Identification of TMEM41B TM6 as a critical domain for coronavirus replication <i>in vitro</i> and <i>in vivo</i>

ABSTRACT Transmembrane protein 41B (TMEM41B) serves as a critical host factor for various coronavirus infections, providing a potential target for developing antiviral gene-edited animals. However, TMEM41B is a gene essential for mammalian embryonic development, with complete knockout resulting in embryonic lethality. Consequently, it is of paramount importance to identify its critical functional domains that are amenable to deletion. In this study, we demonstrated that TMEM41B is involved in the replication stage of porcine deltacoronavirus (PDCoV), an emerging zoonotic coronavirus that infects various animals and even humans, but not in the attachment, internalization, or release steps. Through the construction of truncated mutants and cell lines stably expressing or precisely lacking the functional domains of TMEM41B, we confirm that, in addition to the VTT domain, the sixth transmembrane (TM6) domain of TMEM41B is also required for coronavirus infection. Using CRISPR/Cas9 technology, we generated a C57BL/6J mouse model with a deletion of the TMEM41B TM6 domain. Animal experiments demonstrated that the deletion of the TM6 domain significantly inhibited viral infection in vivo and mitigated liver tissue damage caused by mouse hepatitis virus (MHV), thereby increasing the survival rate of mice after MHV infection. Taken together, this study is the first to report a requirement of TMEM41B TM6 domain for coronavirus infection, deepening our understanding of TMEM41B function and providing novel insights for the development of antiviral gene-edited animals. IMPORTANCE TMEM41B is a proviral host factor for pan-coronaviruses, making it an ideal target for developing antiviral gene-edited animals. However, the essential role of TMEM41B in mammalian embryonic development poses a major obstacle to this goal. Identifying its critical functional domains or key amino acids that are amenable to deletion is pivotal for evaluating TMEM41B as a viable target for developing antiviral gene-edited animals. Our present study demonstrates that the sixth transmembrane (TM6) domain of TMEM41B is critical for the infection of PDCoV and other coronaviruses, without obviously compromising the function of its conserved VTT domain. Furthermore, a mouse infection model provides strong evidence that deletion of TM6 suppresses viral infection and attenuates virus-induced liver damage. Collectively, we identify TM6 as a crucial domain for coronavirus infection both in vitro and in vivo , providing new insights into the structure-function relationship of TMEM41B and proposing a novel strategy for antiviral animal development.