Usutu virus NS4A induces autophagy and is targeted by the selective autophagy receptor p62/SQSTM1 for degradation.

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.