Chandra’s study, featuring Mariana’s MPH thesis work, has been published in the Journal of Virology. Chandra characterized two replication-competent HIV-1 reporter viruses: one encoding the bioluminescent protein nanoluciferase (nLuc) and the other encoding a near-infrared fluorescent protein called iRFP670 (or iRFP). After characterization in human CD4+ T cells, Chandra infected NSG mice engrafted with human CD34+ cells and could detect nLuc or iRFP via whole body imaging. However, the imaging results did not correlate well with plasma viremia, particularly for the iRFP virus, such that reporter signal decreased over time despite stable plasma HIV-1 RNA levels.
We noted that the reporter genes contained high numbers of CG dinucleotides (nearly 40 CGs for the 516 bp nLuc gene and a whopping 138 CGs for the 936 bp iRFP gene!), which others have described as targets for the human antiviral protein ZAP. The CGs in the reporter sequences were removed by synonymous mutations and Chandra infected new sets of mice with them. He found that the reporter signal intensity was maintained longer and now correlated with plasma viremia with these codon-optimized viruses.
In addition, he showed that replication and reporter gene expression of the viruses could be suppressed with antiretroviral therapy (ART) and that animals could be infected with both viruses simultaneously.
Finally, we showed that CG codon optimization improved the reporter gene expression in mouse cells, which Mariana assessed with immunofluorescence imaging of the mouse spleens (results summarized below). In addition, it appeared that viral RNA from the animals infected with the original (OG) reporter viruses had more deletions of the reporter gene than animals infected with the codon-optimized (CO) reporter HIV-1, suggesting that viruses lacking reporter genes were preferentially selected over time.
We have made the plasmids encoding these codon-optimized, replication-competent viruses available upon request at the NIH HIV Reagent Program.
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