Heinrich Hoffmann & Bill Schneider - April 14, 2021

Heinrich Hoffmann, PhD, Research Associate, The Rockefeller University, Bill Schneider, PhD, Research Associate, The Rockefeller University, CRISPR screens identify TMEM41B as a pan-coronavirus and pan-flavivirus host factor

Knock-out CRISP screening was applied to identify host factors that are essential for the virus surviving but not for host cells. They further expect to discover potential drug targets and new biology that unknown before. They screened not only SARS-CoV2 but also other human Coronavirus strains (HCoV-O43, HCoV-229E and HCoV-NL63) to compare and discover any common factors. Each cell was probed with Single Guide RNA (sgRNA) and made a full genome library with a total of 20,000 genes. Coronavirus was then infected to these heterogeneous cells and survived populations from the viral cytotoxicity were sequenced to identify the host factors which may contribute to viral entering or inhibition of viral replication. Bioinformatics study showed TMEM41B was suggested as a pan-coronavirus host factor which is involved early stage of lipid mobilization. Validation was carried out by making TMEM41B – and TMEM41+ constructs and applied to 2 cell lines (Huh-7.5 cells and A549 (ACE2/TMPRSS2). The result showed impaired viral replication with KO while restored the viral replication when re-introduced TMEM41B KO+. the qRT-PCR study showed the viral components (sgN, N, Nsp14) expressions were reduced in KO and recovered after re-introducing of TMEM41B. They also investigated if TMEM41B is involved in viral entry. KO SARS-CoV RNA was directly inserted into the cells to avoid the RNA contact cell surface receptor and the result showed a failure of viral replication indicating TMEM41B is not associated with viral entering but related to viral replication.

A previous study of TMEM41B in Flaviviruses: Knock out CRISP screening was performed and identified TMEM41B and VMP1 as host factors for Zika virus (ZIKV) and Yellow Fever virus (YFV). TMEM41 has an important function in mobilizing lipid in ZIKV. KO study showed that TMEM41B is associated with viral replication. TMEM41B also decreases the viral replication in other members of Flaviviridae including Hepaciviruses (HCV), Pestiviruses, Pegiviruses (BVDV). TMEM41B is not a host factor for non-Coronaviridae and non-Flaviviridae. In flavivirus, TMEM41B interacts with membrane coverture forming proteins (NS4B and NS4A) and contributes to help to build the virus replication complex by shielding the virus and evade from host immunity. In TEME41B KO cells, flavivirus can still translate and replication can start but will be aborted because they are unable to form replication complex without the coverture and the exposed virus will be detected by the host and induced innate immunity. It is interesting to study flavivirus since the Coronavirus life cycle is similar to flavivirus.