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Abstract
Transcription of the HIV-1 provirus generates a viral pre-mRNA, which is alternatively spliced into more than 50 HIV-1 mRNAs encoding all viral proteins. Regulation of viral alternative splice site usage includes the presence of splicing regulatory elements (SREs) which can dramatically impact RNA expression and HIV-1 replication when mutated. Recently, we were able to show that two viral SREs, GI3-2 and ESEtat, are important players in the generation of viral vif, vpr and tat mRNAs. Furthermore, we demonstrated that masking these SREs by transfected locked nucleic acid (LNA) mixmers affect the viral splicing pattern and viral particle production. With regard to the development of future therapeutic LNA mixmer-based antiretroviral approaches, we delivered the GI3-2 and the ESEtat LNA mixmers “nakedly”, without the use of transfection reagents (gymnosis) into HIV-1 infected cells. Surprisingly, we observed that gymnotically-delivered LNA mixmers accumulated in the cytoplasm, and seemed to co-localize with GW bodies and induced degradation of mRNAs containing their LNA target sequence. The GI3-2 and the ESEtat LNA-mediated RNA degradation resulted in abrogation of viral replication in HIV-1 infected Jurkat and PM1 cells as well as in PBMCs.
Highlights
According to the World Health Organization (WHO) in 2017, 36.9 million people were living with the human immunodeficiency virus type 1 (HIV-1) and globally only 21.7 million people received antiretroviral therapy (ART) which combines drugs targeting crucial steps of the HIV-1 replication cycle
We demonstrated that mutating these elements by site-directed mutagenesis or masking these elements by co-transfecting host cells with the proviral DNA and the respective locked nucleic acid (LNA) mixmer successfully interfered with viral pre-mRNA splicing and viral replication [13,15]
Gymnotically-Delivered LNA Mixmers Binding the SREs GI3-2 and ESEtat Induce Degradation of Their Target mRNAs. Both viral splicing regulatory elements (SREs), GI3-2 and ESEtat (Figure 1a), localized within HIV-1 intron 3 and downstream of the viral SA3 respectively, are involved in regulating HIV-1 splice site usage which is essential for the generation of tat as well as vpr and vif mRNA species
Summary
According to the World Health Organization (WHO) in 2017, 36.9 million people were living with the human immunodeficiency virus type 1 (HIV-1) and globally only 21.7 million people received antiretroviral therapy (ART) which combines drugs targeting crucial steps of the HIV-1 replication cycle. The identification of additional and alternative targets within the viral life cycle for antiviral drug development is desirable. The SREs are bound by family members of the serine- and arginine-rich phosphoproteins (SR proteins) or heterogeneous nuclear ribonucleoproteins (hnRNPs), which positively or negatively influence viral splice site selection depending on their position relative to them [5,8,9]. Disruption of the viral splicing process, e.g., by preventing binding of splicing regulatory proteins to their RNA target seems to be a promising approach to impair viral replication. As shown in several mutational analyses of viral SREs, interference with the SREs’ function dramatically impacts viral splicing or RNA expression, and influences HIV-1 particle production [4,10,11,12,13,14,15,16]
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