Mechanisms of Antiviral Action of Human Interferon

1. 已经鉴定了与抗病毒活性相关的23个基因和17个蛋白质。 我们的实验室鉴定了25个基因（通过微阵列），其中23个通过qRT-PCR确认，和17个蛋白质（通过蛋白质印迹）与I型IFN的抗病毒活性相关，使用我们建立的条件，其允许我们在单个MTT测定中区分IFN-α处理的Daudi（伯基特淋巴瘤）细胞的抗增殖和抗病毒活性。 2. IFIT3与IFN-α的抗病毒活性相关 我们的工作集中在这些蛋白质中的两个的检查：第一个是干扰素诱导的蛋白与tetratricopeptide repeats 3（IFIT-3），也被称为视黄酸诱导的基因G（RIG-G），ISG-60或IFI60。 IFIT3在A549细胞中的RNA干扰和IFIT3在VERO细胞中的过表达证实IFIT3是与抗水泡性口炎病毒（VSV）和心内膜心肌炎病毒（EMCV）的抗病毒活性相关的蛋白质之一。 此外，IFN-α 2在贴壁细胞（OVCAR-3、2fTGH和HeLa）和悬浮细胞（B-JAB、U937、Jurkat和人单核细胞）中诱导IFIT3。我们通过显示IFIT3的过表达降低了HUH7细胞中的HCV复制和HEK293细胞中的登革病毒复制，进一步阐明了IFIT3的抗病毒作用。 3. 通过IFN工程化构建体抑制HIV 亲本分子IFN-α 2c和IFN-α 21 b用于产生杂交体以及定点突变体和盒式突变体。 从命名为HY4（IFN-α 21 b（1 - 75）/IFN-α 2c（76 - 81）/IFN-α 21 b（82 - 95）/IFN-α 2c（96 - 166））的杂交体产生三种突变体，命名为SDM-1（S86 Y）、SDM-2（N90 Y）和CM-3（S86 K）（12）。突变体SDM-1和SDM-2在与亲本分子相似的浓度下有效地抑制HIV，并且超过了它们所来源的杂交体（HY4）的活性。与未处理的巨噬细胞相比，所有测试的IFN-α构建体都增加了PKR表达。这种增加的表达与HIV抑制水平之间没有直接相关性。 有趣的是，定点突变体SDM-1导致巨噬细胞和单核细胞中APOBEC3A的上调，其水平远高于亲本IFN、HY4或CM-3突变体。 这些结果指出，酪氨酸和可能的天冬酰胺氨基酸残基在位置86和90分别作为重要的不仅是调节AV活性，而且APOBEC诱导。IFN诱导的STAT（1、2、3、5和6）磷酸化似乎与AV活性和APOBEC3A的调节相关。SDM-1和2的抗逆转录病毒活性和毒性之间的可能联系进行了研究，使用干扰素诱导的吲哚胺2，3-双加氧酶（IDO）作为标记物。发现SDM-1和SDM-2诱导的IDO水平明显低于亲本IFN分子。我们已经发现，尽管亲本IFN-α显著增加了I-κ B-α的磷酸化，这与NF κ B途径一致，但SDM-1没有。 这可以解释SDM-1对AV活性和降低的炎症或毒性特性的明显有利。由于已知IFN与TNF-α协同进行IDO转录，并且TNF-α可促进HIV感染（13），我们研究了TNF-α作为IFN毒性中间环节的可能性。 尽管亲本IFN在基因和蛋白质水平上都导致TNF-α的快速和丰富水平，但对于SDM-1和SDM-2却并非如此。 此外，直接添加外源性TNF-α到巨噬细胞导致增强的IDO表达，即使在低浓度（1 ng/ml）。 然而，TNF-α对APOBEC3A的刺激无效，无论浓度如何，这与两种途径的解离一致。我们想要进一步检查SDM-1和SDM-2与IFN-α 2相比对人单核细胞/巨噬细胞中的其他病毒的作用，并评估IDO、TNF-α和毒性/炎症的其他生物标志物。此外，我们将进一步研究SDM-1和SDM-2的AV解离和毒性途径的机制。最后，将产生新的IFN构建体，以通过评估其生物活性和毒性/炎症标志物来优化治疗指数。我们将研究这些结构进一步产品开发的潜力。

1. Twenty-three genes and 17 proteins associated with antiviral activity have been identified. Our laboratory identified 25 genes (by microarray), 23 of which were confirmed by qRT-PCR, and 17 proteins (by Western Blotting) associated with the antiviral activity of Type I IFN using conditions we established which allows us to distinguish, in a single MTT assay, between the antiproliferative and antiviral activities of IFN-alpha-treated Daudi (Burkitts Lymphoma) cells. 2. IFIT3 is associated with the antiviral activity of IFN-alpha Our work has focused on the examination of two of these proteins: The first is Interferon-induced protein with tetratricopeptide repeats 3 (IFIT-3) which is also known as Retinoic Acid Induced Gene G (RIG-G), ISG-60 or IFI60. RNA interference of IFIT3 in A549 cells and overexpression of IFIT3 in VERO cells confirmed that IFIT3 is one of the proteins associated with antiviral activity against both Vesicular Stomatitis Virus (VSV) and Endomyocarditis Virus (EMCV). In addition, IFIT3 was induced in both adherent cells (OVCAR-3, 2fTGH and HeLa) and in suspension cells (B-JAB, U937, Jurkat and human monocytes) by IFN-alpha2. We have further elucidated the antiviral role of IFIT3 by showing that overexpression of IFIT3 decreases HCV replication in HUH7 cells and dengue virus replication in HEK293 cells. 3. HIV suppression by IFN- engineered constructs Parental molecules IFN-alpha2c and IFN-alpha21b were used to generate hybrids as well as both site-directed and cassette mutants. From the hybrid designated HY4 (IFN-alpha21b (1-75)/IFN-alpha2c (76-81)/IFN-alpha21b (82-95)/ IFN-alpha2c (96-166), three mutants were generated designated SDM-1 (S86Y), SDM-2 (N90Y), and CM-3(S86K) (12). Mutants SDM-1 and-2 effectively inhibited HIV at concentrations similar to the parental molecules and exceeded the activity of the hybrid from which they were derived (HY4). PKR expression was increased by all IFN-alpha constructs tested compared to untreated macrophages. There was no direct correlation between this increased expression and levels of HIV suppression. Interestingly, the site-directed mutant SDM-1 led to up-regulation of APOBEC3A in macrophages and monocytes at much higher levels than that seen with parental IFNs, HY4 or the CM-3 mutant. These results point to the tyrosine and likely asparagine amino acid residues at positions 86 and 90 respectively as being important not only for regulating AV activity but also for APOBEC induction. IFN-induced phosphorylation of STATs (1,2,3,5 and 6) appears to correlate with the regulation of AV activity and APOBEC3A. The possible link between the antiretroviral activity and toxicity of SDM-1 and 2 was investigated using the IFN-induced indoleamine 2,3-dioxygenase (IDO) as a marker. It was found that both SDM-1 and SDM-2 induced appreciably lower levels of IDO than the parental IFN molecules. We have found that whereas parental IFN-alphas substantially increased the phosphorylation of I-kappaB-alpha which is consistent with the NF kappa B pathway, SDM-1 did not. This may explain the apparent favoring of SDM-1 for AV activity and decreased inflammatory or toxic properties. Since IFN is known to synergize with TNF-alpha for IDO transcription and that TNF-alpha can facilitate HIV infection (13), we investigated the possibility of TNF-alpha being an intermediate link in IFN toxicity. Whereas parental IFNs resulted in rapid and plentiful levels of TNF-alpha both at the gene and protein levels, the same was not true for SDM-1 and SDM-2. In addition, direct addition of exogenous TNF-alpha to macrophages led to enhanced IDO expression even at low (1 ng/ml) concentrations. However, TNF-alpha was not effective for the stimulation of APOBEC3A regardless of concentration which is consistent with dissociation of the two pathways. We would like to further examine the effects of SDM-1 and -2 compared to IFN-alpha2 on other viruses in human monocytes/macrophages and evaluate IDO, TNF-alpha and other biomarkers of toxicity/inflammation. Also we will further examine the mechanism of dissociation of the AV and toxicity pathways of SDM-1 and-2. Finally new IFN constructs will be generated to optimize the therapeutic index by evaluating their bioactivities and markers of toxicity/inflammation. We will examine the potential of these constructs for further product development.