Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State Universitygrid.30064.31, Pullman, Washington, USA.
Microbiol Spectr. 2022 Oct 26;10(5):e0311422. doi: 10.1128/spectrum.03114-22. Epub 2022 Sep 29.
Herpes simplex virus 1 (HSV-1) commandeers the host cell proteasome at several steps of its replication cycle, including entry. Here we demonstrate that HSV-2, pseudorabies virus (PRV), and bovine herpesvirus 1 (BoHV-1) entry are blocked by bortezomib, a proteasome inhibitor that is an FDA-approved cancer drug. Proteasome-dependent entry of HSV-1 is thought to be ubiquitin-independent. To interrogate further the proteasomal mechanism of entry, we determined the involvement of the ubiquitin-like molecule NEDD8 and the neddylation cascade in alphaherpesvirus entry and infection. MLN4924 is a small-molecule inhibitor of neddylation that binds directly to the NEDD8-activating enzyme. Cell treatment with MLN4924 inhibited plaque formation and infectivity by HSV-1, PRV, and BoHV-1 at noncytotoxic concentrations. Thus, the neddylation pathway is broadly important for alphaherpesvirus infection. However, the neddylation inhibitor had little effect on entry of the veterinary viruses but had a significant inhibitory effect on entry of HSV-1 and HSV-2 into seven different cell types. Washout experiments indicated that MLN4924's effect on viral entry was reversible. A time-of-addition assay suggested that the drug was acting on an early step in the entry process. Small interfering RNA (siRNA) knockdown of NEDD8 significantly inhibited HSV entry. In probing the neddylation-dependent step in entry, we found that MLN4924 dramatically blocked endocytic uptake of HSV from the plasma membrane by >90%. In contrast, the rate of HSV entry into cells that support direct fusion of HSV with the cell surface was unaffected by MLN4924. Interestingly, proteasome activity was less important for the endocytic internalization of HSV from the cell surface. The results suggest that the NEDD8 cascade is critical for the internalization step of HSV entry. Alphaherpesviruses are ubiquitous pathogens of humans and veterinary species that cause lifelong latent infections and significant morbidity and mortality. Host cell neddylation is important for cell homeostasis and for the infection of many viruses, including HSV-1, HSV-2, PRV, and BoHV-1. Inhibition of neddylation by a pharmacologic inhibitor or siRNA blocked HSV infection at the entry step. Specifically, the NEDD8 pathway was critically important for HSV-1 internalization from the cell surface by an endocytosis mechanism. The results expand our limited understanding of cellular processes that mediate HSV internalization. To our knowledge, this is the first demonstration of a function for the neddylation cascade in virus entry.
单纯疱疹病毒 1 (HSV-1) 在其复制周期的几个步骤中利用宿主细胞蛋白酶体，包括进入。在这里，我们证明疱疹病毒 2、伪狂犬病病毒 (PRV) 和牛疱疹病毒 1 (BoHV-1) 的进入被蛋白酶体抑制剂硼替佐米阻断，硼替佐米是一种获得 FDA 批准的癌症药物。据认为，HSV-1 的蛋白酶体依赖性进入与泛素无关。为了进一步探讨蛋白酶体进入的机制，我们确定了泛素样分子 NEDD8 和 neddylation 级联在α疱疹病毒进入和感染中的作用。MLN4924 是一种小分子量的 neddylation 抑制剂，可直接与 NEDD8 激活酶结合。细胞用 MLN4924 处理可在非细胞毒性浓度下抑制 HSV-1、PRV 和 BoHV-1 的蚀斑形成和感染性。因此，neddylation 途径对α疱疹病毒感染非常重要。然而，neddylation 抑制剂对兽医病毒的进入几乎没有影响，但对 HSV-1 和 HSV-2 进入七种不同细胞类型的进入有显著的抑制作用。冲洗实验表明，MLN4924 对病毒进入的作用是可逆的。添加时间测定表明，该药物作用于进入过程的早期步骤。NEDD8 的小干扰 RNA(siRNA) 敲低显著抑制了 HSV 的进入。在探索进入过程中的 neddylation 依赖性步骤时，我们发现 MLN4924 可使 HSV 从质膜的内吞摄取显著减少 >90%。相比之下，MLN4924 对支持 HSV 与细胞表面直接融合的细胞中 HSV 进入细胞的速率没有影响。有趣的是，蛋白酶体活性对 HSV 从细胞表面的内吞内化不太重要。结果表明，NEDD8 级联对 HSV 进入的内化步骤至关重要。α疱疹病毒是人类和兽医物种的普遍病原体，可引起终身潜伏感染和显著的发病率和死亡率。宿主细胞的 neddylation 对细胞内稳态和许多病毒的感染很重要，包括 HSV-1、HSV-2、PRV 和 BoHV-1。用药理抑制剂或 siRNA 抑制 neddylation 可在进入步骤阻断 HSV 感染。具体而言，NEDD8 途径对 HSV-1 通过胞吞作用从细胞表面内化非常重要。该结果扩展了我们对介导 HSV 内化的细胞过程的有限理解。据我们所知，这是 neddylation 级联在病毒进入中的功能的首次证明。
