The FACT Complex Promotes Avian Leukosis Virus DNA Integration

Abstract

All retroviruses need to integrate a DNA copy of their genome into the host chromatin. Cellular proteins regulating and targeting lentiviral and gammaretroviral integration in infected cells have been discovered, but the factors that mediate alpharetroviral avian leukosis virus (ALV) integration are unknown. In this study, we have identified the FACT protein complex, which consists of SSRP1 and Spt16, as a principal cellular binding partner of ALV integrase (IN). Biochemical experiments with purified recombinant proteins show that SSRP1 and Spt16 are able to individually bind ALV IN, but only the FACT complex effectively stimulates ALV integration activity in vitro. Likewise, in infected cells, the FACT complex promotes ALV integration activity, with proviral integration frequency varying directly with cellular expression levels of the FACT complex. An increase in 2-long-terminal-repeat (2-LTR) circles in the depleted FACT complex cell line indicates that this complex regulates the ALV life cycle at the level of integration. This regulation is shown to be specific to ALV, as disruption of the FACT complex did not inhibit either lentiviral or gammaretroviral integration in infected cells. IMPORTANCEThe majority of human gene therapy approaches utilize HIV-1-or murine leukemia virus (MLV)-based vectors, which preferentially integrate near genes and regulatory regions; thus, insertional mutagenesis is a substantial risk. In contrast, ALV integrates more randomly throughout the genome, which decreases the risks of deleterious integration. Understanding how ALV integration is regulated could facilitate the development of ALV-based vectors for use in human gene therapy. Here we show that the FACT complex directly binds and regulates ALV integration efficiency in vitro and in infected cells.

Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration

“…Based on the difference in their preference for distinct chromatin structure we propose that FACT deregulation could induce different and inverted effect depending on the retrovirus as observed by us with HIV-1 and PFV (see Fig. 8 c) and by other authors using ALV model [ 36 ]. These putative functions of FACT on retroviral integration is recapitulated in Fig.…”

“…Indeed, in vitro integration assays performed in PN templates showed that FACT remodeling activity allowed efficient HIV-1 integration into dense chromatin that was initially refractory to viral DNA insertion. Importantly, this FACT-mediated activation of integration was found to be nucleosome-dependent since no stimulation was detected on naked DNA, in contrast to what was recently observed for ALV [ 36 ]. Our data indicate that FACT generates chromatin structures that are highly preferential for in vitro integration.…”

“…This different regulation of HIV-1 integration by FACT was demonstrated in our work showing a clear stimulation of this replication step when the FACT remodeling activity was stimulated. Interestingly, a stimulation of HIV-1 integration after SSRP1 knockdown was also observed by Winans et al [ 36 ] but was found non-statistically relevant. However, these data were obtained in chicken DT40, which are not naturally permissive to HIV-1 infection, and collected at 24 h post transduction while our data indicate that the increase in integrated DNA amount becomes more relevant a 48 h than 24 h (see Additional file 7 : Figure S7).…”

“…However, the analyses presented by these authors were focused on the viral LTR-dependent gene transcription. In contrast, possible regulation of the retroviral integration mechanisms by FACT has been recently proposed as SSRP1 depletion was shown to inhibit Avian Leukosis Virus (ALV) integration [ 36 ]. In this latest work no inhibition of HIV-1 integration by SSRP1 depletion was observed suggesting that FACT-dependent modulation of integration could depend on the retrovirus family.…”

Selection for avian leukosis virus integration sites determines the clonal progression of B-cell lymphomas

“…Relative to other well studied retroviruses like HIV-1 and MLV, ALV was shown to integrate relatively randomly into the host genomic DNA, with little bias for genomic features [ 4 – 7 ]. The FACT (facilitates chromatin transcription) complex, a chromatin remodeler, was recently reported to promote ALV integration [ 8 ].…”