Reversal of epigenetic silencing rescues integrase-deficient HIV-1 replication

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ABSTRACT After reverse transcription in the cytoplasm, retroviral DNAs enter the nucleus naked and are then detected by currently unknown innate immune factors and coated with repressive chromatin. However, retroviruses encode an integrase (IN) which allows the proviral DNA to be inserted into transcriptionally active areas of the host genome, which then results in the repressive chromatin marks on the retroviral DNA being replaced by the active marks characteristic of the flanking host chromatin. If IN activity is blocked either by mutagenesis or by a drug, then the unintegrated retroviral DNA remains covered with inhibitory chromatin and is transcriptionally silenced. This grant proposes has two goals. Firstly, I hypothesize that if unintegrated HIV-1 DNA is epigenetically silenced then it should be possible to activate that DNA, and rescue the replication of IN- HIV-1, by either directly activating the HIV-1 LTR promoter and/or by directly reversing the epigenetic silencing. We have now shown that expression of the HTLV-1 Tax protein, a potent activator of cellular NK-kB/Rel transcription factors, induces the recruitment of RelA and RelB to the NF-kB sites present in the HIV-1 LTR and also prevents or reverses the epigenetic silencing of unintegrated HIV-1 DNA, allowing IN- HIV-1 mutants to mount a robust, spreading infection in CEM-SS cells. It remains unclear whether transcriptional activation of the HIV-1 LTR occurs before or after the change in epigenetic marks on the viral DNA, and this is something we wish to address, including in primary T cells. We now report preliminary data, obtained in 293T cells, showing that the HSV-1 ICP0 protein can also rescue IN- HIV-1 gene expression, in this case by apparently directly regulating the epigenetic status of unintegrated HIV-1 DNA. We aim to extend this analysis to T cells to see if ICP0 is indeed rescuing IN- HIV-1 gene expression via a different mechanism than Tax. Finally, while the factors that recognize and silence IN- MLV have been identified by the Goff laboratory as the HUSH complex acting in concert with NP220, we show that these factors are not required to silence IN- HIV-1. A key goal of this application is therefore to perform an unbiased CRISPR/Cas genetic screen to identify the human innate immune factors that perform this task and then define their mechanism of action.