Fidelity Variants of RNA Dependent RNA Polymerases Uncover an Indirect, Mutagenic Activity of Amiloride Compounds


In a screen for RNA mutagen resistance, we isolated a high fidelity RNA dependent RNA polymerase (RdRp) variant of Coxsackie virus B3 (CVB3). Curiously, this variant A372V is also resistant to amiloride. We hypothesize that amiloride has a previously undescribed mutagenic activity. Indeed, amiloride compounds increase the mutation frequencies of CVB3 and poliovirus and high fidelity variants of both viruses are more resistant to this effect. We hypothesize that this mutagenic activity is mediated through alterations in intracellular ions such as Mg2+ and Mn2+, which in turn increase virus mutation frequency by affecting RdRp fidelity. Furthermore, we show that another amiloride-resistant RdRp variant, S299T, is completely resistant to this mutagenic activity and unaffected by changes in ion concentrations. We show that RdRp variants resist the mutagenic activity of amiloride via two different mechanisms: 1) increased fidelity that generates virus populations presenting lower basal mutation frequencies or 2) resisting changes in divalent cation concentrations that affect polymerase fidelity. Our results uncover a new antiviral approach based on mutagenesis.


Vyšlo v časopise: Fidelity Variants of RNA Dependent RNA Polymerases Uncover an Indirect, Mutagenic Activity of Amiloride Compounds. PLoS Pathog 6(10): e32767. doi:10.1371/journal.ppat.1001163
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1001163

Souhrn

In a screen for RNA mutagen resistance, we isolated a high fidelity RNA dependent RNA polymerase (RdRp) variant of Coxsackie virus B3 (CVB3). Curiously, this variant A372V is also resistant to amiloride. We hypothesize that amiloride has a previously undescribed mutagenic activity. Indeed, amiloride compounds increase the mutation frequencies of CVB3 and poliovirus and high fidelity variants of both viruses are more resistant to this effect. We hypothesize that this mutagenic activity is mediated through alterations in intracellular ions such as Mg2+ and Mn2+, which in turn increase virus mutation frequency by affecting RdRp fidelity. Furthermore, we show that another amiloride-resistant RdRp variant, S299T, is completely resistant to this mutagenic activity and unaffected by changes in ion concentrations. We show that RdRp variants resist the mutagenic activity of amiloride via two different mechanisms: 1) increased fidelity that generates virus populations presenting lower basal mutation frequencies or 2) resisting changes in divalent cation concentrations that affect polymerase fidelity. Our results uncover a new antiviral approach based on mutagenesis.


Zdroje

1. HorisbergerJD

1998

Amiloride-sensitive Na channels.

Curr Opin Cell Biol

10

443

449

2. NakayamaS

NomuraH

1995

Mechanisms of intracellular Mg2+ regulation affected by amiloride and ouabain in the guinea-pig taenia caeci.

J Physiol (

488

Pt 1

1

12

3. WilsonL

GageP

EwartG

2006

Hexamethylene amiloride blocks E protein ion channels and inhibits coronavirus replication.

Virology

353

294

306

4. PremkumarA

HoranCR

GagePW

2005

Dengue virus M protein C-terminal peptide (DVM-C) forms ion channels.

J Membr Biol

204

33

38

5. EwartGD

MillsK

CoxGB

GagePW

2002

Amiloride derivatives block ion channel activity and enhancement of virus-like particle budding caused by HIV-1 protein Vpu.

Eur Biophys J

31

26

35

6. PremkumarA

WilsonL

EwartGD

GagePW

2004

Cation-selective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride.

FEBS Lett

557

99

103

7. GazinaEV

HarrisonDN

JefferiesM

TanH

WilliamsD

2005

Ion transport blockers inhibit human rhinovirus 2 release.

Antiviral Res

67

98

106

8. HarrisonDN

GazinaEV

PurcellDF

AndersonDA

PetrouS

2008

Amiloride derivatives inhibit coxsackievirus B3 RNA replication.

J Virol

82

1465

1473

9. LoebLA

EssigmannJM

KazaziF

ZhangJ

RoseKD

1999

Lethal mutagenesis of HIV with mutagenic nucleoside analogs.

Proc Natl Acad Sci U S A

96

1492

1497

10. CrottyS

CameronC

AndinoR

2002

Ribavirin's antiviral mechanism of action: lethal mutagenesis?

J Mol Med

80

86

95

11. AiraksinenA

ParienteN

Menendez-AriasL

DomingoE

2003

Curing of foot-and-mouth disease virus from persistently infected cells by ribavirin involves enhanced mutagenesis.

Virology

311

339

349

12. ParienteN

SierraS

LowensteinPR

DomingoE

2001

Efficient virus extinction by combinations of a mutagen and antiviral inhibitors.

J Virol

75

9723

9730

13. Ruiz-JaraboCM

LyC

DomingoE

de la TorreJC

2003

Lethal mutagenesis of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV).

Virology

308

37

47

14. PfeifferJK

KirkegaardK

2003

A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity.

Proc Natl Acad Sci U S A

100

7289

7294

15. ArnoldJJ

VignuzziM

StoneJK

AndinoR

CameronCE

2005

Remote site control of an active site fidelity checkpoint in a viral RNA-dependent RNA polymerase.

J Biol Chem

280

25706

25716

16. PfeifferJK

KirkegaardK

2005

Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice.

PLoS Pathog

1

e11

17. VignuzziM

StoneJK

ArnoldJJ

CameronCE

AndinoR

2006

Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population.

Nature

439

344

348

18. VignuzziM

WendtE

AndinoR

2008

Engineering attenuated virus vaccines by controlling replication fidelity.

Nat Med

14

154

161

19. CrottyS

MaagD

ArnoldJJ

ZhongW

LauJY

2000

The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen.

Nat Med

6

1375

1379

20. Jackson-GrusbyL

LairdPW

MaggeSN

MoellerBJ

JaenischR

1997

Mutagenicity of 5-aza-2′-deoxycytidine is mediated by the mammalian DNA methyltransferase.

Proc Natl Acad Sci U S A

94

4681

4685

21. ArnoldJJ

CameronCE

2000

Poliovirus RNA-dependent RNA polymerase (3D(pol)). Assembly of stable, elongation-competent complexes by using a symmetrical primer-template substrate (sym/sub).

J Biol Chem

275

5329

5336

22. UetaniT

MatsubaraT

NomuraH

MuroharaT

NakayamaS

2003

Ca2+-dependent modulation of intracellular Mg2+ concentration with amiloride and KB-R7943 in pig carotid artery.

J Biol Chem

278

47491

47497

23. SteitzTA

1998

A mechanism for all polymerases.

Nature

391

231

232

24. KorneevaVS

CameronCE

2007

Structure-function relationships of the viral RNA-dependent RNA polymerase: fidelity, replication speed, and initiation mechanism determined by a residue in the ribose-binding pocket.

J Biol Chem

282

16135

16145

25. Ferrer-OrtaC

AriasA

Perez-LuqueR

EscarmisC

DomingoE

2007

Sequential structures provide insights into the fidelity of RNA replication.

Proc Natl Acad Sci U S A

104

9463

9468

26. AriasA

ArnoldJJ

SierraM

SmidanskyED

DomingoE

2008

Determinants of RNA-dependent RNA polymerase (in)fidelity revealed by kinetic analysis of the polymerase encoded by a foot-and-mouth disease virus mutant with reduced sensitivity to ribavirin.

J Virol

82

12346

12355

27. CrottyS

GoharaD

GilliganDK

KarelskyS

CameronCE

2003

Manganese-dependent polioviruses caused by mutations within the viral polymerase.

J Virol

77

5378

5388

28. ArnoldJJ

CameronCE

2004

Poliovirus RNA-dependent RNA polymerase (3Dpol): pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mg2+.

Biochemistry

43

5126

5137

29. ArnoldJJ

GoharaDW

CameronCE

2004

Poliovirus RNA-dependent RNA polymerase (3Dpol): pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mn2+.

Biochemistry

43

5138

5148

30. LanfordRE

ChavezD

GuerraB

LauJY

HongZ

2001

Ribavirin induces error-prone replication of GB virus B in primary tamarin hepatocytes.

J Virol

75

8074

8081

31. SeversonWE

SchmaljohnCS

JavadianA

JonssonCB

2003

Ribavirin causes error catastrophe during Hantaan virus replication.

J Virol

77

481

488

32. CuevasJM

Gonzalez-CandelasF

MoyaA

SanjuanR

2009

The effect of ribavirin on the mutation rate and spectrum of Hepatitis C virus in vivo.

J Virol

83

5760

5764

33. GraciJD

HarkiDA

KorneevaVS

EdathilJP

TooK

2007

Lethal mutagenesis of poliovirus mediated by a mutagenic pyrimidine analogue.

J Virol

81

11256

11266

34. GraciJD

TooK

SmidanskyED

EdathilJP

BarrEW

2008

Lethal mutagenesis of picornaviruses with N-6-modified purine nucleoside analogues.

Antimicrob Agents Chemother

52

971

979

35. MoriyamaK

SuzukiT

NegishiK

GraciJD

ThompsonCN

2008

Effects of introduction of hydrophobic group on ribavirin base on mutation induction and anti-RNA viral activity.

J Med Chem

51

159

166

36. CampagnolaG

WeygandtM

ScogginK

PeersenO

2008

Crystal structure of coxsackievirus B3 3Dpol highlights the functional importance of residue 5 in picornavirus polymerases.

J Virol

82

9458

9464

37. GruezA

SeliskoB

RobertsM

BricogneG

BussettaC

2008

The crystal structure of coxsackievirus B3 RNA-dependent RNA polymerase in complex with its protein primer VPg confirms the existence of a second VPg binding site on Picornaviridae polymerases.

J Virol

82

9577

9590

38. ThompsonAA

PeersenOB

2004

Structural basis for proteolysis-dependent activation of the poliovirus RNA-dependent RNA polymerase.

EMBO J

23

3462

3471

39. ArnoldJJ

BernalA

UcheU

SternerDE

ButtTR

2006

Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity.

Anal Biochem

350

214

221

40. StudierFW

2005

Protein production by auto-induction in high density shaking cultures.

Protein Expr Purif

41

207

234

41. CastroC

SmidanskyE

MaksimchukKR

ArnoldJJ

KorneevaVS

2007

Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases.

Proc Natl Acad Sci U S A

104

4267

4272

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

Článok vyšiel v časopise

PLOS Pathogens


2010 Číslo 10
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Aktuální možnosti diagnostiky a léčby litiáz
nový kurz
Autori: MUDr. Tomáš Ürge, PhD.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa