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

抗病毒限制因子（RF）可以是由病原体感应诱导的I型干扰素级联的效应蛋白。虽然RF具有有效抑制HIV-1复制的能力，但病毒已经进化出多种对抗策略，这些策略由所谓的病毒辅助蛋白发挥作用。Vpr是在病毒体中具有最高丰度的病毒辅助蛋白，并且在病毒蛋白从头产生之前被直接递送到感染细胞的细胞质中。Vpr具有各种描述的但仍然是谜一样的功能，其与增强HIV-1整合和增加病毒基因表达有关，并且最近的研究表明Vpr在逃避HIV-1免疫感应中的作用。因此，我们推测HIV-1 Vpr可能在早期病毒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作用有关。此外，我们假设，这种机制可能是保守的主要患者来源的HIV-1 Vpr等位基因，而他们可能会失去在实验室适应株。在SPP资助期的前两年（2/2017-02/2019），我们通过从一组病毒载量和CD 4 + T细胞计数不同的未经治疗的HIV-1感染者中分离和表征Vpr蛋白来挑战这一假设。虽然我们鉴定了Vpr中迄今未知的与疾病进展相关的残基，但我们发现Vpr对骨髓细胞中的cGAS通路没有抑制作用。此外，Vpr适度地增强而不是抑制了原发性HIV-1感染的巨噬细胞中ISG的诱导，并且与此一致，观察到Vpr介导的NFAT的稳健活化和对NF-κB的促刺激作用。有趣的是，我们最近鉴定了Vpr降解的和染色质相关的蛋白PHF 13，其是抑制HIV-1基因表达的转录调节因子，与SIV/HIV-2 Vpx降解的HUSH复合物高度相似。Vpr通过至少两种机制引发HIV-1感染的细胞进行生产性感染，即（i）通过降解PHF 13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通过产生促炎性整体刺激细胞环境来促进静息非活化免疫细胞的生产性感染。因此，Vpr克服了在静息非活化原代HIV-1靶细胞中对生产性病毒复制的初始限制。我们的目的是分析PHF 13在这一过程中的功能作用，并阐明Vpr靶点及其在HIV-1感染原代CD 4 + T细胞早期的功能作用。基于RNAseq数据和我们自己的初步工作，我们进一步提出了PHF 13将DNA损伤和染色质改变与免疫激活和先天免疫应答相互联系的假设。

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.