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 (Nb190), 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 Nb190 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 Nb190 on the most prevalent polymorphisms occurring at the key epitope positions (K20 and Y23) in Rev. We found that Nb190 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 Nb190 are protected against virus-induced cytopathogenic effect and display a selective survival advantage upon infection. In addition, Nb190 suppresses the replication of a wide range of different HIV-1 subtypes. Large-scale sequence analysis reveals that the Nb190 epitope positions in Rev are well conserved across major HIV-1 subtypes and groups. Altogether, our results indicate that Nb190 may have broad potential as a gene therapeutic agent against HIV-1.