The codon usage of the major 96ZM651.8 gene sequences was transcribed manually to that of highly
expressed human genes by changing third-codon positions from A or T to G or C residues respectively.
A Kozak (GCCGCCGCC) sequence and a methionine codon(ATG) were added immediately before the first
codon of the reverse transcriptase(RT). The protease (PR) gene was excluded because overexpression
of PR is known to be toxic to cells.
Gao, F.; Li, Y.; Decker, J. M.; Peyerl, F. W.; Bibollet-Ruche, F.; Rodenburg, C. M.; Chen, Y.; Shaw, D. R.; Allen, S.; Musonda, R.; Shaw, G. M.; Zajac, A. J.; Letvin, N.; Hahn, B. H.
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Codon usage optimization of HIV type 1 subtype C gag, pol, env, and nef genes: in vitro expression and immune responses in DNA-vaccinated mice
Codon usage optimization of human immunodeficiency virus type 1 (HIV-1) structural genes has been shown to increase protein expression in vitro as well as in the context of DNA vaccines in vivo; however, all optimized genes reported thus far are derived from HIV-1 (group M) subtype B viruses. Here, we report the generation and biological characterization of codon usage-optimized gag, pol, env (gp160, gp140, gp120), and nef genes from a primary (nonrecombinant) HIV-1 subtype C isolate. After transfection into 293T cells, optimized subtype C genes expressed one to two orders of magnitude more protein (as determined by immunoblot densitometry) than the corresponding wild-type constructs. This effect was most pronounced for gp160, gp140, Gag, and Pol (>250-fold), but was also observed for gp120 and Nef (45- and 20-fold, respectively). Optimized gp160- and gp140-derived glycoproteins were processed, incorporated into virus particles, and mediated virus entry when expressed in trans to complement an env-minus HIV-1 provirus. Mice immunized with optimized gp140 DNA developed antibody as well as CD4+ and CD8+ T cell immune responses that were orders of magnitude greater than those of mice immunized with wild-type gp140 DNA. These data confirm and extend previous studies of codon usage optimization of HIV-1 genes to the most prevalent group M subtype. Our panel of matched optimized and wild-type subtype C genes should prove valuable for studies of protein expression and function, the generation of subtype-specific immunological reagents, and the production of DNA-based sub-unit vaccines directed against a broader spectrum of viruses.
AIDS Res Hum Retroviruses. 19(9): 817-23.
The authors reported the generation and biological characterization of codon usage-optimized gag, pol, env (gp160, gp140, gp120), and nef genes from a primary HIV1 subtype C isolate. The codon usage of the major 96ZM651.8 gene sequences was transcribed manually to that of highly expressed human genes by changing third-codon positions from A or T to G or C residues respectively. None of these substitutions resulted in changes of the encoded protein sequences. Experiments were done both in vitro and vivo for the expression levels and for cellular immune responses. The experimental results showed that the codon optimized genes increased the expression levels as well as cellular immune responses.