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.

CD8+ T-cell responses to different HIV proteins have discordant associations with viral load.

Selection of T-cell vaccine antigens for chronic persistent viral infections has been largely empirical. To define the relationship, at the population level, between the specificity of the cellular immune response and viral control for a relevant human pathogen, we performed a comprehensive analysis of the 160 dominant CD8(+) T-cell responses in 578 untreated HIV-infected individuals from KwaZulu-Natal, South Africa. Of the HIV proteins targeted, only Gag-specific responses were associated with lowering viremia. Env-specific and Accessory/Regulatory protein-specific responses were associated with higher viremia. Increasing breadth of Gag-specific responses was associated with decreasing viremia and increasing Env breadth with increasing viremia. Association of the specific CD8(+) T-cell response with low viremia was independent of HLA type and unrelated to epitope sequence conservation. These population-based data, suggesting the existence of both effective immune responses and responses lacking demonstrable biological impact in chronic HIV infection, are of relevance to HIV vaccine design and evaluation.

Acclimatization to chronic hypobaric hypoxia is associated with a differential transcriptional profile between the right and left ventricle.

Acute hypobaric hypoxia induces a transient reactivation of the fetal-metabolic gene program in the rat heart. Although chronic hypobaric hypoxia causes alterations in metabolism and cardiac function, little is known about the transcriptional profile associated with acclimatization to chronic hypoxia. Because in chronic hypoxia only the right ventricle is exposed to pressure overload (pulmonary hypertension), we hypothesized that chronic hypobaric hypoxia induces a differential transcriptional profile in the right and left ventricle. Male Wistar rats were exposed to a hypobaric environment (11% O2) for 4, 10, and 12 weeks. Right and left ventricular tissue was isolated for histology and candidate gene expression. Chronic hypobaric hypoxia induced right ventricular hypertrophy without fibrosis. In the right ventricle, changes in metabolic gene expression suggested a downregulation of fatty acid metabolism and an increase in glucose metabolism, while left ventricular metabolic gene expression suggested restoration of fatty acid metabolism. While myosin heavy chain isoform transcript levels in the right ventricle indicated a progressive reactivation of the fetal iso-gene pattern, there was normalization of myosin iso-gene expression in the left ventricle. Similarly, sarcoendoplasmic reticulum ATPase 2a (SERCA2a) transcript levels in the right ventricle decreased by 12 weeks of chronic hypoxia exposure, whereas, left ventricular SERCA2a expression was unchanged. In conclusion, acclimatization to chronic hypobaric hypoxia induced a differential transcriptional response between the right and left ventricle. We speculate that reactivation of the fetal-metabolic program in the right ventricle is adaptive to pressure overload.

Dynamic changes of gene expression in hypoxia-induced right ventricular hypertrophy.

Hypobaric hypoxia induces right ventricular hypertrophy. The relative contribution of pulmonary hypertension, decreased arterial oxygen, and neuroendocrine stimulation to the transcriptional profile of hypoxia-induced right ventricular hypertrophy is unknown. Whereas both ventricles are exposed to hypoxia and neuroendocrine stimulation, only the right ventricle is exposed to increased load. We postulated that right ventricular hypertrophy would reactivate the fetal gene transcriptional profile in response to increased load. We measured the expression of candidate genes in the right ventricle of rats exposed to hypobaric hypoxia (11% O(2)) and compared the results with the left ventricle. Hypoxia induced right ventricular hypertrophy without fibrosis. In the right ventricle only, atrial natriuretic factor transcript levels progressively increased starting at day 7. Metabolic genes were differentially regulated, suggesting a substrate switch from fatty acids to glucose during early hypoxia and a switch back to fatty acids by day 14. There was also a switch in myosin isogene expression and a downregulation of sarcoplasmic/endoplasmic ATPase 2a during early hypoxia, whereas later, both myosin isoforms and SERCA2a were upregulated. When the right and left ventricle were compared, the transcript levels of all genes, except for myosin isoforms and pyruvate dehydrogenase kinase-4, differed dramatically suggesting that all these genes are regulated by load. Our findings demonstrate that hypoxia-induced right ventricular hypertrophy transiently reactivates the fetal gene program. Furthermore, myosin iso-gene and pyruvate dehydrogenase kinase-4 expression is not affected by load, suggesting that either hypoxia itself or neuroendocrine stimulation is the primary regulator of these genes.

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.