Fusion to Flaviviral Leader Peptide Targets HIV-1 Reverse Transcriptase for Secretion and Reduces Its Enzymatic Activity and Ability to Induce Oxidative Stress but Has No Major Effects on Its Immunogenic Performance in DNA-Immunized Mice

Reverse transcriptase (rt) is a key enzyme in viral replication and susceptibility to art and a crucial target of immunotherapy against drug-resistant hiv-1. rt induces oxidative stress which undermines the attempts to make it immunogenic. we hypothesized that artificial secretion may reduce the stress and make rt more immunogenic. inactivated multidrug-resistant rt (rt1.14opt-in) was n-terminally fused to the signal providing secretion of ns1 protein of tbev (ld) generating optimized inactivated ld-carrying enzyme rt1.14oil. promotion of secretion prohibited proteasomal degradation increasing the half-life and content of rt1.14oil in cells and cell culture medium,drastically reduced the residual polymerase activity,and downmodulated oxidative stress. balb/c mice were dna-immunized with rt1.14opt-in or parental rt1.14oil by intradermal injections with electroporation. fluorospot and elisa tests revealed that rt1.14opt-in and rt1.14oil induced ifn-γ/il-2,rt1.14opt-in induced granzyme b,and rt1.14oil induced perforin production. perforin secretion correlated with coproduction of ifn-γ and il-2 (r=0,97). both dna immunogens induced strong anti-rt antibody response. ldpeptide was not immunogenic. thus,ld-driven secretion inferred little change to rt performance in dna immunization. positive outcome was the abrogation of polymerase activity increasing safety of rt-based dna vaccines. identification of the molecular determinants of low cellular immunogenicity of rt requires further studies. © 2017 anastasia latanova et al.