breaking down the barriers: first steps in herpes simplex virus reactivation · 2018-05-14 ·...

Breaking down the barriers: first steps in herpes simplex virus reactivation

Angus WilsonDepartment of MicrobiologyNew York University School of Medicine

• Widespread human pathogen associated with a broad spectrum of disease states

• Recurrent reactivation of latent virus is a major contributor to persistence and pathogenesis

• Currently lack effective vaccines or drugs to control latency/reactivation

HSV-1 establishes latency in the human peripheral nervous system

• Latent viral genomes reside in the neuronal nucleus, which in turn is hidden in the nerve ganglion.

• The latent genome is circularized and associated with chromatin carrying repressive histone modifications and factors that limit viral gene expression.

GanglionEpithelia/exterior

PRIMARY INFECTION

LATENCY

REACTIVATION

Control of latency is tightly coupled to neuronal physiology

“Stress”

Modeling HSV-1 latency in cultured primary neurons

Camarena et al. 2010 Cell Host & Microbe; Kobayashi et al. 2012 JoVE; Jurak et al. 2013 JVI; Kim et al. 2014 Methods Mol Biol.

Isolate superior cervical ganglia

(SCG)

Dissociateganglia

Establish neuron culturesNerve growth factor (NGF)

+ 5-FU & aphidicolin

Infect with HSV-1 + acyclovir (ACV)

E21 prenatal rats HSV-1Adaptation of Wilcox & Johnson protocols (1988-90)

In vivo infections In vitro infections

Sustained cap-dependent translation governs HSV-1 latency

Camarena et al. 2010 Cell Host & Microbe; Kobayashi et al. 2012 Genes & Dev

VLADIMIRCAMARENA

MARIKOKOBAYASHI

Inhibition of cap-dependent translation using constitutive 4EBP1 repressor

MARIKOKOBAYASHI

Growth of neurons7 days

Collection of samples

1-12 hours

Fold

cha

nge

Fold

cha

nge

Viral genomic DNAAcute infection profileqPCRqRT-PCR

IE E L

hours post infection (h.p.i.)

HSV-

1 ge

ne e

xpre

ssio

n(m

RNA

leve

l)

Acute infections of SCG neurons follow the canonical cascade

establishneuroncultures

infectwith

HSV-1

Ju Youn Kim

Textbook view

Inducing reactivation results in two waves of viral gene transcription


infectwith

HSV-1

supply reactivation

stimulus

DIV 1 DIV 6-7 DIV 12-14

sample

+ NGF

+ ACV

Viral genome amplification

Infectious virus

Plaq

ues

(pfu

/ml)

Fold

cha

nge

Fold

Cha

nge

hours + LY

Lytic mRNAs (RT-qPCR)

Ju Youn Kim et al. 2012 PLoS Pathogens

Fundamental differences between Phase I & Phase II


Fold

Cha

nge

CHX:

Lytic mRNAs (RT-qPCR)

ICP27

UL5UL30

VP16

UL36

- +

4

8

12

20 hours + LY (Phase I)

E & L gene transcription independent of protein synthesis

Fold

Cha

nge

2.5

7.5

10

5

0

‘Late’ mRNAs (RT-qPCR)

- PAA+ PAA

P < 0.045

Ph I Ph II Ph I Ph II

UL36VP16

L gene transcription independent of DNA replication

• Biphasic profile unaffected by DNA encapsidation inhibitor (WAY150138)Phase II not due to spread

VP16-HCF complex drives IE gene transcription

HCF-1

Oct-1

ICP0

ICP27ICP4

ICP47ICP22

20 h + LY (Phase I)

α-VP16+ DAPI

α-VP16

0 15 20 25 480

0.4

0.8

1.2

Nuc

lear

acc

umul

atio

n in

VP

16 p

ositi

ve n

euro

ns(%

)

hours + LY

*

*

48 h + LY (Phase II)


α-VP16+ DAPI

α-VP16

25-30% neurons VP16+

VP16

LY treatment promotes HCF-1 nuclear accumulation

α-HCF-1

0

10

20

30

40

LY: - +

Neu

rons

with

nuc

lear

HC

F-1

(%)

+/- LY (30 hours)


Kristie TM, Vogel JL, Sears AE (1999) Nuclear localization of the C1 factor (host cell factor) in sensory neurons correlates with reactivation of herpes simplex virus from latency. PNAS 96: 1229–1233.

α-ICP27

α-ICP27 α-HCF-1

25 h + LY (Phase I)

shRNA depletion of VP16 results in reduced Phase II lytic mRNA levels


VP16 knockdown

α-VP16blot

α-RhoGDIblot

50

75

shVP16:control: ++

mRNA levels (RT-qPCR)

shVP16:control:

2

1Fo

ld d

iffer

ence

(KD

/con

trol)

20h 48h15h

++

++

++


infectwith

HSV-1

supply reactivation

stimulus

DIV 1 DIV 6 DIV 16

sample

+ NGF

+ ACV

infectwith

lentivirus

DIV 11

JNK activation connects Phase I to neuronal stress response pathways

Explant/axotomy of latently infected murine trigeminal ganglia

ICP27 mRNAPhase I (6h)

Viral DNAPhase II (48h)

Infectious virusPhase II (48h)

SP600125: + + +

Anna Cliffe et al. Deshmukh 2015 Cell Host & Microbe Cliffe & Wilson 2016 JVI

HSV Lytic Promoters

H3K9me3

Phase II/Full Reactivation

H3K27me3

IEE

LPhase I/Animation

IEE L acH3

Histone demethylases

Latency

JNK

H3pS10

DLK/JIP3JNK?

VP16

Histone methyl/phospho switchallows derepression of viral genes without

removal of repressive lysine marks

Hostfactor(s)

miRNAs

ViralmRNAs

ICP0

VP16

ICP27

Latent HSV-1Repression of viral

productive cycle genes

Phase I (‘Animation’)Wide-scale transcription & de

novo synthesis of viral proteins

Phase II (‘Synthesis’)Transcriptional directed by viral proteins & viral DNA replication

Viralepisome

Phase I provides latent HSV-1 with missing tegument factors

incomingvirion

Releasetegumentfactors

Acute infection

stresssignaling pathways

JNK

Latency can be established without ACV by infection at low MOI

DIV = 0 DIV = 7 DIV = 14

Isolate neurons

Infect with HSV-1 Us11-GFPMOI=0.001-0.01

neuron culture (7 days)

Score GFP+ wellsHarvest RNA

Establishment period (7 days)

seed in multi-well dishes

HSV-1 Us11-GFP at MOI of 0.001(7 days post infection without ACV)

DIC GFP

GFP-

GFP+

MOI 0.01 0.001

Perc

enta

ge o

f wel

ls (%

)

0

20

40

60

80

100

1 2

0

20

40

60

80

100

Wells without neurons expressing GFPWells with neurons expressing GFP

Are these wells latently

infected?

Ju Youn Kim, unpublished

Detection of LAT ncRNA in GFP negative wells 7 days after infection

LAT

rat Rpl19

+ -virus

control GFP negative wells

MOI = 0.001 (50 pfu/well) without ACV

Concordance between input virus and # of neurons supporting reactivation

Infection MOI = 0.001without ACV

Num

ber o

f GFP

+ ne

uron

s

Reactivate in presence of WAY150138

to prevent spread

0

5

10

15

20

25

30

35

40

45

Efficient establishment of latency at low MOI


Biphasic reactivation from latency established without ACV

Rela

tive

vira

l gen

omic

DN

A

Rela

tive

vira

l mRN

A le

vels

+ LY294002 inducer (hours)


MOI=1+ 100 μm ACV

1 2 3 4 5 60

5

1050

100150

Rel

ativ

e vi

ral m

RN

A le

vels

0

5

1050

100

150ICP27 (IE)

0 15 20 25 48 72

Data 6

1 2 3 4 5 6

0100200

100000

200000

300000UL30 (E)

2x105

3x105

1x105

0100200

0 15 20 25 48 72

Geno

mic

DN

A (fo

ld c

hang

e)0 15 20 25

Time post-induction (hours)48

Phase I Phase II

MOI=0.01without ACV


GanglionEpithelia/exterior

PRIMARY INFECTION

LATENCY

REACTIVATION

IFN-γ

IFN-α/β

CD8+

memory T cells

GrnB

HSV-specific T-cells infiltrate ganglia and prevent reactivation without destroying the neurons

Exogenous interferon inhibits reactivation in neuron-only cultures

Jessica Linderman et al. 2017 Cell Reports

Not shown: Reactivation signal not impaired by IFN

IFNβ IFNγJESSICA LINDERMANMARIKO KOBAYASHI

Interferon is less effective after Phase I

remove LY+20 h

establishHSV-1latency

reactivationstimulus+LY 0h

+ IFN during & after LY

scorereactivation

Phase I Phase II

+ IFN during LY

+ IFN after LY

Linderman et al. 2017 Cell Reports

Not shown: LY does not interfere with induction of

IFN stimulated genes (ISGs)

HSV-1 encodes multiple IFN antagonists

25 h + LY (Phase I)

ICP0• SUMO-dependent Ub E3 ligase• Disrupts ND10 bodies• Impairs activation of sensors• Blocks induction of ISGsICP34.5• Inhibits IFN-mediated eIF-2α

phosphorylationICP27• Inhibits IFN & ISG expressionUs11• Inhibits 2’-5 OAS synthesis & eIF-2α

phosphorylationVhs• Inhibits STAT signaling & eIF-2α

phosphorylationUs3• Inhibits IFN receptor signaling & ISG

synthesis

Okay immune system: bring it

on!

Ectopic ICP0 allows HSV-1 reactivation in the presence of IFN-γ

Establish HSV-1 latency Ad-ICP0 infectionInduce reactivation

+ IFNβ or IFNγ

score reactivation


IFNβ and IFNγ induce unique profiles of ISG

expression in SCG neurons

Hostfactor(s)

ViralmRNAs

ICP0

VP16

ICP27

Viralepisome

Infectiousvirions

Phase I also provides viral factors that block innate defenses

Interferon-stimulatedlatency-promoting factors


Limitations of the Rat Model• Few molecular reagents, neurons expensive • Molecular mismatches: human virus in non-human cells.

POU

Oct-1

Helix 1 Helix 2 Helix 3

POUS POUH

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----.----1----.----2----.----3----.----4----.----5----.----6

rodents

natu

ral α

-her

pesv

irush

ost s

peci

es

**

***

**

*

*** recurrent shedding, ** some shedding, * no unassisted shedding

rabbits

Cleary et al. 1993 Genes & Dev

Differentiation of human embryonic stem cells into neurons

hESCs

+ Noggin+ TGFβi

RosetteNSCs NSCs

+ EGF/FGF2 + BDNF+ ascorbic acid

Neurons

Aldo Pourchet et al. 2017 Pathogens

nestin

bright field bright field β-3 tubulin

nestin

Establishment of non-productive infections in human neurons using IFN-α in combination with ACV and low pfu/neuron ratio

LAT

RNA

(nor

mal

ized)

LAT intron ncRNA (RT-qPCR)

1 dpi 6 dpi 12 dpi100

102

104

106

108

Infect with HSV-1 GFP-Us11

+/- acyclovir

establishment

+/- IFN-α

Time (days) -1 0 6 15 20

Maintenance


Non-productive infections reactivate to produce infectious virus

A NaBu NaBu B

GFP- (GFP+)GFP-+

NaBu

100

101

102

103

104

105

106

107

Tota

l pfu


Take Home Lessons

• HSV latency in cultured neurons requires sustained NGF signaling and cap-dependent protein synthesis.

• Reactivation involves 2 mechanistically distinct steps, initiated by activation of JNK and release of epigenetic suppression.

• Requirement for Phase I (‘animation’) makes sense for HSV in light of the minimal viral protein expression during latency.

• Controlled localization of VP16 and HCF-1 provides another additional host control.

• IFN suppresses reactivation in a neurons by blocking Phase I transcription.

• Phase I products such as ICP0 antagonize IFN, allowing reactivation.

• Latency can now be modeled in human neurons, the appropriate species matched environment for this human virus.

Acknowledgements

Ju Youn Kim Jessica LindermanMariko Kobayashi

Aldo PourchetAram Modrek

Dimitris Placantonakis

Ian Mohr Moses Chao

breaking down the barriers: first steps in herpes simplex virus reactivation · 2018-05-14 ·...

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