Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody

Abstract

IntroductionThe advent of monoclonal antibodies was a major breakthrough for the development of antibody-based therapies against various diseases, including virus infection. 1 However, expression in the cytoplasm or nucleus of eukaryotic cells of full-length immunoglobulin G or single-chain antibody variable domain fragments aiming at blocking an intracellular target is poorly efficient because of the reducing nature of these compartments. The engineering of single-chain antibody variable domain fragments (scFvs) that consist in a tandem fusion of the VH and VL domains of a specific antibody partially overcame this limitation. Numerous scFvs have shown their potency for inhibition of the function of their target proteins in cells. In particular, a HIV-1 Tat-specific scFv was shown to neutralize the trans-activating function of this viral protein and to prevent full expression of the viral genome in infected cells. 2 ScFvs specific for HIV-1 matrix, integrase, or regulatory proteins such as Tat, Rev, and Vif are also able to interfere with HIV-1 replication. [3][4][5][6][7][8][9] However, disulfide bonds between VH and VL chains of scFvs are unlikely to form when scFvs are targeted to reducing compartments such as the cytoplasm or the nucleus, resulting in scFvs with lower or no affinity for their antigen. 10 Recently, a major improvement was brought by the use of single-domain antibodies (sdAbs) derived from camelids. In addition to conventional antibodies, camelids express antibodies composed of heavy chains only, with a single variable domain (VHH) capable of recognizing their cognate antigens. 11 These variable domains, called sdAbs, are endowed with many attractive features. 12 The absence of requirement for disulfide bond formation in sdAbs is particularly interesting when targeting of proteins found in reducing cell compartments is considered. 13 In contrast to conventional antibodies, these 13-kDa sdAb fragments can penetrate in cavities located on the surface of antigens (for review, see Nguyen et al 14 ). These cryptic sites are often more conserved than exposed epitopes on viral proteins and are a target of choice for blocking antibodies. Interestingly, sdAb fragments targeting the HIV-1 Rev or Vif proteins have been already characterized and have displayed antiviral activity in cell-culture assays. 15,16 In the present study, we characterized an sdAb from a llama immunized with a recombinant form of Nef, an HIV-1 nonstructural protein found both in the cytoplasm of infected cells and in association with cellular membranes. Considering Nef as a potential target for antiviral therapy arose from the findings that this HIV-1 protein is important for AIDS pathogenesis in vivo (for review, see Foster and Garcia 17 ). Nef is abundantly expressed early after virus infection and perturbs the trafficking of several membrane proteins through action on the endocytic pathway. This leads to the modulation of cell surface expression of some receptors, including CD4 and major histocompatibility complex class I (MHC-I...

HIV-1 reprograms the migration of macrophages

“…All other Nef SF2 variants, including 2 Nef variants (AxxA and VGF→AAA) that impact the interaction of Nef with SH3-binding domains, were not found around podosomes and were unable to modify their size (supplemental Figure 5A-B). When wt-Nef SF2 was coexpressed with antiNef single-domain antibodies (sdAbs) and Neffins C1 and B6, 63,64 we observed that Nef SF2 was delocalized from podosomes (data not shown), and F-actin content in podosomes was significantly decreased (supplemental Figure 5C). Neffins C1 and B6 are anti-Nef sdAbs linked to the SH3 domain of the Src family member Hck, which inhibit most Nef biological activities.…”

“…Neffins C1 and B6 are anti-Nef sdAbs linked to the SH3 domain of the Src family member Hck, which inhibit most Nef biological activities. 63,64 The strong inhibitory effect of Neffins on podosome size, together with the effects of Nef mutants AxxA and VGF→AAA, suggested a pivotal role of the Nef-SH3 interaction in podosome modulation. In addition, Nef SF2 AxxA failed to increase the life span of podosomes compared with wt-Nef SF2 (supplemental Figure 5D).…”

Antibody-Dependent Cellular Cytotoxicity against Reactivated HIV-1-Infected Cells

“…ADCC antibodies within HIV-1 ϩ sera primarily recognize CD4-induced Env epitopes concealed as a result of Vpu-and Nef-mediated CD4-downregulation (23). The inhibition of Nef and Vpu might be a possible strategy to increase recognition of Env-expressing cells (49). On a similar note, we show here that CD4mc have potential therapeutic utility because they can efficiently induce the CD4-bound conformation and increase binding of HIV-1 ϩ serum antibodies to ex vivo-expanded primary CD4 ϩ T cells.…”

Recruitment of the Nuclear Form of Uracil DNA Glycosylase into Virus Particles Participates in the Full Infectivity of HIV-1

“…293T cells were seeded into T75 flasks at a density of 4 ϫ 10 6 cells and transfected 24 h later with 10 g of the GFP-tagged murine or human UNG2 expression plasmid in combination with the HA-Vpr expression plasmid. After 24 h, the cells were lysed in NP-40 lysis buffer supplemented with protease inhibitor (Roche) for 30 min under gentle agitation at 4°C, as described previously (7). After centrifugation at 13,200 rpm, immunoprecipitations were carried out on 450 g of cell lysate proteins by incubation with 3 g of anti-GFP antibody (7.1/13.1; Roche) and 30 l of protein A-Sepharose 4B beads (Sigma) for 2 h under gentle agitation at 4°C.…”