Role of PYHIN proteins in retroviral restriction, spread and latency

利用进化基因组学、转录组学和蛋白质相互作用数据，我们确定干扰素诱导蛋白16 （IFI16）是潜在的抗逆转录病毒限制因子。在SPP的第一个资助期，我们对IFI16进行了全面的功能和突变分析。我们发现IFI16通过隔离转录因子Sp1和抑制病毒基因表达来独立地限制HIV-1的免疫感应。抗逆转录病毒活性和Sp1相互作用需要n端pyrin结构域和IFI16的核定位，而不需要参与DNA结合的HIN结构域。有趣的是，我们发现高度流行的C亚型毒株对Sp1的依赖性较低，并且能够逃避ifi16介导的限制。此外，我们的研究结果表明IFI16在维持HIV-1潜伏期和控制LINE 1反转录转座子中发挥作用。IFI16只是人类PYHIN 5个蛋白中的一个，我们的数据表明该家族的另外两个成员（MNDA和IFIX）共享IFI16的抗病毒活性。最后，基因敲除小鼠的实验表明，PYHIN蛋白在体内限制逆转录病毒的传播，独立于对干扰素产生的影响。总之，我们的结果支持PYHIN蛋白在先天免疫防御逆转录病毒病原体中的重要作用。然而，关于潜在机制和PYHIN蛋白与病毒限制的相关性的许多问题仍有待解决。在第二个资助期，我们将确定ifi16介导的Sp1抑制的确切机制，并分析Sp1驱动的细胞基因表达的后果。另一个目标将是阐明C亚型HIV-1毒株如何逃避IFI16抑制，并阐明IFI16和Sp1抑制在HIV潜伏期中的作用。此外，我们将研究人类PYHIN蛋白对内源性逆转录因子的影响。最后，我们将进行合作研究，以确定PYHIN蛋白与体内逆转录病毒限制的相关性。我们的研究结果将阐明PYHIN蛋白是否在外源性逆转录病毒和内源性逆转录元件的转录沉默中发挥关键作用。

Using evolutionary genomics, transcriptomics, and protein interaction data, we identified interferon-inducible protein 16 (IFI16) as potential antiretroviral restriction factor. In the first funding period of this SPP, we performed thorough functional and mutational analyses of IFI16. We found that IFI16 restricts HIV-1 independently of immune sensing by sequestering the transcription factor Sp1 and inhibiting viral gene expression. Antiretroviral activity and Sp1 interaction require the N-terminal pyrin domain and nuclear localization of IFI16, but not the HIN domains involved in DNA binding. Interestingly, we found that highly prevalent subtype C strains that are responsible for about half of all HIV-1 infections worldwide are less dependent on Sp1 and capable of evading IFI16-mediated restriction. In addition, our results suggest that IFI16 plays a role in the maintenance of HIV-1 latency and the control of the LINE 1 retrotransposon. IFI16 is only one of five human PYHIN proteins and our data suggest that two other members of this family (MNDA and IFIX) share the antiviral activity of IFI16. Finally, experiments in knockout mice indicate that PYHIN proteins restrict retroviral spread in vivo, independently of effects on interferon production. Altogether, our results support an important role of PYHIN proteins in the innate immune defense against retroviral pathogens. However, many questions regarding the underlying mechanisms and the relevance of PYHIN proteins for virus restriction remain to be addressed. In the 2nd funding period, we will determine the exact mechanism of IFI16-mediated inhibition of Sp1 and analyze the consequences for Sp1-driven cellular gene expression. Another goal will be to clarify, how subtype C HIV-1 strains evade IFI16 inhibition and to elucidate the role of IFI16 and Sp1 inhibition in HIV latency. In addition, we will examine the effects of human PYHIN proteins on endogenous retroelements. Finally, we will perform collaborative studies to determine the relevance of PYHIN proteins for retroviral restriction in vivo. Our results will clarify whether PYHIN proteins play key roles in transcriptional silencing of exogenous retroviruses and endogenous retroelements.