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sp de nAbstract Membrane fusion between virus and host cells is the key step for enveloped virus entry and is mediated by the viral envelope fusion protein. In murine coronavirus, mouse hepatitis virus (MHV), the spike (S) protein mediates this process. Recently, the forma-tion of anti-parallel 6-helix bundle of the MHV S protein heptad repeat (HR) regions (HR1 and HR2) has been conWrmed, implying coronavirus has a class I fusion protein. This bundle is also called fusion core. To facilitate the solution of the crystal structure of this fusion core, we deployed an Escherichia coli in vitro expression system to express the HR1 and HR2 regions linked together by a Xex-ible linker as a single chain (named 2-helix). This 2-helix polypeptide subsequently assembled into a typical 6-helix bundle. This bun-dle has been analyzed by a series of biophysical and biochemical techniques and conWrmed that the design technique can be used for coronavirus as we successfully used for members of paramyxoviruses