A novel TaqMan real-time PCR assay to estimate ex vivo human immunodeficiency virus type 1 fitness in the era of multi-target (pol and env) antiretroviral therapy
Despite numerous studies on human immunodeficiency virus type 1 (HIV-1) fitness, many key conceptual and technical questions are still unsolved. For example, the proper system to determine virus fitness of HIV-1 is still unknown. In this study, an assay was developed to estimate HIV-1 fitness based on growth competition experiments and TaqMan real-time PCR. This novel technique was compared with several methods (i.e. virus growth kinetics, growth competition/heteroduplex-tracking analysis and single-cycle replication capacity assay) in order to analyse the impact of various genomic regions and overall genetic background on virus fitness. HIV-1 primary isolates and three different sets of recombinant viruses [i.e. recombinant clones carrying protease (PR), reverse transcriptase (RT) or the 3′ end of Gag, PR and RT (3′Gag/PR/RT), sequences amplified by PCR from the same primary isolates)] were evaluated. Here, it is demonstrated that, in spite of intrinsic differences, both growth competition/TaqMan and single-cycle replication assays detected a significant reduction in HIV-1 fitness as a consequence of drug-resistant mutations in pol. However, this new assay, based on HIV-1 isolates, may be useful to quantify replicative fitness in viruses from patients treated simultaneously with antiretroviral drugs targeting different genomic regions of HIV-1 (e.g. pol and env).
BallS. C.,
AbrahaA.,
CollinsK. R.11 other authors2003; Comparing the ex vivo fitness of CCR5-tropic human immunodeficiency virus type 1 isolates of subtypes B and C. J Virol 77:1021–1038
BarbourJ. D.,
WrinT.,
GrantR. M.,
MartinJ. N.,
SegalM. R.,
PetropoulosC. J.,
DeeksS. G.2002; Evolution of phenotypic drug susceptibility and viral replication capacity during long-term virologic failure of protease inhibitor therapy in human immunodeficiency virus-infected adults. J Virol 76:11104–11112
BleiberG.,
MunozM.,
CiuffiA.,
MeylanP.,
TelentiA.2001; Individual contributions of mutant protease and reverse transcriptase to viral infectivity, replication, and protein maturation of antiretroviral drug-resistant human immunodeficiency virus type 1. J Virol 75:3291–3300
BrennerB. G.,
RoutyJ. P.,
PetrellaM.9 other authors2002; Persistence and fitness of multidrug-resistant human immunodeficiency virus type 1 acquired in primary infection. J Virol 76:1753–1761
CabanaM.,
ClotetB.,
MartinezM. A.1999; Emergence and genetic evolution of HIV-1 variants with mutations conferring resistance to multiple reverse transcriptase and protease inhibitors. J Med Virol 59:480–490
CroteauG.,
DoyonL.,
ThibeaultD.,
McKercherG.,
PiloteL.,
LamarreD.1997; Impaired fitness of human immunodeficiency virus type 1 variants with high-level resistance to protease inhibitors. J Virol 71:1089–1096
de BaarM. P.,
TimmermansE. C.,
BakkerM.,
de RooijE.,
van GemenB.,
GoudsmitJ.2001; One-tube real-time isothermal amplification assay to identify and distinguish human immunodeficiency virus type 1 subtypes A, B, and C and circulating recombinant forms AE and AG. J Clin Microbiol 39:1895–1902
DeeksS. G.,
BarbourJ. D.,
MartinJ. N.,
SwansonM. S.,
GrantR. M.2000; Sustained CD4+ T cell response after virologic failure of protease inhibitor-based regimens in patients with human immunodeficiency virus infection. J Infect Dis 181:946–953
DeeksS. G.,
WrinT.,
LieglerT.8 other authors2001; Virologic and immunologic consequences of discontinuing combination antiretroviral-drug therapy in HIV-infected patients with detectable viremia. N Engl J Med 344:472–480
de RondeA.,
van DoorenM.,
van Der HoekL.,
BouwhuisD.,
de RooijE.,
van GemenB.,
de BoerR.,
GoudsmitJ.2001; Establishment of new transmissible and drug-sensitive human immunodeficiency virus type 1 wild types due to transmission of nucleoside analogue-resistant virus. J Virol 75:595–602
DesireN.,
DeheeA.,
SchneiderV.,
JacometC.,
GoujonC.,
GirardP. M.,
RozenbaumW.,
NicolasJ. C.2001; Quantification of human immunodeficiency virus type 1 proviral load by a TaqMan real-time PCR assay. J Clin Microbiol 39:1303–1310
Garcia-LermaJ. G.,
GerrishP. J.,
WrightA. C.,
QariS. H.,
HeneineW.2000; Evidence of a role for the Q151L mutation and the viral background in development of multiple dideoxynucleoside-resistant human immunodeficiency virus type 1. J Virol 74:9339–9346
GrantR. M.,
LieglerT.,
BonhoefferS.9 other authors2001; Large fitness differences between protease inhibitor susceptible and resistant HIV-1 in vivo
. 8th Annual Meeting on HIV Dynamics and Evolution , p. 28 (Paris, France: 27 29 April, 2001
Gutierrez-RivasM.,
IbanezA.,
MartinezM. A.,
DomingoE.,
Menendez-AriasL.1999; Mutational analysis of Phe160 within the ‘palm’ subdomain of human immunodeficiency virus type 1 reverse transcriptase. J Mol Biol 290:615–625
HallT. A.1999; BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98
HanceA. J.,
LemialeV.,
IzopetJ.7 other authors2001; Changes in human immunodeficiency virus type 1 populations after treatment interruption in patients failing antiretroviral therapy. J Virol 75:6410–6417
HarriganP. R.,
BloorS.,
LarderB. A.1998; Relative replicative fitness of zidovudine-resistant human immunodeficiency virus type 1 isolates in vitro
. J Virol 72:3773–3778
HertogsK.,
de BethuneM. P.,
MillerV.14 other authors1998; A rapid method for simultaneous detection of phenotypic resistance to inhibitors of protease and reverse transcriptase in recombinant human immunodeficiency virus type 1 isolates from patients treated with antiretroviral drugs. Antimicrob Agents Chemother 42:269–276
HollandJ. J.,
de la TorreJ. C.,
ClarkeD. K.,
DuarteE.1991; Quantitation of relative fitness and great adaptability of clonal populations of RNA viruses. J Virol 65:2960–2967
KellamP.,
LarderB. A.1994; Recombinant virus assay: a rapid, phenotypic assay for assessment of drug susceptibility of human immunodeficiency virus type 1 isolates. Antimicrob Agents Chemother 38:23–30
KosalaraksaP.,
KavlickM. F.,
MarounV.,
LeR.,
MitsuyaH.1999; Comparative fitness of multi-dideoxynucleoside-resistant human immunodeficiency virus type 1 (HIV-1) in an in vitro competitive HIV-1 replication assay. J Virol 73:5356–5363
LaurendeauI.,
BahuauM.,
VodovarN.,
LarramendyC.,
OliviM.,
BiecheI.,
VidaudM.,
VidaudD.1999; TaqMan PCR-based gene dosage assay for predictive testing in individuals from a cancer family with INK4 locus haploinsufficiency. Clin Chem 45:982–986
LewinS. R.,
VesanenM.,
KostrikisL.,
HurleyA.,
DuranM.,
Zhangl.,
HoD. D.,
MarkowitzM.1999; Use of real-time PCR and molecular beacons to detect virus replication in human immunodeficiency virus type 1-infected individuals on prolonged effective antiretroviral therapy. J Virol 73:6099–6103
MaedaY.,
VenzonD. J.,
MitsuyaH.1998; Altered drug sensitivity, fitness, and evolution of human immunodeficiency virus type 1 with pol gene mutations conferring multi-dideoxynucleoside resistance. J Infect Dis 177:1207–1213
MasA.,
PareraM.,
BrionesC.,
SorianoV.,
MartinezM. A.,
DomingoE.,
Menendez-AriasL.2000; Role of a dipeptide insertion between codons 69 and 70 of HIV-1 reverse transcriptase in the mechanism of AZT resistance. EMBO J 19:5752–5761
MascheraB.,
FurfineE.,
BlairE. D.1995; Analysis of resistance to human immunodeficiency virus type 1 protease inhibitors by using matched bacterial expression and proviral infection vectors. J Virol 69:5431–5436
PetropoulosC. J.,
ParkinN. T.,
LimoliK. L.8 other authors2000; A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1. Antimicrob Agents Chemother 44:920–928
Quiñones-MateuM. E.,
ArtsE. J.2001; HIV-1 fitness: implications for drug resistance, disease progression, and global epidemic evolution. In HIV Sequence Compendium 2001 pp 134–170 Edited by
KuikenC.,
FoleyB.,
HahnB.,
MarxP.,
McCutchanF.,
MellorsJ.,
WolinskyS.,
KorberB.
Los Alamos, NM: Los Alamos National Laboratory, Theoretical Biology and Biophysics Group;
Quiñones-MateuM. E.,
BallS. C.,
MarozsanA. J.,
TorreV. S.,
AlbrightJ. L.,
VanhamG.,
van Der GroenG.,
ColebundersR. L.,
ArtsE. J.2000; A dual infection/competition assay shows a correlation between ex vivo human immunodeficiency virus type 1 fitness and disease progression. J Virol 74:9222–9233
Quiñones-MateuM. E.,
TadeleM.,
PareraM.8 other authors2002; Insertions in the reverse transcriptase increase both drug resistance and viral fitness in a human immunodeficiency virus type 1 isolate harboring the multi-nucleoside reverse transcriptase inhibitor resistance 69 insertion complex mutation. J Virol 76:10546–10552
ReschW.,
ZiermannR.,
ParkinN.,
GamarnikA.,
SwanstromR.2002; Nelfinavir-resistant, amprenavir-hypersusceptible strains of human immunodeficiency virus type 1 carrying an N88S mutation in protease have reduced infectivity, reduced replication capacity, and reduced fitness and process the Gag polyprotein precursor aberrantly. J Virol 76:8659–8666
RobinsonL. H.,
MyersR. E.,
SnowdenB. W.,
TisdaleM.,
BlairE. D.2000; HIV type 1 protease cleavage site mutations and viral fitness: implications for drug susceptibility phenotyping assays. AIDS Res Hum Retroviruses 16:1149–1156
ShiC.,
MellorsJ. W.1997; A recombinant retroviral system for rapid in vivo analysis of human immunodeficiency virus type 1 susceptibility to reverse transcriptase inhibitors. Antimicrob Agents Chemother 41:2781–2785
ThompsonJ. D.,
GibsonT. J.,
PlewniakF.,
JeanmouginF.,
HigginsD. G.1997; The clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882
TorreV. S.,
MarozsanA. J.,
AlbrightJ. L.,
CollinsK. R.,
HartleyO.,
OffordR. E.,
Quiñones-MateuM. E.,
ArtsE. J.2000; Variable sensitivity of CCR5-tropic human immunodeficiency virus type 1 isolates to inhibition by RANTES analogs. J Virol 74:4868–4876
WeberJ.,
RangelH. R.,
ChakrabortyB.12 other authors2003; Role of baseline pol genotype in human immunodeficiency virus type 1 fitness evolution. J AIDS (in press)
ZhaoY.,
YuM.,
MillerJ. W.,
ChenM.,
BremerE. G.,
KabatW.,
YogevR.2002; Quantification of human immunodeficiency virus type 1 proviral DNA by using TaqMan technology. J Clin Microbiol 40:675–678
A novel TaqMan real-time PCR assay to estimate ex vivo human immunodeficiency virus type 1 fitness in the era of multi-target (pol and env) antiretroviral therapy