Quantitative effect of suboptimal codon usage on translational efficiency of mRNA encoding HIV-1 gag in intact T cells.

BACKGROUND: The sequences of wild-isolate strains of Human Immunodeficiency Virus-1 (HIV-1) are characterized by low GC content and suboptimal codon usage. Codon optimization of DNA vectors can enhance protein expression both by enhancing translational efficiency, and by altering RNA stability and export. Although gag codon optimization is widely used in DNA vectors and experimental vaccines, the actual effect of altered codon usage on gag translational efficiency has not been quantified. METHODOLOGY AND PRINCIPAL FINDINGS: To quantify translational efficiency of gag mRNA in live T cells, we transfected Jurkat cells with increasing doses of capped, polyadenylated synthetic mRNA corresponding to wildtype or codon-optimized gag sequences, measured Gag production by quantitative ELISA and flow cytometry, and estimated the translational efficiency of each transcript as pg of Gag antigen produced per microg of input mRNA. We found that codon optimization yielded a small increase in gag translational efficiency (approximately 1.6 fold). In contrast when cells were transfected with DNA vectors requiring nuclear transcription and processing of gag mRNA, codon optimization resulted in a very large enhancement of Gag production. CONCLUSIONS: We conclude that suboptimal codon usage by HIV-1 results in only a slight loss of gag translational efficiency per se, with the vast majority of enhancement in protein expression from DNA vectors due to altered processing and export of nuclear RNA.

Counter-regulatory effects of incremental hypoxia on the transcription of a cardiac fatty acid oxidation enzyme-encoding gene.

Cardiac fatty acid oxidation (FAO) enzyme gene expression is known to be downregulated during hypoxia in concordance with reduced FAO rates. To evaluate this metabolic switch, the transcriptional control of a cardiac FAO enzyme-encoding gene (medium-chain acyl-CoA dehydrogenase, MCAD) was characterized in response to hypobaric hypoxia. Transgenic mice harboring 560-bp of the human MCAD gene promoter fused to the bacterial chloramphenicol acetyl transferase (CAT) reporter gene were exposed to moderate (14% O2) or severe (8% O2) hypoxia for 2 or 7 days. MCAD-CAT activity and gene expression were significantly downregulated following 7 days of moderate hypoxia versus normoxic controls (p < 0.05). In parallel two known transcriptional regulators of MCAD expression, PPARalpha and Sp3, were concordantly downregulated at 7 days hypoxia. In contrast, severe hypoxia increased MCAD-CAT activity by 31 +/- 1.4% after 2 days hypoxia, returning to base +/- 4% after 2 days (p < 0.001) and returned to control levels after 7 days of hypoxia. These data demonstrate that MCAD gene expression is downregulated after 7 days of moderate hypoxia and inversely regulated with severe hypoxia. The known MCAD transcriptional regulators PPARalpha and Sp3 mirror MCAD expression. These data indicate that the transcriptional regulatory circuits involved in the control of MCAD gene expression under hypoxic conditions are modulated by upstream factors that are sensitive to the levels of oxygen.