HIV-1 gag EXPRESSION IS QUANTITATIVELY DEPENDENT ON THE RATIO OF NATIVE AND
OPTIMIZED CODONS
Alexander Kofman,1 Marcus Graf, Alexandra Bojak, Ludwig Deml, Kurt Bieler, Alexandra Kharazova,
Hans Wolf, Ralf Wagner
Institute for Medical Microbiology and Hygiene, University of Regensburg, D-93053 Regensburg, Germany; and St.
Petersburg State University;
1 Current address: Center for Gene Therapy, Tulane University School of Medicine, 333 S.Saratoga St.
JBJ Building, Rm. 650, New Orleans, 70112, LA, USA;
e-mail: akofman@tulane.edu
There is a significant variation of codon usage bias among different species and even among genes within the same
organisms. Codon optimization, this is, gene redesigning with the use of codons preferred for the specific expression
system, results in improved expression of heterologous genes in bacteria, plants, yeast, mammalian cells, and
transgenic animals. The mechanisms preventing expression of genes with rare or low-usage codons at adequate levels
are not completely elucidated. Human immunodeficiency virus (HIV) represents an interesting model for studying how
differences in codon usage affect gene expression in heterologous systems. Construction of svnfhetic genes with
optimized codons demonstrated that the codon-usage effects might be a major impediment to the efficient expression of
HIV gag/pol and env gene products in mammalian cells. According to another hypothesis, the poor
expression of HIV structural proteins even without HIV contex is attributed to the so-called cis-acting inhibitory
elements (INS), which are located within the protein-coding region. They ;onsist of AU-rich sequences and may be
inactivated through the introduction of multiple mutations over the large regions of gag gene. In our work, we
evaluated expression of hybrid HIV-1 gag mRNAs where wild-type (A-rich) gag sequences were combined
with artificial sequences. In such "humanized" gag fragments with adapted codon usage, AT-content was
significantly reduced in favor of G and Ñ nucleotides without any changes in protein sequence. We show that wild-type
gag sequences negatively influence expre ssion of gag-reporter, and the addition of fragments with
optimized codons to gag mRNA partially rescues its expression. The results demonstrate that the expression of
HIV-1 gag is determined by the ratio of optimized and rare codons within mRNA. Our data also indicates that some
wtgag fragments counteract the influence of the other wtgag sequences, which cause the inhition of gag
expression. The presented data do not contradict the concepof INS; yet, it makes the definition of INS more complex. This supports the idea of a broader role of the selected
codon usage in influencing the expression of HIV proteins in mammalian cells.
Key words: codon, HIV, gag, chimeric mRNA, synthetic gene
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