Camelized rabbit-derived VH single-domain Intrabodies Against vif strongly neutralize HIV-1 infectivity

被引:71
作者
da Silva, FA
Santa-Marta, M
Freitas-Vieira, A
Mascarenhas, P
Barahona, I
Moniz-Pereira, J
Gabuzda, D
Goncalves, J [1 ]
机构
[1] Univ Lisbon, URIA, Ctr Patogenese Mol, Fac Farm, P-1649019 Lisbon, Portugal
[2] Inst Super Ciencias Saude Sul, P-2829511 Caparica, Portugal
[3] Dana Farber Canc Inst, Dept Canc Immunol & AIDS, Boston, MA 02115 USA
[4] Harvard Univ, Sch Med, Dept Neurol, Boston, MA 02115 USA
关键词
intracellular antibodies; VH single-domains; camelization; Vif neutralization; HIV infectivity;
D O I
10.1016/j.jmb.2004.04.062
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We recently developed a specific single-chain antibody from immunized rabbits to HIV-1 Vif protein that was expressed intracellularly and inhibited reverse transcription and viral replication. The Vif of HIV-1 overcomes the innate antiviral activity of a cytidine deaminase Apobec3G (CEM15) that induces G to A hypermutation in the viral genome, resulting in enhancement of viral replication infectivity. Here, we have developed a minimal scaffold VH fragment with intrabody properties derived from anti-Vif single-chain antibody that was engineered to mimic camelid antibody domains. Non-specific binding of VH by its interface for the light chain variable domain (VL) was prevented through amino acid mutations in framework 2 and 4 (Val37F, G44E, L45R, W47G and W103R). Our results demonstrate that all constructed anti-Vif VH single-domains preserve the antigen-binding activity and specificity in the absence of the parent VL domain. However, only the most highly camelized domains had high levels of intracellular expression. The expression in eukaryotic cells showed that VH single-domains could correctly fold as soluble proteins in the reducing environment. The results demonstrated an excellent correlation between improvements in protein solubility with gradually increasing camelization. Camelized single-domains efficiently bound Vif protein and neutralized its infectivity enhancing function, by reducing late reverse transcripts and proviral integration. The activity of the anti-Vif single-domains was shown to be cell-specific, with inhibitory effects only in cells non-permissive that require Vif for HIV-1 replication. Moreover, cell specificity of anti-Vif intrabodies was correlated with an increase of Apobec3G, which potentiates viral inhibition. The present study strongly suggests that camelization of rabbit VH domains is a potentially useful approach for engineering intrabodies for gene therapy. (C) 2004 Elsevier Ltd. All rights reserved.
引用
收藏
页码:525 / 542
页数:18
相关论文
共 58 条
[1]   Inhibition of Tat-mediated transactivation and HIV-1 replication by human anti-hCyclinT1 intrabodies [J].
Bai, JR ;
Sui, JH ;
Zhu, RY ;
Tallarico, AS ;
Gennari, F ;
Zhang, DS ;
Marasco, WA .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2003, 278 (03) :1433-1442
[2]   SINGLE-CHAIN ANTIGEN-BINDING PROTEINS [J].
BIRD, RE ;
HARDMAN, KD ;
JACOBSON, JW ;
JOHNSON, S ;
KAUFMAN, BM ;
LEE, SM ;
LEE, T ;
POPE, SH ;
RIORDAN, GS ;
WHITLOW, M .
SCIENCE, 1988, 242 (4877) :423-426
[3]   A quantitative assay for HIV DNA integration in vivo [J].
Butler, SL ;
Hansen, MST ;
Bushman, FD .
NATURE MEDICINE, 2001, 7 (05) :631-634
[4]   Engineering antibodies for imaging and therapy [J].
Carter, P ;
Merchant, AM .
CURRENT OPINION IN BIOTECHNOLOGY, 1997, 8 (04) :449-454
[5]   The selection of intracellular antibodies [J].
Cattaneo, A ;
Biocca, S .
TRENDS IN BIOTECHNOLOGY, 1999, 17 (03) :115-121
[6]   Characterization of a new intrabody directed against the N-terminal region of human p53 [J].
Cohen, PA ;
Mani, JC ;
Lane, DP .
ONCOGENE, 1998, 17 (19) :2445-2456
[7]   Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs [J].
Conrath, KE ;
Lauwereys, M ;
Wyns, L ;
Muyldermans, S .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (10) :7346-7350
[8]   Efficient tumor targeting by single-domain antibody fragments of camels [J].
Cortez-Retamozo, V ;
Lauwereys, M ;
Gh, GH ;
Gobert, M ;
Conrath, K ;
Muyldermans, S ;
De Baetselier, P ;
Revets, H .
INTERNATIONAL JOURNAL OF CANCER, 2002, 98 (03) :456-462
[9]   CAMELISING HUMAN-ANTIBODY FRAGMENTS - NMR-STUDIES ON VH DOMAINS [J].
DAVIES, J ;
RIECHMANN, L .
FEBS LETTERS, 1994, 339 (03) :285-290
[10]   Antigen-independent selection of stable intracellular single-chain antibodies [J].
der Maur, AA ;
Escher, D ;
Barberis, A .
FEBS LETTERS, 2001, 508 (03) :407-412