A novel substrate-based HIV-1 protease inhibitor drug resistance mechanism.
BACKGROUND
HIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors.


RESULTS
We have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with virological failure during PI therapy.


CONCLUSIONS
HIV can use an alternative mechanism to become resistant to PI by changing the substrate instead of the protease. Further studies are required to determine to what extent cleavage site mutations may explain virological failure during PI therapy.
Nijhuis
Monique
M
Eijkman Winkler Center, Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, Netherlands.
van Maarseveen
Noortje M
NM
Lastere
Stephane
S
Schipper
Pauline
P
Coakley
Eoin
E
Glass
Bärbel
B
Rovenska
Mirka
M
de Jong
Dorien
D
Chappey
Colombe
C
Goedegebuure
Irma W
IW
Heilek-Snyder
Gabrielle
G
Dulude
Dominic
D
Cammack
Nick
N
Brakier-Gingras
Lea
L
Konvalinka
Jan
J
Parkin
Neil
N
Kräusslich
Hans-Georg
HG
Brun-Vezinet
Francoise
F
Boucher
Charles A B
CA
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003062:Codon / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics*; D016368:Frameshift Mutation; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism; D016679:Genome, Viral; D016333:HIV Protease / Q000235:genetics / Q000378:metabolism; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation; D012367:RNA, Viral / Q000235:genetics; D019438:Ritonavir / Q000494:pharmacology; D013379:Substrate Specificity; D014162:Transfection
D003062:Codon; D015683:Gene Products, gag; D017320:HIV Protease Inhibitors; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1; D019438:Ritonavir