Irwan, I. D. | bioRxiv Science

bioRxiv2021

Tax induces the recruitment of NF-kB to unintegrated HIV-1 DNA to rescue viral gene expression and replication

We have previously reported that the normally essential step of integration of the HIV-1 proviral DNA intermediate into the host cell genome becomes dispensable in T cells that express the Human T cell leukemia virus 1 (HTLV-1) Tax protein. The rescue of integrase (IN) deficient HIV-1 replication by Tax results from the strong activation of transcription from the long terminal repeat (LTR) promoter on episomal HIV-1 DNA, an effect that is closely correlated with the recruitment of activating epigenetic marks, such as H3Ac, and depletion of repressive epigenetic marks, such as H3K9me3, from chromatinized unintegrated proviruses. In addition, activation of transcription from unintegrated HIV-1 DNA coincides with the recruitment of NF-kB to the two NF-kB binding sites found in the HIV-1 LTR enhancer. Here we report that the recruitment of NF-kB to unintegrated viral DNA precedes, and is a prerequisite for, Tax-induced changes in epigenetic marks, so that an IN-HIV-1 mutant lacking both LTR NF-kB sites is entirely non-responsive to Tax and fails to undergo the epigenetic changes listed above. We also report that heterologous promoters introduced into IN-HIV-1-based vectors are transcriptionally active even in the absence of Tax. Finally, we failed to reproduce a recent report arguing that heterologous promoters introduced into IN-vectors based on HIV-1 are more active if the HIV-1 promoter and enhancer, located in the LTR U3 region, are deleted, in a so-called self inactivating or SIN lentivector design. ImportanceIntegrase-deficient expression vectors based on HIV-1 are becoming increasingly popular as tools for gene therapy in vivo due to their inability to cause insertional mutagenesis. However, many IN-lentiviral vectors are able to achieve only low levels of gene expression and methods to increase this low level have not been extensively explored. Here we analyze how the HTLV-1 Tax protein is able to rescue the replication of IN-HIV-1 in T cells and describe IN-lentiviral vectors that are able to express a heterologous gene effectively.

molecular biology↗

bioRxiv2020

Robust HIV-1 Replication in the Absence of Integrase Function.

Integration of the proviral DNA intermediate into the host cell genome represents an essential step in the retroviral life cycle. While the reason(s) for this requirement remains unclear, it is known that unintegrated proviral DNA is epigenetically silenced. Here, we demonstrate that HIV-1 mutants lacking functional integrase can mount a robust, spreading infection in cells expressing the Tax transcription factor encoded by human T-cell leukemia virus 1. In these cells, HIV-1 forms episomal DNA circles, analogous to Hepatitis B virus covalently closed circular DNAs (cccDNAs), that are transcriptionally active and fully capable of supporting viral replication. This rescue correlates with the loss of inhibitory epigenetic marks, and the acquisition of activating marks, on histones bound to unintegrated HIV-1 DNA. Thus retroviral DNA integration may have evolved, at least in part, as a mechanism to avoid the epigenetic silencing of extrachromosomal viral DNA by host innate antiviral factors. SignificanceWhile retroviral DNA is synthesized normally after infection by integrase-deficient viruses, the resultant episomal DNA is then epigenetically silenced. Here, we show that expression of the Tax transcription factor encoded by a second human retrovirus, HTLV-1, prevents the epigenetic silencing of unintegrated HIV-1 DNA and instead induces the addition of activating epigenetic marks, and the recruitment of NF-kB/Rel proteins, to the HIV-1 LTR promoter. Moreover, in the presence of Tax, the HIV-1 DNA circles that form in the absence of integrase function are not only efficiently transcribed but also support a spreading, pathogenic IN- HIV-1 infection. Thus, retroviruses have the potential to replicate without integration, as is indeed seen with HBV.

microbiology↗