Counteraction of innate sensing and retroviral restriction by patient-derived HIV-1 Vpr.

抗病毒限制因子（RFs）可能是I型干扰素级联诱导的病原体感应效应蛋白。虽然RFs有能力有效地抑制HIV-1的复制，但病毒已经进化出了多种对抗策略，这些策略是由所谓的病毒辅助蛋白施加的。Vpr是病毒粒子中丰度最高的病毒辅助蛋白，在病毒蛋白重新生成之前直接进入被感染细胞的细胞质。Vpr具有多种已被描述但仍不清楚的功能，这些功能与增强HIV-1整合和增加病毒基因表达有关，最近的研究表明Vpr在逃避HIV-1免疫感知中起作用。因此，我们假设HIV-1 Vpr可能在病毒早期周期中具有重要功能，可能与病毒复制和生产性基因表达的初始阻断的抵消有关。此外，我们假设这种机制可能在原始患者衍生的HIV-1 Vpr等位基因中保守，而在实验室适应的菌株中可能丢失。在SPP资助期的前两年（2017年2月至2019年2月），我们通过从治疗组naïve hiv -1感染个体中分离和表征Vpr蛋白来挑战这一假设，这些患者的病毒载量和CD4+ T细胞计数不同。虽然我们在Vpr中发现了迄今未知的与疾病进展相关的残基，但我们发现Vpr对髓细胞中的cgas通路没有抑制作用。此外，在原代hiv -1感染的巨噬细胞中，Vpr适度增强而不是抑制isg的诱导，与此一致，观察到Vpr介导的NFAT的强大激活和对NF-κB的促刺激作用。有趣的是，我们最近发现了一种vpr降解的染色质相关蛋白PHF13，它是一种抑制HIV-1基因表达的转录调节因子，与SIV/HIV-2 Vpx降解的hsh复合物高度相似。Vpr至少通过两种机制启动hiv -1感染细胞的生产感染，即(i)通过降解PHF13减轻转录抑制和（ii）通过诱导NFAT激活细胞。总的来说，我们的主要假设是Vpr通过产生促炎的整体刺激细胞环境来促进静止的非激活免疫细胞的生产性感染。因此，Vpr克服了在静止的非激活的原代HIV-1靶细胞中产生病毒复制的初始限制。我们旨在分析PHF13在这一过程中的功能作用，并阐明vpr靶点及其在HIV-1原发CD4+ T细胞感染早期的功能作用。基于RNAseq数据和我们自己的前期工作，我们进一步提出了PHF13将dna损伤和染色质改变与免疫激活和先天免疫反应联系起来的假设。

Antiviral restriction factors (RFs) can be effector proteins of the type I interferon cascade induced by pathogen sensing. While RFs have the capacity to potently suppress HIV-1 replication, the virus has evolved multiple counteraction strategies which are exerted by the so-called viral accessory proteins. Vpr is the viral accessory protein with the highest abundance in the virion and is delivered directly into the cytoplasm of the infected cell before de novo production of viral proteins. Vpr has various described but still enigmatic functions related to the enhancement of HIV-1 integration and increased viral gene expression and recent studies suggested a role of Vpr in evasion of HIV-1 immune sensing. Hence, we postulate that HIV-1 Vpr might have important functions in the early viral cycle, potentially related to the counteraction of initial blocks in viral replication and productive gene expression. Furthermore, we hypothesize that such mechanisms are likely to be conserved in primary patient-derived HIV-1 Vpr alleles while they could be lost in lab-adapted strains. During the first two years of the SPP funding period (2/2017-02/2019) we challenged this hypothesis by isolation and characterization of Vpr proteins from a cohort of therapy naïve HIV-1-infected individuals differing in viral loads and CD4+ T cell counts. While we identified thus far unknown residues in Vpr associated with disease progression, we found no inhibitory effect of Vpr on the cGAS-pathway in myeloid cells. Furthermore, Vpr modestly enhanced rather than suppressed induction of ISGs in primary HIV-1-infected macrophages and in line with this, robust Vpr-mediated activation of NFAT and a pro-stimulatory effect on NF-κB was observed.Intriguingly, we recently identified a Vpr-degraded and chromatin-associated protein, PHF13, which is a transcriptional regulator that inhibits HIV-1 gene expression, highly reminiscent to the HUSH-complex that is degraded by SIV/HIV-2 Vpx. Vpr primes HIV-1-infected cells for productive infection by at least two mechanisms, i.e. (i) alleviation of transcriptional repression through degradation of PHF13 and (ii) cellular activation via induction of NFAT. Overall, our major hypothesis is that Vpr boosts productive infection of resting non-activated immune cells by generating a pro-inflammatory overall stimulating cellular environment. Thereby, Vpr overcomes the initial restriction to productive viral replication in resting non-activated primary HIV-1 target cells. We aim here to analyze the functional role of PHF13 in this process and elucidate Vpr-targets and their functional role early in HIV-1 infection of primary CD4+ T cells. Based on RNAseq data and our own preliminary work we furthermore put forward the hypothesis that PHF13 interconnects DNA-damage and alterations of the chromatin to immune activation and innate immune response.