Laboratory of Retrovirology, Department of Medical Biology and BioMed Research Group, Université du Québec à Trois-Rivières, 3351 Boulevard des Forges, CP500, Trois-Rivières, QC G9A 5H7, Canada.
Virus Res. 2014 May 12;184:30-8. doi: 10.1016/j.virusres.2014.02.013. Epub 2014 Feb 25.
TRIM5α is a type I interferon-stimulated anti-retroviral restriction factor expressed in most primates and homologous proteins are expressed in other mammals. Through its C-terminal PRYSPRY (B30.2) domain, TRIM5α binds to incoming and intact post-fusion retroviral cores in the cytoplasm. Following this direct interaction, the retroviral capsid core is destabilized and progression of the virus life cycle is interrupted. Specific recognition of its viral target by TRIM5α also triggers the induction of an antiviral state involving the activation of transcription factors NF-κB- and AP-1. In addition to PRYSPRY, several other TRIM5α domains are important for anti-retroviral function, including a RING zinc-binding motif. This domain has "E3" ubiquitin ligase activity and is involved in both the direct inhibition of incoming retroviruses and innate immune activation. A highly conserved sumoylation consensus site is present between the RING motif and the N-terminal extremity of TRIM5α. No clear role in restriction has been mapped to this sumoylation site, and no sumoylated forms of TRIM5α have been observed. Here we confirm that mutating the putatively sumoylated lysine (K10) of the Rhesus macaque TRIM5α (TRIM5αRh) to an arginine has only a small effect on restriction. However, we show that the mutation significantly decreases the TRIM5α-induced generation of free K63-linked ubiquitin chains, an intermediate in the activation of innate immunity pathways. Accordingly, K10R decreases TRIM5α-mediated activation of both NF-κB and AP-1. Concomitantly, we find that K10R causes a large increase in the levels of ubiquitylated TRIM5α. Finally, treatment with the nuclear export inhibitor leptomycin B shows that K10R enhances the nuclear localization of TRIM5αRh, while at the same time reducing its level of association with nuclear SUMO bodies. In conclusion, the TRIM5α sumoylation site appears to modulate the E3 ubiquitin ligase activities of the adjacent RING domain, promoting K63-linked ubiquitin chains at the expense of auto-ubiquitylation which is probably K48-linked. Consistently, we find this sumoylation site to be important for innate immune activation by TRIM5α. In addition, lysine 10 regulates TRIM5α nuclear shuttling and nuclear localization, which may also be related to its role in innate immunity activation.
TRIM5α 是一种 I 型干扰素刺激的抗逆转录病毒限制因子，在大多数灵长类动物中表达，同源蛋白在其他哺乳动物中表达。通过其 C 端 PRYSPRY（B30.2）结构域，TRIM5α 与细胞质中传入的完整融合后逆转录病毒核心结合。在这种直接相互作用之后，逆转录病毒衣壳核心失稳，病毒生命周期的进展被中断。TRIM5α 对其病毒靶标的特异性识别还触发了涉及转录因子 NF-κB 和 AP-1 激活的抗病毒状态的诱导。除了 PRYSPRY 外，TRIM5α 的其他几个结构域对抗病毒功能也很重要，包括一个 RING 锌结合基序。该结构域具有“E3”泛素连接酶活性，参与直接抑制传入的逆转录病毒和先天免疫激活。在 RING 基序和 TRIM5α 的 N 端之间存在一个高度保守的 SUMO 化保守位点。该 SUMO 化位点在限制中的作用尚未明确映射，也未观察到 SUMO 化的 TRIM5α 形式。在这里，我们证实将恒河猴 TRIM5α（TRIM5αRh）中假定的 SUMO 化赖氨酸（K10）突变为精氨酸对限制的影响很小。然而，我们表明，该突变显著降低了 TRIM5α 诱导的游离 K63 连接的泛素链的产生，这是先天免疫途径激活的中间产物。因此，K10R 降低了 TRIM5α 介导的 NF-κB 和 AP-1 的激活。相应地，我们发现 K10R 导致泛素化 TRIM5α 的水平大量增加。最后，用核输出抑制剂莱普霉素 B 处理表明，K10R 增强了 TRIM5αRh 的核定位，同时降低了其与核 SUMO 体的关联水平。总之，TRIM5α 的 SUMO 化位点似乎调节相邻 RING 结构域的 E3 泛素连接酶活性，促进 K63 连接的泛素链的形成，而不是可能是 K48 连接的自身泛素化。一致地，我们发现该 SUMO 化位点对于 TRIM5α 的先天免疫激活很重要。此外，赖氨酸 10 调节 TRIM5α 的核穿梭和核定位，这可能也与其在先天免疫激活中的作用有关。
