Structure-function analysis of HIV-1 gp120 amino acid mutations

51 Pages·2009·0.65 MB·English

JVI Accepts, published online ahead of print on 23 September 2009
J. Virol. doi:10.1128/JVI.01351-09
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Structure-function analysis of HIV-1 gp120 amino acid mutations
associated with resistance to the CCR5 co-receptor antagonist vicriviroc

Robert A. Ogert1, Lei Ba1, Yan Hou1, Catherine Buontempo1, Ping Qiu2, Jose Duca3,
Nicholas Murgolo2, Peter Buontempo1, Robert Ralston1 and John A. Howe1* D
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Schering-Plough Research Institute, 1Departments of Biological Sciences-Virology, lo
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2Molecular Design and Informatics, and 33-D Drug Design, 2015 Galloping Hill Road d
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K-15-4945, Kenilworth, N.J. 07033 d
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*Address correspondence to: 20
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John A. Howe, Ph.D.
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Schering-Plough Research Institute b
2015 Galloping Hill Road, K-15-E403C, 4945 y

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Kenilworth, N.J. 07033 u
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Phone: [phone]
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FAX: [phone] t
Email: [email protected]

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Abstract
Vicriviroc (VCV) is a small molecule CCR5 co-receptor antagonist currently in
clinical trials for treatment of R5-tropic HIV-1 infection. With this drug in development,
identification of resistance mechanisms to VCV is needed to allow optimal outcomes in
clinical practice. In this study we further characterized VCV resistance in a lab-adapted,
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VCV resistant RU570 virus (RU570-VCV ). We show that K305R, R315Q, and K319T o
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amino acid changes in the V3 loop, along with P437S in C4 completely reproduced the lo
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resistance phenotype in a chimeric ADA envelope containing the C2-V5 region from e
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RU570 passage control gp120. The K305R amino acid change primarily impacted the o
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degree of resistance, whereas K319T contributed to both resistance and virus infectivity. h
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The P437S mutation in C4 had more influence on the relative degree of virus infectivity, //
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while the R315Q mutation contributed to the virus concentration-dependent phenotypic s
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resistance pattern observed for RU570-VCV . RU570-VCV pseudovirus entry with o
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VCV-bound CCR5 was dramatically reduced by Y10A, D11A, Y14A and Y15A o
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mutations in the N-terminus of CCR5, whereas these mutations had less impact on entry p
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in the absence of VCV. Notably, an additional Q315E/I317F substitution in the crown 4
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region of the V3 loop enhanced resistance to VCV resulting in a stronger dependence on 1
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the N-terminus for viral entry. By fitting the envelope mutations to a recently described y

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docked CCR5 2-15-N-terminus/HIV-1 gp120 CD4 molecular model, potential new e
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interactions in gp120 with the N-terminus of CCR5 were uncovered. The cumulative
results of this study suggest that as the RU570 VCV resistant virus adapted to use the
drug-bound receptor it also developed an increased reliance on the N-terminus of CCR5.
Word count: 263
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Introduction
CCR5 antagonists inhibit HIV-1 entry by binding within a pocket formed by the
transmembrane domains of CCR5. The binding of these agents locks the receptor in a
conformation the virus is unable to recognize (14, 25, 32, 37, 50, 54). The CCR5 co-
receptor antagonists most advanced in development are maraviroc (MVC) and vicriviroc
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(VCV). MVC marketed as Selzentry is approved for use in treatment experienced adult o
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patients with R5-tropic HIV-1 infection having resistance to multiple antiretroviral agents lo
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(18) and VCV is currently being evaluated in phase II and phase III clinical trials (19, e
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49). With the ongoing clinical development of HIV-1 co-receptor antagonists, further o
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studies are needed regarding the biology of HIV-1 resistance to these agents and the h
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ability to assess resistance based on changes within the envelope glycoprotein. //
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The CCR5 co-receptor antagonists are unique in that they bind to the CCR5 co- s
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receptor on the surface of the host cell, whereas most HIV-1 medicines interfere with o
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virus propagation by inhibiting one of the essential viral encoded enzymes. Signature o
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mutations associated with resistance to HIV-1 reverse transcriptase, protease and p
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integrase inhibitors as well, as well as compensatory mutations allowing the virus to 4
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overcome a loss in fitness, have been identified (6, 21, 43). However, similar information 1
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on resistance mutations has not been identified with respect to the CCR5 co-receptor y

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antagonists. e
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It has been established that CCR5 co-receptor antagonists block HIV-1 entry after
the virus has bound to CD4. The initial interaction between CD4 and the envelope
glycoprotein gp120-gp41 homotrimers induces a conformational change in gp120 (47,
48) that enables binding to CCR5 (53, 58). The interaction of the V3 loop and bridging
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sheet region of gp120 with CCR5 (22, 23, 45, 46, 59, 60) is believed to induce a series of
further rearrangements in gp120 that expose the gp41 ectodomain and trigger fusion of
viral and cell membranes (4, 5). Thus, both the complexity of the entry process and the
sequence heterogeneity of the envelope glycoprotein complicate the identification of
resistance mechanisms for CCR5 co-receptor antagonists.
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Mutations associated with resistance to CCR5 co-receptor antagonists for in-vitro o
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derived HIV-1 R5 resistant variants have, in most cases, mapped to the V3 loop region of lo
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gp120 (2, 26, 35, 40, 56); however, one resistant variant with no mutations in the V3 e
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loop (33) was recently shown to have mutations in the N-terminus fusion peptide of gp41 o
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that conferred resistance (1). In clinical trials of MVC (52) and VCV (55), subjects that h
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experienced virologic failure and demonstrated phenotypic resistance to the CCR5 co- //
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receptor antagonists based on the Phenosense Entry assay for co-receptor tropism (57) all s
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developed resistance mutations that mapped to amino acid substitutions in the V3 loop o
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region during therapy. o
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In this study, we have further examined resistance mutations in the V3 and p
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bridging sheet regions of a lab adapted RU570 VCV-resistant (RU570-VCV ) variant 4
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(40) using site-directed mutagenesis to reintroduce the amino acid changes into an ADA 1
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chimeric envelope containing the cognate C2-V5 region from a passage control envelope. y

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We then sought to analyze the effect of these mutations, either alone or in various e
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combinations, on pseudovirus infectivity, susceptibility to VCV and interaction with
CCR5.

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Materials and methods
Reagents. VCV was synthesized at Schering-Plough Research Institute, Kenilworth, N.J.
Lectin from Galanthus nivalis insolublized on 4% cross-linked beaded agarose was
obtained from Sigma-Aldrich, St. Louis, MO. pCD4 plasmid was obtained from Origene
Technologies Inc., Rockville, MD.
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HIV-1 plasmids and HIV-1 primary isolates. The HIV-1 clade G RU570 primary o
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isolate was obtained from the National Institutes of Health (NIH) AIDS Research and lo
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Reference Reagent program and was passaged in PM-1 cells in the presence of escalating e
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concentrations of VCV to generate VCV-resistant cultures as described previously (40). o
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The pNL4-3E-Luc+ and pSV7d-ADA gp160 plasmids were obtained from Dr. John h
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Moore, at the Weill Cornell Medical College of Cornell University, in New York, N.Y. //
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Homologous recombination of HIV-1 RU570 gp120 fragments into pSV7d-ADAgp160 s
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was performed as previously described (40). o
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Cell lines. The neoplastic T-cell line, PM-1, was obtained from the NIH AIDS Research o
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and Reference Reagent program. U87 astroglioma cells expressing CD4 and CCR5 were p
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obtained from Dr. Dan Littman, at New York University, and were maintained in 4
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Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine 1
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sermum (FBS), 500 µg/ml G418, and 1µg/ml puromycin. 293T cells (CRL-11268) were y

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purchased from American Type Culture Collection (ATCC; Manassas, VA) and e
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maintained in DMEM supplemented with 10% FBS and 300 µg/ml G418.
HIV-1 RU570 VCV resistance generated in PM-1 cells. Generation of RU570 HIV-1
VCV was previously described (40). RU570 stocks for phenotypic analysis were
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prepared by infecting PM-1 cells in the presence of 10 µM VCV. VCV was removed
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from the cultures 48 hours post-infection followed by incubation in culture media for 72
hours. Virus stocks were stored as 1 ml aliquots at -70° C.
HIV-1 single-round virus infection assay. This assay is based on a previously
described method (34) and was modified as follows. U87 cells were seeded into 96-well
collagen coated plates 24 hours prior to assay. CCR5 antagonists were prepared as 100x
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stocks in 100% DMSO and diluted to 1x assay concentration in medium (1% DMSO o
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final). Cells were treated with compound 2 h prior to infection. Pre-treatment medium lo
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was aspirated, replaced with virus pool supplemented with compound. Virus was e
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allowed to adsorb in the presence of compound for 2 hours at 37°. Cultures were washed o
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with PBS, and incubation was continued in culture medium supplemented with h
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compound and 2µM amprenavir for 48 hours. Cells were harvested from plates by //
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trypsinization and after neutralization with growth medium, transferred to V-bottom s
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plates. Cells were centrifuged at 700xg for 3 minutes, washed with PBS and fixed with o
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4% paraformaldehyde (CytofixTM; BD Biosciences) for 15 minutes. Following o
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fixation, cells were washed twice with PBS supplemented with 0.2% bovine serum p
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albumin (Stain BufferTM; BD Biosciences). 4
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For intracellular staining of p24-Ag, cells were centrifuged and suspended in 1
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permeabilization buffer (Perm/Wash BufferTM; BD Biosciences) for 15 minutes. y

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Phycoerythrin (PE)-conjugated mouse anti-p24 mAb (KC57-RD1; Beckman Coulter) e
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was diluted to a final concentration of 1:160 in permeabilization buffer. Following
incubation at 4° for 1 hour, cells were washed 3 times, 5 minutes between washes.
Stained cells were analyzed with a FACScalibur flow cytometer (BD Biosciences) and
data analysis was performed with CellQuest Pro Software (BD Biosciences). The ratio of
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p24+ cells was determined by using a bivariate plot of FL-2 versus FL-1 fluorescence,
with the gate being set on mock infected cells. Ten-thousand events were acquired per
data point, giving a limit of quantification (LOQ, 99% CI) of ten p24+ cells. Percent
inhibition for each virus concentration was defined as the ratio of p24 positive cells in
CCR5 antagonist treated culture normalized to the ratio of p24 positive cells in non-
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treated control x 100%. Inhibition greater than 100% indicates that the fraction of p24+ o
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cells in the CCR5 antagonist treated cultures was greater than fraction of p24+ of the lo
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non-treated control. EC and maximal percent inhibition (MPI) for the passaged viruses e
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were determined using GraphPad Prism Software (v.4, GraphPad Software, Inc., San o
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Diego). h
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Site-directed mutagenesis (SDM) of gp120 amino acids. SDM of individual, gp120 //
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amino acids in the pADA-C2-V5pc clone was performed using the QuikChange® SDM s
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kit (Stratagene, La Jolla, CA). Amino acid changes corresponding to HXB2 gp120 amino o
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acid coordinates were as follows: K305R, R315Q, K319T, and P437S. All sequence o
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changes were verified by DNA sequence analysis. SDM of gp120 amino acids (Q315E p
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and I317F) in the pADA-C2-V5 clone were also performed as described above. 4
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SDM of CCR5. The pcDNA3.1 CCR5 expression plasmid was obtained from Dr. Dan 1
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Littman, New York University. The N-terminus-∆ 2-17 CCR5 construct was generated y

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using overlapping PCR with the forward primer: e
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5’-CTAAGCTTACCATGGATGAGCCCTGCCAAAAAATCAATG – 3’ and
reverse primer 5’ – CCACCACCCAAGTGATCACACTTG – 3’. The resulting PCR
product was cloned into the parental expression vector pcDNA3.1-CCR5 using HindIII
and AleI restriction sites. Individual mutations in the N-terminus of CCR5 (Y3A, Y10A,
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D11A, N13A, Y14A, and Y15A) and in the extracellular loop 2 of CCR5 (R168A,
K171A, E172A, L174A, and C178A) were generated in pcDNA3.1-CCR5 using the
QuikChange® SDM Kit (Stratagene, La Jolla, CA). All mutations were confirmed by
DNA sequence analysis.
Generation and characterization of HIV-1 pseudoviruses. HIV-1 pseudoviruses were
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produced in 293T cells by calcium phosphate transfection of pNL4-3E-Luc+ and HIV-1 o
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envelope expression vectors using the ProFection® Mammalian Transfection System lo
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(Promega Corp., Madison, WI). HIV-1 pseudovirus was harvested in culture e
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supernatants 48 hrs post-transfection. Supernantants were clarified of cell debris by o
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centrifuging at 1500xg for 10 min. Single-cycle infection assays were generally h
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performed on the same day as harvesting of HIV-1 pseudovirus. HIV-1 p24 //
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concentrations in pseudovirus stocks were measured using a commercial enzyme-linked s
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immunoassay (Alliance HIV-1 p24 Ag Kit, Perkin Elmer; Waltham, MA). Pseudovirus o
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stocks were normalized by p24 prior to testing. In addition, some assays were also o
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performed by normalizing RLU to the amount of p24 input per well and the linearity of p
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results to virus input verified for each assay. To assess susceptibility of pseudoviruses to 4
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CCR5 co-receptor antagonists, U87-CD4-CCR5 were seeded into 96-well luminometer 1
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plates (Perkin Elmer, Inc., Waltham, MA) using 5,000 cells/well. Plates were incubated y

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at 37ºC. The next day serial 10-fold dilutions of inhibitor in cell culture medium (10 µM e
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→ 0.01 nM) were added to wells 1 hour prior to the addition of HIV-1 pseudovirus plus
inhibitor. Antibody neutralization of HIV-1 pseudovirus infection with CTC5 anti-N
terminus CCR5 MAb (R& D Systems Inc., Minneapolis, MN; Catalog # MAB1802) was
also performed in the same manner using serial 2-fold dilutions of Ab (25 µg/ml) in the
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presence and absence of 10 µM VCV. Plates were incubated for 72 hrs, and luciferase
activity was analyzed by adding 50 µl of BrightGloTM luciferase assay buffer (Promega
Corp., Madison, WI). Plates were read on a Dynex luminometer (300 mSec/well).
Relative light units (RLU) were normalized to virus dose, measured as ng p24, and
percent inhibition was calculated as follows: 100- [average normalized RLU for HIV-1
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pseudovirus plus drug/average normalized RLU for HIV-1 pseudovirus from control o
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wells without drug] x 100. Dose-response data was analyzed using a non-linear lo
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regression 4-parameter logistic curve fit program with GraphPad Prism® Software e
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Version 4.0 (GraphPad Software Inc., San Diego, CA). o
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Expression of CCR5 mutants in 293T Cells. 293T cells were transfected with 10 µg h
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pcDNA3.1-CD4 and 20 µg of pcDNA3.1-CCR5 per 10-cm plate using ProFection® //
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Mammalian Transfection System (Promega Corp., Madison, WI). The CCR5 and CD4 s
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cell surface expression in 293T cells was analyzed by fluorescence activated cell sorting o
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(FACS) using murine anti-human CD195 (clone 2D7) CCR5 antibody (Ab) and anti- o
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human CD4 Ab (BD PharmingenTM, San Diego, CA; Catalog #555346). 293T cells 24 p
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hrs post transfection were seeded into 96-well luminometer plates (Perkin Elmer) at 4
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5,000 cells/well and plates were incubated overnight at 37ºC. HIV-1 pseudovirus 1
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infection of 293T cells was performed as described above. y

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Western blot analysis of gp120 incorporated into HIV-1 pseudovirus. HIV-1 e
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pseudovirus stocks were generated as described above. p24 levels in pseudovirus stocks
was measured by enzyme-linked immunosorbent assay. Equivalent levels of p24 (3 µg)
from each stock was batch absorbed using lectin-agarose from Galanthus nivalis
overnight at 4ºC. Lectin-agarose beads were washed with 0.25 % Tween in phosphate-
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buffered saline (PBS). HIV-1 envelope incorporated into eluted pseudovirus was
analyzed by Western blot with rabbit anti-gp120 polyclonal antibody
(ImmunoDiagnostics, Inc., Woburn, MA).

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