Nelemans Tessa, Tas Ali, de Beijer Nina L, Janssen George M C, van Veelen Peter A, van Hemert Martijn J, Kikkert Marjolein
Molecular Virology Laboratory, Center for Infectious Diseases (LUCID), Leiden University, Leiden University Medical Center, Albinusdreef 2, Leiden, 2333 ZA, The Netherlands.
Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands.
Virol J. 2025 Apr 17;22(1):103. doi: 10.1186/s12985-025-02719-5.
Usutu virus (USUV) is an emerging orthoflavivirus, which mainly affects birds but in rare cases can cause severe neuroinvasive disease in humans. The virus relies on a multitude of host cell proteins, molecules and cellular processes for its replication, and must subvert host antiviral responses to establish a successful infection. Studying the complex network of virus-host protein interactions by proteomics approaches can therefore provide new insights in the replication cycle of USUV and its pathogenesis. We have previously shown that the USUV protein NS4A acts as an antagonist of the antiviral interferon response, and here we further map the host interaction partners of USUV NS4A using proximity labeling coupled to mass spectrometry. The resulting NS4A interactome revealed many host proteins involved in the autophagy pathway. We showed that both USUV infection and overexpression of USUV NS4A can indeed induce the autophagy pathway. However, stimulation or inhibition of the autophagy pathway in general did not affect USUV replication. Therefore, we decided to specifically analyze the role of the selective autophagy receptor sequestosome 1 (p62/SQSTM1), since we identified this protein as an important interaction partner of USUV NS4A. We found that p62 is involved in the degradation of USUV NS4A. In agreement with this, the knockdown of p62 enhanced replication of USUV in A549 cells. P62 thus plays an antiviral role during USUV infection, although this antiviral effect might also be related to its functions outside the autophagy pathway, such as modulation of the immune response. In conclusion, this study showed that USUV NS4A induces autophagy and is then targeted by p62 for degradation by the autophagic machinery, uncovering a new role of p62 in the antiviral defense against USUV.
乌苏图病毒（USUV）是一种新出现的正黄病毒，主要感染鸟类，但在罕见情况下可导致人类严重的神经侵袭性疾病。该病毒的复制依赖于多种宿主细胞蛋白、分子和细胞过程，并且必须颠覆宿主的抗病毒反应才能成功感染。因此，通过蛋白质组学方法研究病毒与宿主蛋白相互作用的复杂网络，可以为USUV的复制周期及其发病机制提供新的见解。我们之前已经表明，USUV蛋白NS4A作为抗病毒干扰素反应的拮抗剂，在此我们进一步使用与质谱联用的邻近标记技术来绘制USUV NS4A的宿主相互作用伙伴。由此产生的NS4A相互作用组揭示了许多参与自噬途径的宿主蛋白。我们表明，USUV感染和USUV NS4A的过表达确实都能诱导自噬途径。然而，一般情况下刺激或抑制自噬途径并不影响USUV的复制。因此，我们决定专门分析选择性自噬受体隔离小体1（p62/SQSTM1）的作用，因为我们将该蛋白鉴定为USUV NS4A的重要相互作用伙伴。我们发现p62参与了USUV NS4A的降解。与此一致的是，敲低p62可增强USUV在A549细胞中的复制。因此，p62在USUV感染期间发挥抗病毒作用，尽管这种抗病毒作用也可能与其在自噬途径之外的功能有关，例如免疫反应的调节。总之，本研究表明，USUV NS4A诱导自噬，然后被p62靶向，通过自噬机制进行降解，揭示了p62在抗USUV病毒防御中的新作用。
