title: Changes in human immunodeficiency virus type 1 Gag at positions L449 and P453 are linked to I50V protease mutants in vivo and cause reduction of sensitivity to amprenavir and improved viral fitness in vitro.
abstract: Human immunodeficiency virus type 1 (HIV-1) Gag protease cleavage sites (CS) undergo sequence changes during the development of resistance to several protease inhibitors (PIs). We have analyzed the association of sequence variation at the p7/p1 and p1/p6 CS in conjunction with amprenavir (APV)-specific protease mutations following PI combination therapy with APV. Querying a central resistance data repository resulted in the detection of significant associations (P < 0.001) between the presence of APV protease signature mutations and Gag L449F (p1/p6 LP1'F) and P453L (p1/p6 PP5'L) CS changes. In population-based sequence analyses the I50V mutant was invariably linked to either L449F or P453L. Clonal analysis revealed that both CS mutations were never present in the same genome. Sequential plasma samples from one patient revealed a transition from I50V M46L P453L viruses at early time points to I50V M46I L449F viruses in later samples. Various combinations of the protease and Gag mutations were introduced into the HXB2 laboratory strain of HIV-1. In both single- and multiple-cycle assay systems and in the context of I50V, the L449F and P453L changes consistently increased the 50% inhibitory concentration of APV, while the CS changes alone had no measurable effect on inhibitor sensitivity. The decreased in vitro fitness of the I50V mutant was only partially improved by addition of either CS change (I50V M46I L449F mutant replicative capacity approximately 16% of that of wild-type virus). Western blot analysis of Pr55 Gag precursor cleavage products from infected-cell cultures indicated accumulation of uncleaved Gag p1-p6 in all I50V viruses without coexisting CS changes. Purified I50V protease catalyzed cleavage of decapeptides incorporating the L449F or P453L change 10-fold and 22-fold more efficiently than cleavage of the wild-type substrate, respectively. HIV-1 protease CS changes are selected during PI therapy and can have effects on both viral fitness and phenotypic resistance to PIs.
authors.affiliation: Department of Clinical Virology, GlaxoSmithKline Research and Development, Stevenage SG1 2NY, United Kingdom.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Maguire
authors.forename: Rosario
authors.initials: R
authors.lastname: Guinea
authors.forename: Philip
authors.initials: P
authors.lastname: Griffin
authors.forename: Sarah
authors.initials: S
authors.lastname: Macmanus
authors.forename: Robert C
authors.initials: RC
authors.lastname: Elston
authors.forename: Josie
authors.initials: J
authors.lastname: Wolfram
authors.forename: Naomi
authors.initials: N
authors.lastname: Richards
authors.forename: Mary H
authors.initials: MH
authors.lastname: Hanlon
authors.forename: David J T
authors.initials: DJ
authors.lastname: Porter
authors.forename: Terri
authors.initials: T
authors.lastname: Wrin
authors.forename: Neil
authors.initials: N
authors.lastname: Parkin
authors.forename: Margaret
authors.initials: M
authors.lastname: Tisdale
authors.forename: Eric
authors.initials: E
authors.lastname: Furfine
authors.forename: Chris
authors.initials: C
authors.lastname: Petropoulos
authors.forename: B Wendy
authors.initials: BW
authors.lastname: Snowden
authors.forename: Jörg-Peter
authors.initials: JP
authors.lastname: Kleim
mesh_terms: D001665:Binding Sites; D002219:Carbamates; D024882:Drug Resistance, Viral; D004359:Drug Therapy, Combination; D005663:Furans; D015683:Gene Products, gag / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016333:HIV Protease / Q000235:genetics* / Q000378:metabolism; D017320:HIV Protease Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation; D013379:Substrate Specificity; D013449:Sulfonamides / Q000494:pharmacology* / Q000627:therapeutic use; D014779:Virus Replication; D054301:gag Gene Products, Human Immunodeficiency Virus
chemical_list: D002219:Carbamates; D005663:Furans; D015683:Gene Products, gag; D017320:HIV Protease Inhibitors; D013449:Sulfonamides; D054301:gag Gene Products, Human Immunodeficiency Virus; C083868:gag protein p1, Human immunodeficiency virus; C083002:p6 gag protein, Human immunodeficiency virus 1; C095108:amprenavir; D016333:HIV Protease