A small protein encoded by PCBP1-AS1 is identified as a key regulator of influenza virus replication via enhancing autophagy.

Many annotated long noncoding RNAs (lncRNAs) contain small open reading frames (sORFs), some of which have been demonstrated to encode small proteins or micropeptides with fundamental biological importance. However, functions of lncRNAs-encoded small proteins or micropeptides in viral pathogenesis remain largely unexplored. Here, we identified a 110-amino acid small protein as a key regulator of influenza A virus (IAV) replication. This small protein that we call PESP was encoded by the putative lncRNA PCBP1-AS1. It was observed that both PCBP1-AS1 and PESP were significantly upregulated by IAV infection. Furthermore, they were markedly induced by treatment with either type I or type III interferon. Overexpression of either PCBP1-AS1 or PESP alone significantly enhanced IAV replication. In contrast, shRNA-mediated knockdown of PCBP1-AS1 or CRISPR/Cas9-mediated knockout of PESP markedly inhibited the viral production. Moreover, the targeted deletion or mutation of the sORF within the PCBP1-AS1 transcript, which resulted in the disruption of PESP expression, significantly diminished the capacity of PCBP1-AS1 to enhance IAV replication, underscoring the indispensable role of PESP in the facilitation of IAV replication by PCBP1-AS1. Interestingly, overexpression of PESP enhanced the IAV-induced autophagy by increasing the expression of ATG7, an essential autophagy effector enzyme. We also found that the 7–22 amino acids at the N-terminus of PESP were crucial for its functionality in modulating ATG7 expression and action as an enhancer of IAV replication. Additionally, HSP90AA1, a protein identified previously as a facilitator of autophagy, was found to interact with PESP, resulting in the stabilization of PESP and consequently an increase in the production of IAV. These data reveal a critical lncRNA-encoded small protein that is induced and exploited by IAV during its infection, and provide a significant insight into IAV-host interaction network. Author summary: Emerging evidence has shown that some lncRNAs encode biologically active small proteins or micropeptides involved in diverse cellular processes. Here, we identified a previously unrecognized small protein derived from the lncRNA PCBP1-AS1 and established a role for this protein in influenza A virus (IAV) infection. We named it as PCBP1-AS1-encoded small protein (PESP) and found it to be crucially implicated in replication of IAV. Expression of both PESP and PCBP1-AS1 were significantly increased in response to IAV infection or upon interferon treatment. By overexpressing PESP, we observed a substantial enhancement in IAV replication, which was counteracted by reducing PESP level. We further demonstrated that PESP coding sequences were essential for PCBP1-AS1 to facilitate IAV replication, as evidenced by significantly reduced functionality of PCBP1-AS1 with deletion of the sequences. Intriguingly, PESP appeared to promote viral replication by modulating autophagy. This was achieved by significant upregulation of ATG7, a key enzyme in autophagy. Additionally, we revealed that interaction of PESP with HSP90AA1, a protein known to support autophagy, contributed to the protein's stability of PESP and consequently to increased IAV replication. This study sheds light on the intricate interplay between host factors and viral pathogens, offering potential targets for antiviral strategies. [ABSTRACT FROM AUTHOR]