEQGLTSVALRDALTLRQS. constricta MAAEQGLTSVALRDALTLRQDTSALRLS KGS.H constrictaVALWD MAAEQGLTSVALRDALTLRQDTSALRLS KGHVALWD DTSALRLS KGHVALWD E. complanata MELLYFTIFFVLGDLVSAL DTSALRLS VGHVALWD E. complanata MELLYFTIFFVLGDLVSALE. complanata MELLYFTIFFVLGDLVSALDTSALRLS E.VG complanataHVALWD MELLYFTIFFVLGDLVSALDTSALRLS VGHVALWD DTSALRLS VGHVALWD M. galloprovincialis MELHSTILVILLIAEYVVGD DTSALRLS RGHIVLWD M. galloprovincialis MELHSTILVILLIAEYVVGDM. galloprovincialis MELHSTILVILLIAEYVVGDDTSALRLSM. galloprovincialisRGHIVLWD MELHSTILVILLIAEYVVGDDTSALRLS RGHIVLWD DTSALRLS RGHIVLWD C. gigas MDYLKCLPVLLSCAIASLSETV DTSALRMS KGHIALWN C. gigas MDYLKCLPVLLSCAIASLSETVC. gigas MDYLKCLPVLLSCAIASLSETVDTSALRMS KGHIALWNC. gigas MDYLKCLPVLLSCAIASLSETVDTSALRMS KGHIALWN DTSALRMS KGHIALWN C. teleta MVFSLDDAVLLLLTTLAACWTTTTAF NTAPMRMS SGHMGLWD C. teleta MVFSLDDAVLLLLTTLAACWTTTTAFC. teleta MVFSLDDAVLLLLTTLAACWTTTTAFNTAPMRMS SGHMGLWDC. teleta MVFSLDDAVLLLLTTLAACWTTTTAFNTAPMRMS SGHMGLWD NTAPMRMS SGHMGLWD

ChouChou etet al,al, FigureFigure S6S6 Chou et al, Figure S6 Chou et al, Figure S6 a d 3 2 RC ControlNymphAdult MG SG HL -1 α-Actin Bs, WT 0 α-Dae2 Bs, C43A Ec, WT iability (log c.f.u.) -1 V Ec, C43A Dae-mediated antibacterial% -2 immunity in vivo b 0.001 0.01 0.1 1 7 [Dae2] mM e a d 3 Ixodes scapularis (deer tick)11 is the vector for the Lyme P < 0.005 6 disease pathogen (Borrelia10 burgdorferi) actin)

2 4 /min/mol) RC ControlNymphAdult MG SG HL 9 -1 600 α-Actin ysis rate Lyme disease is the most prevalent vector-borne illness in L OD 5 Bs, WT 8 Δ

0 expression α-Dae2 the US Bs, C43A Ec, WT 7 iability (log c.f.u.) -1(log N/D V 4 dae2 per 10 Ec, C43A dae2 2 6

% ys -2 L WT 5 C43A (log b 0.001 0.01 0.1 1 Control dae2 7 c RNAi[Dae2] knockdown mM 2 weeks post-engorgement ) f

0 e 0 ) 6 11 2 D P < 0.005 P < 0.0005

6 10 actin)

2 -1

4 0 /min/mol) trospA

g Cyto-WT load 4 o 9

l 600

( Cyto-C43A -2 ysis rate -2

t L OD 5 8 Peri-WT

i Δ

expression Peri-C43A n -4 7 e -3

(log N/D

l dae2 per 10 4 l dae2 -6 flaB per 10

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ys C L WT 0 5 50 100 150 C43A (log -8 (log c Time Control (min) dae2 Control dae2

) f

0 0 ) 6 2 D P < 0.0005 Chou*, Daugherty*,…Malik, Mougous Nature 2015

2 -1 0 trospA

g Cyto-WT load 4

( Cyto-C43A -2 -2

y t Peri-WT

i s Peri-C43A n -4

e -3

l l -6 flaB per 10 2 e -4 B. burgdorferi C 0 50 100 150 -8 Time (min) (log Control dae2 Horizontal gene transfer as a mechanism to acquire immunity

Bacteria

Eukaryotes Bacteria

By stealing well-honed interbacterial toxins, eukaryotes get ‘out-of- the-box’ antibacterial immunity factors GENOMIC VIEW OF SELECTION

2500

2000 Non-Immunity 1500

1000

500 number ofgenes number 0 0.5 1.0 1.5 >2 dN/dS purifying neutral positive selection

Siepel Lab beyond the usual suspects

Genetic conﬂicts between components of the same cell and even same genome beyond the usual suspects

Genetic conﬂicts between components of the same cell and even same genome

“selﬁsh” mtDNA replication/ mitochondria transcription proteins

mtRNAPol beyond the usual suspects

Genetic conﬂicts between components of the same cell and even same genome

a/A A >> a Meiosis

• Alleles are evolutionary competitors - fight fair (promote fitness; fitter allele ‘wins’) - cheat to win during/after meiosis; unfit allele can sometimes ‘win’ Mitosis ‘Cheating’ after Male meiosis chromosome segregation

What are possible suppressors?

Meiosis I Meiosis II

What is the nature of this poison? ‘Cheating’ during oocyte chromosome segregation oocyte

Female meiosis

Meiosis I Meiosis II