Rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction
Rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction
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DOI：
10.1128/jvi.75.22.10623-10629.2001
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发表时间：
2001-11-01
影响因子：
5.4
通讯作者：
Schnell, MJ
中科院分区：
文献类型：
--
作者：
Harty, RN;Brown, ME;Schnell, MJ
水泡性口炎病毒（VSV）和狂犬病病毒（RV）的基质（M）蛋白在子代病毒颗粒的组装和出芽中发挥着关键作用。 PPPY 基序（PY 基序或晚出芽结构域）在 VSV 和 RV 的 M 蛋白中保守。这些 PY 基序对于病毒出芽以及介导与包含 WW 结构域的特定细胞蛋白的相互作用非常重要。 PY 基序和 VSV M 蛋白的侧翼序列用作诱饵，筛选小鼠胚胎 cDNA 文库中的细胞相互作用因子。小鼠 Nedd4 蛋白是一种包含多个 WW 结构域的膜定位泛素连接酶，是从该筛选中鉴定出来的。泛素连接酶 Rsp5 是 Nedd4 的酵母同源物，能够以 PY 依赖性方式与全长 VSV M 蛋白在物理和功能上相互作用。事实上，在体外泛素化测定中，VSV M 蛋白被 Rsp5 多泛素化。为了进一步证明泛素可能参与弹状病毒的出芽过程，使用蛋白酶体抑制剂（例如 MG132）来降低 VSV 和 RV 感染细胞中游离泛素的水平。经 MG132 处理的细胞测量的病毒滴度可重复地比未处理的对照细胞测量的病毒滴度低 10 至 20 倍，这表明游离泛素对于有效的病毒出芽很重要。最后，在 MG132 存在的情况下，VSV PY 突变体的释放不受抑制，这表明 MG132 表现出的抑制作用需要 VSV 的功能性 L 结构域。这些数据表明细胞泛素-蛋白酶体机制参与了 VSV 和 RV 的出芽过程。
The matrix (M) proteins of vesicular stomatitis virus (VSV) and rabies virus (RV) play a key role in both assembly and budding of progeny virions. A PPPY motif (PY motif or late-budding domain) is conserved in the M proteins of VSV and RV. These PY motifs are important for virus budding and for mediating interactions with specific cellular proteins containing WW domains. The PY motif and flanking sequences of the M protein of VSV were used as bait to screen a mouse embryo cDNA library for cellular interactors. The mouse Nedd4 protein, a membrane-localized ubiquitin ligase containing multiple WW domains, was identified from this screen. Ubiquitin ligase Rsp5, the yeast homolog of Nedd4, was able to interact both physically and functionally with full-length VSV M protein in a PY-dependent manner. Indeed, the VSV M protein was multiubiquitinated by Rsp5 in an in vitro ubiquitination assay. To demonstrate further that ubiquitin may be involved in the budding process of rhabdoviruses, proteasome inhibitors (e.g., MG132) were used to decrease the level of free ubiquitin in VSV- and RV-infected cells. Viral titers measured from MG132-treated cells were reproducibly 10-to 20-fold lower than those measured from untreated control cells, suggesting that free ubiquitin is important for efficient virus budding. Last, release of a VSV PY mutant was not inhibited in the presence of MG132, signifying that the functional L domain of VSV is required for the inhibitory effect exhibited by MG132. These data suggest that the cellular ubiquitin-proteasome machinery is involved in the budding process of VSV and RV.