Coronavirus (CoV) nucleocapsid (N) protein contains two structurally independent RNA binding domains. These are denoted N-terminal domain (NTD) and C-terminal domain and are joined by a charged linker region rich in serine and arginine residues (SR linker). In mouse hepatitis virus (MHV), the NTD binds the transcriptional regulatory sequence (TRS) RNA, a conserved hexanucleotide sequence required for subgenomic RNA synthesis. The NTD is also capable of disrupting a short RNA duplex. We show here that three residues on the β3 (Arg-125 and Tyr-127) and β5 (Tyr-190) strands play key roles in TRS RNA binding and helix destabilization with Ala substitutions of these residues lethal to the virus. NMR studies of the MHV NTD·TRS complex revealed that this region defines a major RNA binding interface in MHV with site-directed spin labeling studies consistent with a model in which the adenosine-rich 3'-region of TRS is anchored by Arg-125, Tyr-127, and Tyr-190 in a way that is critical for efficient subgenomic RNA synthesis in MHV. Characterization of CoV N NTDs from infectious bronchitis virus and from severe acute respiratory syndrome CoV revealed that, although detailed NTD-TRS determinants are distinct from those of MHV NTD, rapid helix destabilization activity of CoV N NTDs is most strongly correlated with CoV function and virus viability.