Camelized rabbit-derived VH single-domain intrabodies against Vif strongly neutralize HIV-1 infectivity.
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.
Aires da Silva
Frederico
F
URIA - Centro de Patogénese Molecular, Faculdade de Farmácia, Universidade de Lisboa, Lisboa 1649-019, Portugal.
Santa-Marta
Mariana
M
Freitas-Vieira
Acilino
A
Mascarenhas
Paulo
P
Barahona
Isabel
I
Moniz-Pereira
José
J
Gabuzda
Dana
D
Goncalves
Joao
J
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D016346:Gene Products, vif / Q000276:immunology*; D005816:Genetic Complementation Test; D015497:HIV-1 / Q000472:pathogenicity* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D014774:Virulence / Q000276:immunology; D014779:Virus Replication; D054320:vif Gene Products, Human Immunodeficiency Virus
D000906:Antibodies; D017931:DNA Primers; D016346:Gene Products, vif; D054320:vif Gene Products, Human Immunodeficiency Virus