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 LaboratóriumČlánok vyšiel v časopise
PLOS Pathogens
2010 Číslo 10
- Parazitičtí červi v terapii Crohnovy choroby a dalších zánětlivých autoimunitních onemocnění
- Očkování proti virové hemoragické horečce Ebola experimentální vakcínou rVSVDG-ZEBOV-GP
- Koronavirus hýbe světem: Víte jak se chránit a jak postupovat v případě podezření?
Najčítanejšie v tomto čísle
- Retroviral RNA Dimerization and Packaging: The What, How, When, Where, and Why
- Viral Replication Rate Regulates Clinical Outcome and CD8 T Cell Responses during Highly Pathogenic H5N1 Influenza Virus Infection in Mice
- Antimicrobial Peptides: Primeval Molecules or Future Drugs?
- Crystal Structure of DotD: Insights into the Relationship between Type IVB and Type II/III Secretion Systems