| 其他摘要 | 宿主限制因子是新发现的一类以种属特异性的方式限制HIV-1复制的胞质蛋白。目前发现的限制因子包括APOBEC3G、TRIM5α、BST-2、SAMHD1、MxB和SERINC3/5等，它们除了限制HIV-1外，还能限制其它病毒如N-MLV、HBV和HCV的复制。这些限制因子当中，除了SAMHD1作用于宿主细胞的dNTPs外，其它限制因子都是作用于HIV-1的结构蛋白或者核酸，从而限制病毒的复制；而HIV-1能够编码一些辅助蛋白或者通过快速进化保留下对自身复制有利的氨基酸突变来拮抗宿主限制因子的限制作用，完成对宿主细胞的有效感染。因此，在宿主与病毒相互作用的过程中，体内限制因子的表达水平对于限制HIV-1的复制可能具有重要作用，而HIV-1的持续复制可能会对这些限制因子的表达量产生影响。这种病毒与宿主间的博弈结果是影响宿主细胞对HIV-1易感性和疾病进程的主要决定因素，然而对体内宿主限制因子表达与HIV-1复制关系的研究目前依然较少。由于HIV-1和HCV具有相似的感染途径，因此HIV-1感染者中常会伴有HCV的共感染，反之亦然。HIV-1或者HCV感染宿主细胞后，对机体的固有免疫和获得性免疫都会产生影响，这可能导致HIV-1/HCV共感染病人罹患肝脏相关疾病的风险增加或者病程进程加快，但是对于HIV-1和HCV是如何相互作用进而影响机体免疫和疾病进程的，其机制一直都不清楚。考虑到宿主限制因子和I型干扰素在抗病毒感染中的重要作用，我们推测HIV-1和HCV感染可能对宿主细限制因子和I型干扰素的表达产生影响，而后者表达量的变化可能与疾病进展相关。通过实时定量PCR的方法我们检测了TRIM5α、TRIM22、APOBEC3G和IFN-α, -β在HIV-1，HCV单独及共感染人群PBMC中的表达水平，以此来评估病毒感染与体内限制因子和I型干扰素表达量的关系。研究发现，HIV-1和HCV单独以及共感染者体内TRIM22和APOBEC3G的表达显著高于健康对照；HCV单独感染者体内TRIM5α的表达显著高于健康对照，而HIV-1单独感染对TRIM5α几乎不产生影响；相比于HCV和HIV-1单独感染，HIV-1/HCV共感染者表达更低的TRIM5α和APOBEC3G；IFN-α和IFN-β在HIV-1和HCV单独以及共感染者中都显著较高，表明HIV-1和HCV单独及共感染确实能够对宿主限制因子和I型干扰素的表达产生影响。此外，我们检测了HIV-1和HCV感染者血浆中的病毒载量，发现相比于HIV-1或HCV单独感染者，HIV-1/HCV共感染者血浆中具有更高的HIV-1和HCV病毒载量。限制因子TRIM5α和TRIM22与I型干扰素在体内存在显著的正相关性，而HIV-1和HCV单独以及共感染会破坏它们之间的正相关关系，这种内在相互调节关系的破坏间接地反映了HIV-1和HCV感染对机体免疫平衡的影响。我们发现，在HCV单独感染者中TRIM22的表达和HCV病毒载量负相关，而在HIV-1单独感染者中，APOBEC3G的表达与HIV-1病毒载量正相关，表明TRIM22在抑制HCV的复制中可能具有重要作用。因此，研究HIV-1和HCV的复制水平与宿主限制因子和I型干扰素在体内的表达变化可能对HIV-1和HCV的治疗提供一个新的方向。静脉吸毒是HIV-1传播的重要途径之一，它不仅能调节机体免疫细胞的活化和免疫分子的产生，而且会影响病毒的快速进化，导致病毒与宿主相互作用的模式或者强度改变，影响机体对病毒的易感性和相关疾病进程。然而到目前为止，对于毒品本身是加速还是延缓疾病进程尚未有统一的说法。由于HIV-1、HBV和HCV感染可能对宿主限制因子和I型干扰素表达量的影响，因此我们在没有感染这三种病毒的海洛因使用者中检测了TRIM5α、TRIM22、APOBEC3G和IFN-α, -β的表达水平，同时我们也在HIV-1单独感染的静脉吸毒者和非静脉吸毒者的血浆中检测了HIV-1的病毒载量，以此来评估海洛因滥用对限制因子和I型干扰素表达以及HIV-1复制的影响。我们发现海洛因使用者体内TRIM5α、TRIM22、APOBEC3G和IFN-α, -β的表达量显著低于健康人群，而且HIV-1感染的海洛因使用者血浆中具有更高的病毒载量，表明海洛因可能抑制宿主限制因子和I型干扰素的表达，而且对HIV-1的复制具有促进作用。然而，我们并没有发现限制因子或I型干扰素与病毒载量负相关，而且海洛因使用也会破坏TRIM5α和TRIM22与IFN-α, -β内在的正相关关系，可能影响机体的免疫平衡。此外，我们通过分析海洛因使用时间的长短与限制因子和I型干扰素表达量的关系发现，TRIM5α、TRIM22和IFN–β的表达受海洛因使用时间的影响。这些结果显示了海洛因滥用对宿主限制因子和I型干扰素表达的不利影响，增强了HIV-1在宿主细胞中的复制，在一定程度上解释了HIV-1感染的吸毒者疾病进展较快的原因。TRIMCyp是cyclophilinA基因在LINE-1介导的逆转座作用下插入到TRIM5基因座后经选择性剪切产生的一个融合蛋白。鹰猴TRIMCyp不仅能够通过与HIV-1的CA蛋白直接相互作用，限制HIV-1的复制，它还能作为模式识别受体，激活NF-κB和AP-1，调节宿主的固有免疫应答。在之前的研究中，我们首次发现了不同于鹰猴TRIMCyp的另一种插入模式的北平顶猴TRIMCyp，而且发现后者不能够与CA直接相互作用，因而不能够限制HIV-1的复制，但对于北平顶猴TRIMCyp是否具有固有免疫调节功能，至今尚未报道。本研究我们发现，相比于鹰猴TRIMCyp，北平顶猴TRIMCyp不能够激活NF-κB，而且对AP-1和IFN-β的活化调节作用也比较弱。我们采用免疫印记的方法证明了北平顶猴TRIMCyp不能够激活NF-κB可能与它不能活化IκBα有关。通过构建不同的TRIMCyp嵌合体，我们发现鹰猴CypA结构域在NF-κB的激活中起重要作用，该结构域对AP-1和IFN-β的激活也有促进作用。另外我们发现，尽管鹰猴TRIMCyp和平顶猴TRIMCyp对IFN-β的转录激活都具有正调节的作用，但这种调节对VSV复制的影响不大。总的说来，本研究揭示了HIV-1, HCV单独及共感染和海洛因使用对人体内限制因子、I型干扰素表达和它们之间相互调节关系的影响，以及HIV-1/HCV共感染和海洛因使用对病毒载量的影响，在一定程度上解释了HIV-1/HCV共感染者和HIV-1感染的海洛因使用者疾病进程较快或者病情较重的原因。我们先后发现北平顶猴TRIMCyp不能限制HIV-1复制，同时也不能激活NF-κB，这可能是导致北平顶猴易感HIV-1而且能够形成潜伏感染的一个重要原因。; Host restriction factors, newly named cytoplasmic proteins, have been recently identified with a potent activity against HIV-1 infection in a species-specific dependent manner, which contain APOBEC3G, TRIM5α, BST-2, SAMHD1, MxB and SERINC3/5. Besides blocking HIV-1, other viruses such as N-MLV, HBV and HCV are also limited by some restriction factors. On the one hand, host restriction factors block HIV-1 replication through direct interaction with the viral proteins or nucleotides, except for SAMHD1 that inhibits HIV-1 by decreasing dNTPs of host cells. On the other hand, HIV-1 encodes some accessary proteins or conserves the key amino acids mutation that facilitates to its infection or replication during fast evolution, protecting from the restriction factors. As a result, the level of restriction factors expression may play an important role in restricting HIV-1 infection, and vice versa，persistent HIV-1 replication would affect the restriction factors expression. The consequence of the game between the host and the virus will decide the susceptibility to HIV-1 infection and the disease progression. However, studies on the relationship between host restriction factors expression and HIV-1 replication in vivo are much less.Because of sharing the routes of transmission, HIV-1/HCV co-infection is very common. Infection of either HIV-1 or HCV can affect both innate and adaptive immunity of host, which may associate with an increased risk of the liver-related disease and rapid disease progression, but the mechanism underlying this knows a little. Considering the importance of host restriction factors and type I interferon in antiviral activities, we hypothesize that HIV-1 and/or HCV infection may have an influence on these antiviral factors expression, therby affecting the disease progression. In this study, we measured the levels of TRIM5α, TRIM22, APOBEC3G, and IFN-α, -β mRNA expression in peripheral blood mononuclear cells of HIV, HCV mono- and co-infected patients along with healthy controls by utilizing a quantitative real-time PCR assay, to evaluate the influence of HIV-1, HCV mono- and co-infection on these intracellular restriction factors and type I interferon expression. The results showed that HIV-1, HCV mono- and co-infected patients associated with significantly higher levels of TRIM22, APOBEC3G and IFN-α, -β mRNA expression, whereas only HCV but not HIV-1 mono-infected patients had significantly higher levels of TRIM5α. Moreover, HIV-1/HCV co-infected patients had significantly lower levels of TRIM5α and APOBEC3G expression compared with HCV and HIV-1 mono-infected patients respectively. In addition, we examined the plasma viral load of HIV-1 and HCV in the patients, and found HIV-1/HCV co-infected patients were associated with significantly higher viral load, compared to either HIV-1 or HCV mono-infected patients, suggesting co-infection with HIV-1 and HCV facilitates the two viruses replication. Additionally, we showed that TRIM5 and TRIM22 positively correlated with IFN-α, -β, but these intracellular positive associations were dysregulated by HIV-1, HCV mono- and co-infection, which indirectly reflects disruption in the balance of host immunoregulation. Furthermore, we found TRIM22 negatively correlated with HCV viral load in mono-infected patients and APOBEC3G positively correlated with HIV-1 viral load in co-infected patients, suggesting an important role of TRIM22 in restricting HCV replication. Our findings suggest the potential role of restriction factors in restricting HIV, HCV mono- and co-infection in vivo, which appears to be a therapeutic target for potential drug discovery.Injection drug use, one of the primary routes of HIV-1 transmission around the world, plays an important role both in regulating host immune response such as immunocyte activation and immune molecules production and fast viral evolution, leading to the change of interaction between the host and the virus, which in turn affects HIV-1 acquisition and disease progression. However, whether drug abuse accelerates or decreases the disease progression ramains inconsistent. Since HIV-1, HBV or HCV mono- and co-infection affect host restriction factors and type I interferon expression, we similarly measured TRIM5α, TRIM22, and APOBEC3G as well as IFN-α, -β levels in intravenous drug users (IDUs, heroin abuse) and non-IDUs, who are not infected with HIV-1, HBV or HCV. The impact of heroin abuse on HIV-1 viral load was also investigated. We found that IDUs had significantly lower levels of TRIM5α, TRIM22, APOBEC3G, and IFN-α, -β mRNA expression as compared to non-IDUs, and significant increase of HIV-1 viral load in IDUs versus non-IDUs was observed, suggesting heroin abuse plays an important role in down-regulating host restriction factors and type I interferon expression and enhancing HIV-1 replication. However, neither of restriction factors and type I interferon showed any correlation with plasma viral loads. Moreover, we also found the positive correlation between TRIM5α, TRIM22 and type I interferon was dysregulated followed heroin abuse. The years of heroin use correlated negatively with TRIM5α, TRIM22 and IFN-β expression. The results reveal the adverse effects of heroin abuse on restriction factors and type I interferon expression, enhancing HIV-1 replication, which may associate with rapid disease progression, to a certain extent.TRIMCyp, an alternatively spliced isoform of TRIM5, arose via a LINE-1 mediated retrotransposion of cyclophilinA gene inserting into TRIM5 locus. Owl monkey TRIMCyp (omTRIMCyp) not only potently restricts HIV-1 replication through direct interaction with HIV-1 capsid protein, but activates NF-κB and AP-1 transcription, thereby regulating host innate immune response. In previous studies, we firstly reported another mode of TRIMCyp from northern pig-tailed macaques (npmTRIMCyp) that is not able to interact with HIV-1 capsid protein directly, leading to inability of restricting HIV-1 replication. Both modes of TRIMCyp were independently generated, and no studies were reported whether npmTRIMCyp can regulate innate immune response through activating NF-κB and AP-1 thus far. In the present study we found npmTRIMCyp was not able to activate NF-κB, but moderately enhanced AP-1 and IFN-β activation, as compared with omTRIMCyp. We further found npmTRIMCyp was not able to phosphorylate IκBα, the key regulator in canonical NF-κB signaling pathway, which may lead to the inability of npmTRIMCyp in regulating NF-κB activation. Based on different chimeric TRIMCyps testing, we found the CypA domain of omTRIMCyp play an important in regulating NF-κB activation, which also facilitate to AP-1 and INF-β activation. Although npmTRIMCyp and omTRIMCyp positively regulated INF-β transcription, the inhibition of VSV-GFP replication was not observed.Collectively, these data suggest both HIV-1, HCV mono- and co-infection or heroin abuse in vivo affect restriction factors and type I interferon expression and their intrinsic association, which may have an influence on the balance of innate immune regulation, thereby contributing to HIV-1 or HCV replication and rapid disease progression. npmTRIMCyp neither restricts HIV-1 replication nor activates NF-κB, which may contribute to the susceptibility to HIV-1 infection and its latency for northern pig-tailed macaques. |
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