title: A stably expressed llama single-domain intrabody targeting Rev displays broad-spectrum anti-HIV activity.
abstract: The HIV Rev protein mediates the transport of partially and unspliced HIV mRNA from the nucleus to the cytoplasm. Rev multimerizes on a secondary stem-loop structure present in the viral intron-containing mRNA species and recruits the cellular karyopherin CRM1 to export viral mRNAs from the nucleus to the cytoplasm. Previously we have identified a single-domain intrabody (Nb(190)), derived from a llama heavy-chain antibody, which efficiently inhibits Rev multimerization and suppresses the production of infectious virus. We recently mapped the epitope of this nanobody and demonstrated that Rev residues K20 and Y23 are crucial for interaction while residues V16, H53 and L60 are important to a lesser extent. Here, we generated cell lines stably expressing Nb(190) and assessed the capacity of these cell lines to suppress the replication of different HIV-1 subtypes. These cells stably expressing the single-domain antibody are protected from virus-induced cytopathogenic effect even in the context of high multiplicity of infection. In addition, the replication of different subtypes of group M and one strain of group O is significantly suppressed in these cell lines. Next, we analysed the natural variations of Rev amino acids in sequence samples from HIV-1 infected patients worldwide and assessed the effect of Nb(190) on the most prevalent polymorphisms occurring at the key epitope positions (K20 and Y23) in Rev. We found that Nb(190) was able to suppress the function of these Rev variants except for the K20N mutant, which was present in only 0.7% of HIV-1 sequence populations (n = 4632). Cells stably expressing the single-domain intrabody Nb(190) are protected against virus-induced cytopathogenic effect and display a selective survival advantage upon infection. In addition, Nb(190) suppresses the replication of a wide range of different HIV-1 subtypes. Large-scale sequence analysis reveals that the Nb(190) epitope positions in Rev are well conserved across major HIV-1 subtypes and groups. Altogether, our results indicate that Nb(190) may have broad potential as a gene therapeutic agent against HIV-1.
authors.affiliation: Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, KU Leuven, Minderbroedersstraat 10, Leuven B-3000, Belgium. Electronic address: Eline.boons@rega.kuleuven.be.
authors.forename: Eline
authors.initials: E
authors.lastname: Boons
authors.affiliation: Rega Institute for Medical Research, Laboratory for Clinical and Epidemiological Virology, Department of Microbiology and Immunology, KU Leuven, Minderbroedersstraat 10, Leuven B-3000, Belgium. Electronic address: Guangdi.Li@rega.kuleuven.be.
authors.forename: Guangdi
authors.initials: G
authors.lastname: Li
authors.affiliation: Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, KU Leuven, Minderbroedersstraat 10, Leuven B-3000, Belgium. Electronic address: Els.vanstreels@rega.kuleuven.be.
authors.forename: Els
authors.initials: E
authors.lastname: Vanstreels
authors.affiliation: Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, KU Leuven, Minderbroedersstraat 10, Leuven B-3000, Belgium. Electronic address: Thomas.vercruysse@rega.kuleuven.be.
authors.forename: Thomas
authors.initials: T
authors.lastname: Vercruysse
authors.affiliation: Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, KU Leuven, Minderbroedersstraat 10, Leuven B-3000, Belgium. Electronic address: Christophe.pannecouque@rega.kuleuven.be.
authors.forename: Christophe
authors.initials: C
authors.lastname: Pannecouque
authors.affiliation: Rega Institute for Medical Research, Laboratory for Clinical and Epidemiological Virology, Department of Microbiology and Immunology, KU Leuven, Minderbroedersstraat 10, Leuven B-3000, Belgium; Centro de Malária e Outras Doenças Tropicais and Unidade de Microbiologia, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa 1349-008, Portugal. Electronic address: Annemie.vandamme@uzleuven.be.
authors.forename: Anne-Mieke
authors.initials: AM
authors.lastname: Vandamme
authors.affiliation: Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, KU Leuven, Minderbroedersstraat 10, Leuven B-3000, Belgium. Electronic address: Dirk.daelemans@rega.kuleuven.be.
authors.forename: Dirk
authors.initials: D
authors.lastname: Daelemans
mesh_terms: D000818:Animals; D002161:Camelids, New World; D002460:Cell Line; D003588:Cytopathogenic Effect, Viral; D015870:Gene Expression; D014644:Genetic Variation; D015483:HIV Antibodies / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects*; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors* / Q000235:genetics
chemical_list: D015483:HIV Antibodies; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies; D054321:rev Gene Products, Human Immunodeficiency Virus; C514860:rev protein, Human Immunodeficiency Virus-1
keywords: HIV-1; Intrabody; Rev; Single-domain antibody