HIV gp120 blocking peptide

We aimed to evaluate the possibility the nuclear transformation of lettuce and tobacco to produce recombinant anti-HIV microbicide griffithsin under impact of Zera signal peptide. For this purpose, the codon optimized GRFT fused with KDEL retention signal was used with and without the Zera (-zein ER-accumulating domain) signal peptide. Integration of GRFT into the nuclear genome of lettuce and tobacco transgenic lines was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Subsequent reverse transcription-quantitative PCR (RT-qPCR) experiments showed highly divergent GRFT expression patterns, inherent to the applied transformation procedure. The recombinant GRFT was successfully detected by means of western blot and quantified by ELIZA. According to ELIZA results, fusion of GRFT with Zera signal peptide resulted in higher accumulation of the recombinant protein in both species once compared with transgenic line without signal peptide. Lettuce showed higher transgene transcripts and accumulated more the recombinant protein of interest (up to 8.942 mu g/100mg) than tobacco. Both lettuce- and tobacco-derived GRFT (GRFTL and GRFTT, respectively) captured gp120 in a way comparable to E. coli expressed GRFT (GRFTE). Our results suggest that lettuce as a leafy vegetable crop and tobacco as a model plant in transgenic research studies can be used as suitable candidate hosts for the production of recombinant GRFT, augmented by recruitment of plant optimized codon compositions and suitable signal peptide.

The entry of human immunodeficiency virus into host cells is mediated by the envelope glycoprotein (Env) trimeric spike, which consists of three exterior gp120 subunits and three transmembrane gp41 subunits. The trimeric Env undergoes extensive conformational rearrangement upon interaction with the CD4 receptor, transitioning from the unliganded, "closed" State 1 to more-open downstream State 2 and State 3 conformations. Changes in "restraining" amino acid residues, such as leucine 193 and isoleucine 423, destabilize State 1 Env, which then assumes entry-competent, downstream conformations. The introduction of an artificial disulfide bond linking the gp120 and gp41 subunits (SOS) in combination with the I559P (IP) change has allowed structural characterization of soluble gp140 (sgp140) trimers. The conformation of these SOSIP-stabilized sgp140 trimers has been suggested to represent the closed native State 1 conformation. Here we compare the impact on the membrane Env conformation of the SOSIP changes with that of the well-characterized changes (L193R and I423A) that shift Env to downstream States 2 and 3. The results presented here suggest that the SOSIP changes stabilize Env in a conformation that differs from State 1 but also from the downstream Env conformations stabilized by L193R or I423A. IMPORTANCE The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer is triggered by receptor binding to mediate the entry of the virus into cells. Most structural studies of Env trimers have utilized truncated soluble gp140 Envs stabilized with the I559P and SOS changes. Here we present evidence indicating that these stabilizing changes have a profound impact on the conformation of Env, moving Env away from the native pretriggered Env conformation. Our studies underscore the need to acquire structural information on the pretriggered Env conformation, which is recognized by most broadly reactive neutralizing antibodies.