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

Ngumbela KC, Ryan KP, Sivamurthy R, Brockman MA, Gandhi RT, Bhardwaj N, Kavanagh DG - PLoS ONE (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: HIV Pathogenesis Programme, Doris Duke Medical Research Institute, University of KwaZulu Natal, Durban, South Africa.

ABSTRACT

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.

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Related in: MedlinePlus

Gag expression in Jurkat cells transfected with gag mRNA.A. Gag production correlates with mRNA concentration. Jurkat cells were transfected by electroporation with increasing doses of synthetic codon-optimized gag mRNA. After 24 hours, the amount of Gag in the supernatant was determined by ELISA. The line shows linear fit (R2>0.99). B. The amount of Gag in cell supernatants is representative of the median Gag production in electroporated cells. Jurkat cells were transfected with varying doses of gag mRNA; after 24 hours, cell supernatants were harvested for ELISA, and cell pellets were fixed, permeabilized, and stained with anti-Gag/p24 antibody. Gag production as measured by ELISA is shown on the Y axis; Gag production as measured by MFI (median fluorescence intensity) is shown on the X axis. Line shows linear fit (R2 = 0.93). ELISA values are the average of triplicate wells.
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pone-0002356-g001: Gag expression in Jurkat cells transfected with gag mRNA.A. Gag production correlates with mRNA concentration. Jurkat cells were transfected by electroporation with increasing doses of synthetic codon-optimized gag mRNA. After 24 hours, the amount of Gag in the supernatant was determined by ELISA. The line shows linear fit (R2>0.99). B. The amount of Gag in cell supernatants is representative of the median Gag production in electroporated cells. Jurkat cells were transfected with varying doses of gag mRNA; after 24 hours, cell supernatants were harvested for ELISA, and cell pellets were fixed, permeabilized, and stained with anti-Gag/p24 antibody. Gag production as measured by ELISA is shown on the Y axis; Gag production as measured by MFI (median fluorescence intensity) is shown on the X axis. Line shows linear fit (R2 = 0.93). ELISA values are the average of triplicate wells.

Mentions: To measure Gag production by transfected cells, we generated synthetic mRNA encoding HIV-1 gag. Capped, polyadenylated mRNA was transfected into Jurkat cells by electroporation. Gag protein production was quantified by measuring the concentration of Gag p24 in cell supernatant by ELISA and by staining transfected cells with Gag-p24 specific fluorescent antibody. We found that the amount of Gag released into the culture medium was proportional to the amount of mRNA added to the transfection reaction (Fig 1A, R2>0.99). The amount of Gag detected by ELISA was proportional to the median fluorescence intensity (MFI) of cells stained with p24-specific fluorescent antibody (Figure 1B, R2 = 0.93), demonstrating that the amount of secreted Gag detected in cell supernatants was indicative of protein production by the majority of cells in the population.


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

Ngumbela KC, Ryan KP, Sivamurthy R, Brockman MA, Gandhi RT, Bhardwaj N, Kavanagh DG - PLoS ONE (2008)

Gag expression in Jurkat cells transfected with gag mRNA.A. Gag production correlates with mRNA concentration. Jurkat cells were transfected by electroporation with increasing doses of synthetic codon-optimized gag mRNA. After 24 hours, the amount of Gag in the supernatant was determined by ELISA. The line shows linear fit (R2>0.99). B. The amount of Gag in cell supernatants is representative of the median Gag production in electroporated cells. Jurkat cells were transfected with varying doses of gag mRNA; after 24 hours, cell supernatants were harvested for ELISA, and cell pellets were fixed, permeabilized, and stained with anti-Gag/p24 antibody. Gag production as measured by ELISA is shown on the Y axis; Gag production as measured by MFI (median fluorescence intensity) is shown on the X axis. Line shows linear fit (R2 = 0.93). ELISA values are the average of triplicate wells.
© Copyright Policy
Related In: Results  -  Collection

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pone-0002356-g001: Gag expression in Jurkat cells transfected with gag mRNA.A. Gag production correlates with mRNA concentration. Jurkat cells were transfected by electroporation with increasing doses of synthetic codon-optimized gag mRNA. After 24 hours, the amount of Gag in the supernatant was determined by ELISA. The line shows linear fit (R2>0.99). B. The amount of Gag in cell supernatants is representative of the median Gag production in electroporated cells. Jurkat cells were transfected with varying doses of gag mRNA; after 24 hours, cell supernatants were harvested for ELISA, and cell pellets were fixed, permeabilized, and stained with anti-Gag/p24 antibody. Gag production as measured by ELISA is shown on the Y axis; Gag production as measured by MFI (median fluorescence intensity) is shown on the X axis. Line shows linear fit (R2 = 0.93). ELISA values are the average of triplicate wells.
Mentions: To measure Gag production by transfected cells, we generated synthetic mRNA encoding HIV-1 gag. Capped, polyadenylated mRNA was transfected into Jurkat cells by electroporation. Gag protein production was quantified by measuring the concentration of Gag p24 in cell supernatant by ELISA and by staining transfected cells with Gag-p24 specific fluorescent antibody. We found that the amount of Gag released into the culture medium was proportional to the amount of mRNA added to the transfection reaction (Fig 1A, R2>0.99). The amount of Gag detected by ELISA was proportional to the median fluorescence intensity (MFI) of cells stained with p24-specific fluorescent antibody (Figure 1B, R2 = 0.93), demonstrating that the amount of secreted Gag detected in cell supernatants was indicative of protein production by the majority of cells in the population.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: HIV Pathogenesis Programme, Doris Duke Medical Research Institute, University of KwaZulu Natal, Durban, South Africa.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus