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.

Approved Antiviral Drugs over the Past 50 Years

“…drug resistance mutations remain a major cause of treatment failure (10,466,468,469). Second, it is difficult to eradicate viral reservoirs using antiviral agents, because DNA viruses and retroviruses can integrate their genomes into human genomes (470,471).…”

Use, Applications and Mechanisms of Intracellular Actions of Camelid VHHs

“…The nanobody strongly reduced p24 levels for infected cells compared to a control nanobody. More specifically, p24 levels were reduced by >10 folds for subtype A, > 100 folds for subtypes C and G, and >10,000 folds for subtypes B, D, and H [45]. The cells proved to be resistant to viral replication and survived infection.…”

A novel intracellularly expressed NS5B-specific nanobody suppresses bovine viral diarrhea virus replication

“…Intrabodies represent an important class of therapeutic drugs with potential uses for microbial diseases therapy, and they can interrupt early and late events of the viral life cycle (Marasco, 2001;Rondon and Marasco, 1997). Recent studies have shown that several camel single domain intrabodies targeting the HIV-1 Rev protein, the IFV nucleoprotein, and the HCV envelope protein could exert antiviral activity (Ashour et al, 2015;Boons et al, 2014;Serruys et al, 2009), indicating that intracellularly-expressed Nbs are very effective in protecting the host against viral infection. BVD is of great concern worldwide to the H. Duan, et al Veterinary Microbiology 240 (2020) 108449 bovine industry, due to its complex genetic variability and lack of effective therapeutic drugs that can be used to date (Curti and Jaeger, 2013).…”