CG dinucleotide removal in bioluminescent and fluorescent reporters improves HIV-1 replication and reporter gene expression for dual imaging in humanized mice

Visualizing the transmission and dissemination of human immunodeficiency virus type 1 (HIV-1) in real time in humanized mouse models is a robust tool to investigate viral replication during treatments and in tissue reservoirs. However, the stability and expression of HIV-1 reporter genes are obstacles for long-term serial imaging in vivo . Two replication-competent CCR5-tropic HIV-1 reporter constructs were created that encode either nanoluciferase (nLuc) or a near - infrared fluorescent protein (iRFP) upstream of nef . HIV-1 reporter virus replication and reporter gene expression was measured in cell culture and in humanized mice. While reporter gene expression in vivo correlated initially with plasma viremia, expression decreased after 4-5 weeks despite high plasma viremia. The reporter genes were codon-optimized to remove cytosine/guanine (CG) dinucleotides and new CO-nLuc and CO-iRFP viruses were reconstructed. Removal of CG dinucleotides in HIV-1 reporter viruses improved replication in vitro and reporter expression in vivo and ex vivo . Both codon optimized reporter viruses could be visualized during co-infection and in vivo reporter gene expression during treatment failure preceded detection of plasma viremia. While the dynamic range of CO-iRFP HIV-1 was lower than that of CO-nLuc HIV-1, both viruses could have utility in studying and visualizing HIV-1 infection in humanized mice. Importance Animal models are important for studying HIV-1 pathogenesis and treatments. We developed two viruses each encoding a reporter gene that can be expressed in cells after infection. This study shows that HIV-1 infection can be visualized by noninvasive, whole body imaging in mice with human immune cells over time by reporter expression. We improved reporter expression to reflect HIV-1 replication and showed that two viral variants can be tracked over time in the same animal and can predict failure of antiretroviral therapy to suppress virus.