Gu Qinyong, Zhang Zeli, Gertzen Christoph G W, Häussinger Dieter, Gohlke Holger, Münk Carsten
Clinic for Gastroenterology, Hepatology, and Infectiology, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
J Virol. 2018 Feb 26;92(6). doi: 10.1128/JVI.01697-17. Print 2018 Mar 15.
Members of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3 [A3]) family of DNA cytidine deaminases are intrinsic restriction factors against retroviruses. In felids such as the domestic cat (), the A3 genes encode the A3Z2, A3Z3, and A3Z2Z3 antiviral cytidine deaminases. Only A3Z3 and A3Z2Z3 inhibit viral infectivity factor (Vif)-deficient feline immunodeficiency virus (FIV). The FIV Vif protein interacts with Cullin (CUL), Elongin B (ELOB), and Elongin C (ELOC) to form an E3 ubiquitination complex to induce the degradation of feline A3s. However, the functional domains in FIV Vif for the interaction with Cullin are poorly understood. Here, we found that the expression of dominant negative CUL5 prevented the degradation of feline A3s by FIV Vif, while dominant negative CUL2 had no influence on the degradation of A3. In coimmunoprecipitation assays, FIV Vif bound to CUL5 but not CUL2. To identify the CUL5 interaction site in FIV Vif, the conserved amino acids from positions 47 to 160 of FIV Vif were mutated, but these mutations did not impair the binding of Vif to CUL5. By focusing on a potential zinc-binding motif (K175-C161-C184-C187) of FIV Vif, we found a conserved hydrophobic region (174IR175) that is important for the CUL5 interaction. Mutation of this region also impaired the FIV Vif-induced degradation of feline A3s. Based on a structural model of the FIV Vif-CUL5 interaction, the 52LW53 region in CUL5 was identified as mediating binding to FIV Vif. By comparing our results to the human immunodeficiency virus type 1 (HIV-1) Vif-CUL5 interaction surface (120IR121, a hydrophobic region that is localized in the zinc-binding motif), we suggest that the CUL5 interaction surface in the diverse HIV-1 and FIV Vifs is evolutionarily conserved, indicating a strong structural constraint. However, the FIV Vif-CUL5 interaction is zinc independent, which contrasts with the zinc dependence of HIV-1 Vif. Feline immunodeficiency virus (FIV), which is similar to human immunodeficiency virus type 1 (HIV-1), replicates in its natural host in T cells and macrophages that express the antiviral restriction factor APOBEC3 (A3). To escape A3s, FIV and HIV induce the degradation of these proteins by building a ubiquitin ligase complex using the viral protein Vif to connect to cellular proteins, including Cullin 5. Here, we identified the protein residues that regulate this interaction in FIV Vif and Cullin 5. While our structural model suggests that the diverse FIV and HIV-1 Vifs use conserved residues for Cullin 5 binding, FIV Vif binds Cullin 5 independently of zinc, in contrast to HIV-1 Vif.
载脂蛋白B信使核糖核酸编辑酶催化多肽样（APOBEC3 [A3]）家族的DNA胞苷脱氨酶成员是针对逆转录病毒的内在限制因子。在诸如家猫等猫科动物中，A3基因编码A3Z2、A3Z3和A3Z2Z3抗病毒胞苷脱氨酶。只有A3Z3和A3Z2Z3能抑制病毒感染性因子（Vif）缺陷型猫免疫缺陷病毒（FIV）。FIV Vif蛋白与Cullin（CUL）、Elongin B（ELOB）和Elongin C（ELOC）相互作用形成E3泛素化复合物，以诱导猫A3s的降解。然而，FIV Vif中与Cullin相互作用的功能结构域尚不清楚。在此，我们发现显性负性CUL5的表达可阻止FIV Vif对猫A3s的降解，而显性负性CUL2对A3的降解没有影响。在免疫共沉淀试验中，FIV Vif与CUL5结合，但不与CUL2结合。为了确定FIV Vif中与CUL5的相互作用位点，对FIV Vif第47至160位的保守氨基酸进行了突变，但这些突变并不影响Vif与CUL5的结合。通过聚焦于FIV Vif潜在的锌结合基序（K175 - C161 - C184 - C187），我们发现了一个对CUL5相互作用很重要的保守疏水区域（174IR175）。该区域的突变也损害了FIV Vif诱导的猫A3s的降解。基于FIV Vif - CUL5相互作用结构模型，确定CUL5中的52LW53区域介导与FIV Vif的结合。通过将我们的结果与1型人类免疫缺陷病毒（HIV - 1）Vif - CUL5相互作用表面（120IR121，位于锌结合基序中的一个疏水区域）进行比较，我们认为不同的HIV - 1和FIV Vifs中的CUL5相互作用表面在进化上是保守的，这表明存在很强的结构限制。然而，FIV Vif - CUL5相互作用不依赖锌，这与HIV - 1 Vif对锌的依赖性形成对比。猫免疫缺陷病毒（FIV）与人类免疫缺陷病毒1型（HIV - 1）相似，在其天然宿主的表达抗病毒限制因子APOBEC3（A3）的T细胞和巨噬细胞中复制。为了逃避A3s，FIV和HIV通过利用病毒蛋白Vif构建一个泛素连接酶复合物来连接包括Cullin 5在内的细胞蛋白，从而诱导这些蛋白的降解。在此，我们确定了FIV Vif和Cullin 5中调节这种相互作用的蛋白残基。虽然我们的结构模型表明不同的FIV和HIV - 1 Vifs使用保守残基与Cullin 5结合，但与HIV - 1 Vif不同，FIV Vif不依赖锌与Cullin 5结合。
