Frameshifting in HIV genes and chemotaxis of NEF protein isoforms
Olubajo, Babatunde Adetola
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Ribosomal frameshifting is a translational mechanism commonly exploited by RNA viruses in an effort to overcome the constraints of their limited genome size. Frameshifting requires a suitable heptanucleotide “slippery” sequence and a RNA pseudoknot, and the process allows viruses to translate genes hidden in any of the three frames that exist for all genes. Previous analysis of the HIV-1 genome in our laboratory indicates the potential for viral selenoproteins to be encoded by frameshifting through re-coding of UGA stop codons as selenocysteine. One of the main goals of this study was to test the validity of predicted frameshift sequences in the envelope, protease and nef sequences of HIV-1. Furthermore, there was an interest in determining the role intracellular selenium and sulfur concentrations might play in frameshifting when tRNA-lys is involved, as well as the effects of arginine on a proposed hungry arginine codon in the envelope frameshift sequence. A frameshift assay was developed to test the validity of hypothesized frameshift sequences. Green fluorescent protein (GFP) and luciferase genes were cloned and arranged in a eukaryotic expression vector, in a manner that allowed putative frameshift sequences to be inserted into the vector between the two reporter genes, with luciferase in a –1 frame of an upstream GFP gene. The vectors were expressed in 293-T cells and the ratio of reporter protein activity was assessed to determine the extent and efficiency of frameshifting. The envelope and protease genes had significant frameshift activity, which was found to decrease in the presence of selenium and sulfur donor compounds. The hungry arginine codon in the envelope gene also exhibited an increase in frameshifting in arginine deficient media. Previous analysis of the nef frameshift resultant reading frame in our laboratory, indicated amino acid similarity to gamma chemokines. Furthermore, several conserved UGA codons were evident in this reading frame with potential for selenocysteine incorporation. To test this hypothesis, several mutant constructs were made that induced the proposed frameshift reading frames. When these vectors were expressed in 293-T cells, the lysates of recombinant nef and one of the frameshift induced nef mutants displayed chemoattractant ability for Jurkat cells.