Both linear and discontinuous ribosome scanning are used for translation initiation from

bicistronic HIV-1 env mRNAs.

Adam T. Johnson1, Jenny L. Anderson1, Jane L. Howard1, Jörg Krummheuer2, Ilona Hauber3, Susanne Kammler1, Joachim Hauber3, Heiner Schaal2 and Damian F. J. Purcell1

1University of Melbourne, Microbiology and Immunology, Parkville Australia, 2Heinrich-Heine-University, Institute for Virology, Dusseldorf, Germany, 3Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany

HIV-1 Alternative splicing

tat

envtat

rev

polgag

rev

poly(A) 3’

5’ Cap

9kb genome

9kb Unspliced

polgag

poly(A) 3’

5’ Cap

Spliced mRNA

HIV-1 Alternative splicing

tat

envtat

rev

polgag

4kb Singly spliced

rev

poly(A) 3’

5’ Cap

poly(A) 3’

env5’ Cap

SD1 SA4b

9kb genome

9kb Unspliced

polgag

poly(A) 3’

5’ Cap

Spliced mRNA

HIV-1 Alternative splicing

tat

envtat

rev

polgag

4kb Singly spliced

rev

poly(A) 3’

5’ Cap

poly(A) 3’

env

2kb Multiply spliced

rev

5’ Cap

poly(A) 3’

9kb genome

9kb Unspliced

polgag

poly(A) 3’

5’ Cap

Spliced mRNA

SD4 SA7

5’ Cap

4aE4cE

env 3 1 / 4aEenv 4 1 / 4cEenv 5 1 / 2 / 5Eenv 6 1 / 2 / 4bEenv 7 1 / 2 / 4aEenv 9 1 / 2 / 4cEenv 8 1 / 3 / 5Eenv 10 1 / 3 / 4bEenv 11 1 / 3 / 4aEenv 12 1 / 3 / 4cEenv 13 1 / 2 / 3 / 5Eenv 14 1 / 2 / 3 / 4bEenv 15 1 / 2 / 3 / 4aEenv 16 1 / 2 / 3 / 4cE

4bE4aE4cE

5E

4bE4aE4cE

5E

4bE4aE4cE

5E

113612<1822<13<1<1<1

Primary mRNA

AAACap

1

54b

vpu

env4c

7 SA:

2 3

32 44a

SD: 4 5

6

HIV-1 generates 16 env mRNAs that differ in their 5’ UTR

env mRNAs

4bE

% abundance5Eenv 1 1 / 5E

env 2 1 / 4bE5110

HIV-1 env mRNAs: RNA elements, structures and RNA-protein interactions

AA(A)n

5’ UTR ORFs (All env mRNAs)

vpuenv

nefExon 2Exon 3

PBS DIS

Poly A

RRETAR

7mGpppN

TAR

PKR

Tat

La

Cyclin T

TRBPeIF2

PABPPABPRev

tRNAlys3

rev1

Exon 1

Weak kozak

Strong kozak

vpu

env

nefrev 2

Alternative env mRNAs code gp160 with equal efficiency, but Vpu with different efficiency.

Env

Ribosome Shunt

Translation controlled by “leaky ribosome scanning”?

Vpu

M. MOCK

e1. 1/5E

e2. 1/4bE

e3. 1/4aE

e4. 1/4cE

e5. 1/2/5E

e6. 1/2/4bE

e7. 1/2/4aE

e8. 1/3/5E

e10. 1/3/4bE

e11. 1/3/4aE

e13. 1/2/3/5E

e14. 1/2/3/4bE

M e1 e2 e3 e4 e5 e6 e7 e8 e10 e11 e13 e14

rev AUG +- + + + + + + +- - -

Ex 3?

Ex 2?

rev 1

4c 4a 4b 5SArev AUG + + + -

m7GG/ACCAUGG AAA

AUG

Ribosome Shunting

uORF

Requires RNA secondary structures to act as a launching & landing pad

Shunting elements may also require the presence of a short uORF to position the 40S ribosome at the correct site for shunting

Env expression and reporter constructs

vpu nef 4b 5

AAA

SA

rev 1

rev 2

m7G cap

env

RRE

Env (E)

Strong Kozak AUG

Weak Kozak AUG

R38 Mutation

egfp

vpu nef 4b 5

AAA

SA

rev 1

rev 2

m7G cap

RRE

EnvGFP (EG)

env

Mock

E1R

E2R

E1RG

E2RG

gp41 (TM)

gp160 (FL)

TM-GFP

FL-GFP

Env1 (1/5E)

Env2 (1/4bE)

GFP

Env (E)EnvGFP (EG)

Env-GFP expressed as a chimeric fusion protein

Env mAb C8

LTR vpu

envsj 1.5

LTR vpu

envsj 1.4b

LTR vpu

env gfpsj 1.5

LTR vpu

env gfpsj 1.4b

LTR vpu

env gfpsj 1.5

LTR vpu

env gfpsj 1.4b

0 100 200 300 400 500 600 700 800

Relative Fluorescence

Mock

E1R

E2R

E1(env)RG

E2(env)RG

E1RG

E2RG

Expression of GFP is a measure of translation initiation at the env AUG

Vpu

pNL4-3 CAAAGCAGTAA..........GTAGTACATGTAATGcAACCT...............ATA CONSENSUS_01_AE -----------GTA......ATA----T--------tC----...............T-GCONSENSUS_A1+A2(15) -----------GTATTAgTAAT-----T--------aCT---...............T-- CONSENSUS_B(33) -----------..........------------------T--...............T-- CONSENSUS_C(41) -----------.............---T--------tT-GA-ttactagcaaaagtaGAT CONSENSUS_D(8) -----------..........---------------------...............T-- CONSENSUS_F1+F2(6) -----------...GTGtTAA------T--------tC-TA-...............T-G CONSENSUS_G(5) -----------GTAGTAaTAAT-----T-------------A...............T-- CONSENSUS_H(3) -----------.............---TC-T-----.....................--- J.SE.SE91733 --G--------GTA.ACtTAAT---C-T--------gT-T--...............T-- K.CM.MP535C G--------G-GTAGTGcTAA---------------gTGT-C...............T-- O.CM.ANT70 -----------.............---ACGC-------T-A-...............-GG

uORF

Conservation of a uORF within the 5’UTR of env mRNA’s

SD4

16nt between SD4 and the Vpu AUG is the longest region of non-coding sequence within HIV-1 genome

Does this conserved uORF play a role in maintaining consistent Env levels?

0 20 40 60 80 100 120

Env-Expression [%]

E1eRG

E1sRG

E1RG

E1eRG

E1sRG

E1RG

UAGUACAAGUAAUGC

UAGUACAUGCAAUGCaacctaUAA

UAGUACAUGUAAUGC

uORF vpu

LTR vpu

env gfpsj 1.5

Disrupting a minimal uORF reduces the level of Env expression

VpuVpu-Expression [%]

0 25 50 75 100 125

E1(VK)RG

E1(VT)RG

E1RG

UAGUACAAGAAAUGG

UAGUACAUGUAAUGG

UAGUACAUGUAAUGC

uORF vpu

LTR vpu

env gfpsj 1.5

+4+1-3

0 20 40 60 80 100 120

Env-Expression [%]

E1(VT)RG

E1(VK)RG

E1RG

Increasing the strength of the vpu initiation codon further reduces the levels of Env expression

Vpu Kozak Strength

+/-

++

+++

VpuVpu-Expression [%]

0 50 100 150 200

LTR vpu

env gfpsj 1.4b

UAGUACAAGUAAUGC

UAGUACAUGCAAUGCaacctaUAA

UAGUACAUGUAAUGC

uORF vpu

E2eRG

E2sRG

E2RG

0 20 40 60 80 100 120

Env-Expression [%]

E2eRG

E2sRG

E2RG

An upstream Rev AUG alleviates the effect of uORF mutations on Env Expression

LTR vpu

env gfpsj 1.4b

UAGUACAAGAAAUGG

UAGUACAUGUAAUGG

UAGUACAUGUAAUGC

uORF vpu

E2(VK)RG

E2(VT)RG

E2RG

+1 +4-3

0 20 40 60 80 100 120

Env-Expression [%]

E2(VT)RG

E2(VK)RG

E2RG

Summary

Env is translated from the downstream env ORF using both an unusual discontinuous translation mechanism, and a linear reinitiation mechanism

Mutations that extend or remove an short uORF before the authentic Vpu start strongly inhibit Env expression

This uORF may pause the 40S ribosome at an RNA secondary structure responsible for ribosome shunting.

Recognition of the uORF requires initiation competent 40S ribosomes.

An upstream rev ORF reduces the interaction with uORF before Vpu.

Novel translation mechanisms suggests Env is a candidate for translation modulation that could impact upon viral infectivity

Cell cycle, cell stress, miRNA can modulate translation of select mRNA.

Acknowledgements

Adam Johnson Jenny AndersonJane Howard Marina Alexander

Heiner Schaal Jörg KrummheuerIlona HauberSusanne KammlerJoachim Hauber

Protein expression from env mRNA isoforms mutated to equalise for Rev activity (R38)

Rev

Fold CAT

Activation above

Mock

gp160

0

5

10

15

20N

LM

E1R

E2R

E3R

E1R

E4R

E2R

E3R

E4R

vpu

env

nefrev 2

rev 1

4c 4a 4b 5SArev AUG + + + -

R38

wt: AGU UUG GAA AGG ACC AGC AAA KAGG: AGU GGC ACC AGG GCC AGC UAA KAUG: AGU GGC ACC AUG GCC AGC UAA

SA5

SA4b

envvpu

SD4

rev1exon 2exon 1m7G

start

stop

Vpu Expression [%]

E5

E6

E5KAGG

E6KAGG

E5KAUG

E6KAUG

Rev

Vpu

0

20

40

60

80

100

120

Env Expression

[%]

gp160

0

20

40

60

80

100

120

140

E5

E6

E5KAGG

E6KAGG

E5KAUG

E6KAUG

Rev

Inclusion of a strong AUG in non-coding exon 2 does not effect Env expression