Zhu, Shouhai

ABSTRACT Lactylation, a protein post-translational modification driven by lactate, plays crucial roles in various physiological and pathological processes. However, the function and underlying mechanisms of lactylation in infection and pathogenesis of influenza A virus (IAV) remain poorly understood. Here, we demonstrated that sirtuin 1 (SIRT1) suppressed IAV replication by mediating delactylation of viral proteins. We found that IAV infection significantly downregulated the expression of SIRT1. Functional studies revealed that depletion of SIRT1 markedly enhanced replication of IAV, whereas overexpression of SIRT1 attenuated the viral replication. Mechanistically, we observed that SIRT1 interacted with IAV NP, PA, PB1, and PB2 proteins, components of the viral ribonucleoprotein (vRNP) complex, and restrained the activity of the vRNP complex, thereby repressing transcription and replication of the IAV genome. Furthermore, we identified that viral NP, PA, PB1, and PB2 proteins underwent lactylation during IAV infection, and SIRT1 facilitated the delactylation of NP, PA, and PB2. Loss of SIRT1 enhanced the lactylation of NP, PA, and PB2, while overexpression of SIRT1 impaired lactylation of these viral proteins during IAV infection. Moreover, inhibition or depletion of lactate dehydrogenase A (LDHA) robustly reduced the lactylation of NP, PA, and PB2, thereby abolishing the promotion of IAV replication caused by SIRT1 deficiency, suggesting that SIRT1 repressed IAV replication via delactylation of viral NP, PA, and PB2 proteins. Together, these results uncover a mechanism that SIRT1 suppresses IAV replication via delactylation of the vRNP components and elicit a critical role for the lactylation of viral proteins in regulating IAV infection and pathogenesis. IMPORTANCE Lactylation plays vital roles in diverse pathological processes. However, the relationship between lactylation and influenza A virus (IAV) infection remains largely unexplored. Particularly, little information is available about the lactylation of viral proteins and their roles in the IAV pathogenesis. Here, viral proteins (NP, PA, PB1, and PB2) were identified to become lactylated during IAV infection. SIRT1 interacted with and promoted the delactylation of viral NP, PA, and PB2 proteins, thereby restraining the vRNP activity and inhibiting the viral transcription and replication. Interestingly, the expression of SIRT1 decreased following IAV infection, suggesting that IAV had evolved a mechanism to downregulate the SIRT1 expression, thereby ensuring appropriate lactylation of viral proteins and facilitating the IAV replication. Together, these findings reveal a critical role for the lactylation of viral proteins in regulating the replication of IAV and provide an important insight into the complicated interplay between the host and IAV.

CDK12 primarily functions as a transcription regulatory cyclin-dependent kinase (CDK) that controls mRNA elongation, splicing, and polyadenylation. The CDK12 gene is implicated in human cancers since it is frequently mutated and/or deleted in prostate and ovarian cancer but paradoxically amplified in breast cancer. Here, we demonstrate that CDK12 promotes serine-933 phosphorylation of DNA2, a nuclease/helicase critical for replication fork stress regulation, and the phosphorylation subsequently facilitates DNA2 polyubiquitination and degradation mediated by the APC/C CDC20 E3 ubiquitin ligase. CDK12 inactivation induces but amplification suppresses genome-wide expression of interferon response and antigen processing and presentation machinery genes in ovarian and breast cancer cells, respectively. Besides causing aberrant DNA2 stabilization, replication stress, genomic instability, and cytosolic double-stranded DNA (dsDNA) accumulation, CDK12 loss also triggers cGAS–STING activation and innate immune response, which can be reversed by forced expression of replication protein A (RPA) subunits or DNA2 depletion. Our findings identify DNA2 as a phosphorylation substrate of CDK12, connecting CDK12 to cell cycle regulation. These data also reveal DNA2 protein destruction as a critical mechanism that dictates genomic instability, cGAS–STING signaling activation, and innate immune response in CDK12-deregulated cancers.

Triple-negative breast cancer (TNBC) represents the most malignant subtype of breast cancer. The clinical application of PARP inhibitors (PARPi) is limited by the low frequency of BRCA1/2 mutations in TNBC. Here, we identified that MTAP deletion sensitized genotoxic agents in our clinical cohort of metastatic TNBC. Further study demonstrated that MTAP deficiency or inhibition rendered TNBC susceptibility to chemotherapeutic agents, particularly PARPi. Mechanistically, targeting MTAP that synergized with PARPi by disrupting the METTL16-MAT2A axis involved in methionine metabolism and depleting in vivo s-adenosylmethionine (SAM) levels. Exhausted SAM in turn impaired PARPi-induced DNA damage repair through attenuation of MRE11 recruitment and end resection by diminishing MRE11 methylation. Notably, brain metastatic TNBC markedly benefited from a lower dose of PARPi and MTAP deficiency/inhibition synergy due to the inherently limited methionine environment in the brain. Collectively, our findings revealed a feed-forward loop between methionine metabolism and DNA repair through SAM, highlighting a therapeutic strategy of PARPi combined with MTAP deficiency/inhibition for TNBC.