Department of Experimental Medicine, University of Insubria, Varese, Italy.
J Transl Med. 2011 Jul 7;9:106. doi: 10.1186/1479-5876-9-106.
MHC class II transactivator CIITA inhibits the function of HTLV-2 Tax-2 viral transactivator and, consequently, the replication of the virus in infected cells. Moreover overexpression of the nuclear factor NF-YB, that cooperates with CIITA for the expression of MHC class II genes, results also in inhibition of Tax-2 transactivation. The purpose of this investigation was to assess the cellular and molecular basis of the CIITA-mediated inhibition on Tax-2, and the relative role of NF-YB in this phenomenon.
By co-immunoprecipitation of lysates from 293T cells cotransfected with CIITA or fragments of it, and Tax-2 it was assessed whether the two factors interact in vivo. A similar approach was used to assess Tax-2-NF-YB interaction. In parallel, deletion fragments of CIITA were tested for the inhibition of Tax-2-dependent HTLV-2 LTR-luciferase transactivation. Subcellular localization of CIITA and Tax-2 was investigated by immunofluorescence and confocal microscopy.
CIITA and Tax-2 interact in vivo through at least two independent regions, at the 1-252 N-term and at the 410-1130 C-term, respectively. Interestingly only the 1-252 N-term region mediates Tax-2 functional inhibition. CIITA and Tax-2 are localized both in the cytoplasm and in the nucleus, when separately expressed. Instead, when coexpressed, most of Tax-2 colocalize with CIITA in cytoplasm and around the nuclear membrane. The Tax-2 minor remaining nuclear portion also co-localizes with CIITA. Interestingly, when CIITA nucleus-cytoplasm shuttling is blocked by leptomycin B treatment, most of the Tax-2 molecules are also blocked and co-localize with CIITA in the nucleus, suggesting that CIITA-Tax-2 binding does not preclude Tax-2 entry into the nucleus.Finally, the nuclear factor NF-YB, also strongly binds to Tax-2. Notably, although endogenous NF-YB does not inhibit Tax-2-dependent HTLV-2 LTR transactivation, it still binds to Tax-2, and in presence of CIITA, this binding seems to increase.
These results strongly suggest that CIITA inhibit Tax-2 by binding the viral transactivator both directly or through a tripartite interaction with NF-YB in. CIITA is therefore a viral restriction factor for HTLV-2 and this open the possibility to control HTLV-2 viral replication and spreading by the controlled induction of CIITA in infected cells.
MHC 类 II 转录激活物 CIITA 抑制 HTLV-2 Tax-2 病毒转录激活物的功能，从而抑制病毒在感染细胞中的复制。此外，核因子 NF-YB 的过度表达与 CIITA 合作表达 MHC 类 II 基因，也会抑制 Tax-2 的转录激活。本研究的目的是评估 CIITA 介导的对 Tax-2 的抑制的细胞和分子基础，以及 NF-YB 在这种现象中的相对作用。
通过共免疫沉淀 293T 细胞裂解物，其中转染了 CIITA 或其片段，以及 Tax-2，评估这两个因子是否在体内相互作用。类似的方法用于评估 Tax-2-NF-YB 相互作用。同时，CIITA 的缺失片段被测试用于抑制 Tax-2 依赖性 HTLV-2 LTR-荧光素酶转录激活。通过免疫荧光和共聚焦显微镜研究 CIITA 和 Tax-2 的亚细胞定位。
CIITA 和 Tax-2 通过至少两个独立的区域在体内相互作用，分别在 1-252 N 端和 410-1130 C 端。有趣的是，只有 1-252 N 端区域介导 Tax-2 的功能抑制。CIITA 和 Tax-2 分别表达时，都定位于细胞质和细胞核中。然而，当共表达时，大多数 Tax-2 与 CIITA 共定位于细胞质和核膜周围。Tax-2 的一小部分剩余核部分也与 CIITA 共定位。有趣的是，当用莱普霉素 B 处理阻断 CIITA 的核质穿梭时，大多数 Tax-2 分子也被阻断，并与 CIITA 在核内共定位，这表明 CIITA-Tax-2 结合并不妨碍 Tax-2 进入细胞核。最后，核因子 NF-YB 也与 Tax-2 强烈结合。值得注意的是，尽管内源性 NF-YB 不抑制 Tax-2 依赖性 HTLV-2 LTR 转录激活，但它仍然与 Tax-2 结合，并且在 CIITA 的存在下，这种结合似乎增加。
这些结果强烈表明，CIITA 通过直接结合病毒转录激活物或通过与 NF-YB 的三部分相互作用抑制 Tax-2。因此，CIITA 是 HTLV-2 的病毒限制因子，这为通过在感染细胞中受控诱导 CIITA 来控制 HTLV-2 病毒复制和传播开辟了可能性。
