Phylogenet. from other CoV Mpros, the HCoV-HKU1 Mpro structure reported here provides insights into both substrate preference and the design of antivirals targeting CoVs. Coronaviruses (CoVs) are positive-strand RNA viruses that have been identified as the main etiologic agents responsible for a vast number of enteric, gastric, and respiratory syndromes of both humans and animals (14-17, 19, 21, 23, 26, 30, 31, 34, 45). CoVs can be divided into three groups: group 1 (including human CoV 229E [HCoV 229E] and transmissible gastric enteritis virus [TGEV]), group 2 (including HCoV-OC43, murine hepatitis virus [MHV], and bovine CoV [BCoV), and group 3 (including avian infectious bronchitis virus [IBV]). Shortly after the emergence of severe acute respiratory syndrome CoV (SARS-CoV) in 2003, group RCBTB1 2 CoVs were further divided into two subgroups, termed 2A and 2B (46). The classical group 2 viruses constitute subgroup 2A, while the newly emergent SARS-CoV and its animal counterparts (37) form subgroup 2B. Group 1 and group 2 CoVs have more impact on human health than group 3, since group 3 CoVs (such as avian IBV) can only infect avian species. Following the outbreak of SARS, group 2 CoVs have continued to attract greater attention for two reasons. First, they consist of human viruses (SARS-CoV and HCoV-OC43) as well as several important animal viruses (MHV and BCoV) that serve as useful models for CoV-host interactions. Second, group 2 CoVs are reported to have crossed the animal-to-human species barrier in two instances: one bat-to-human transmission in group 2B (27, 37) and one transmission event in group 2A CoVs, in which BCoV led to the emergence of HCoV-OC43 (36). Group 2A HCoVs were less widely studied prior to the global SARS epidemic in 2003. However, they are closely associated with a wide Coluracetam range of acute or chronic respiratory syndromes (3, 4, 7-9, 11, 12, 15, 20, 22, 35, 39, 40, 47). In the wake of the SARS outbreak, several novel HCoVs have been discovered, one of which is HCoV-HKU1 (9, 39). HCoV-HKU1 has achieved global distribution since it was first identified in 2005: infections were first characterized in Hong Kong (26), followed by the identification of several strains of the virus in Korea (9), Europe (5, 17), Australia (31), and North America (14). In contrast to the lethal SARS-CoV, infection by HCoV-HKU1 usually leads to self-limiting syndromes affecting the lower respiratory tract. Nevertheless, the consequences could be more severe in patients with a compromised or immature immune system, such as asthma sufferers or newborn infants (24). Genome sequencing has confirmed that the HCoV-HKU1 virus belongs to CoV group 2A and shares high sequence homology with MHV and BCoV (39). The functional components of the CoV replication machinery are released via posttranslational cleavage by two or three proteases. These proteases were first designated the Coluracetam papain-like protease (PLP) and 3C-like protease (3CL) for their respective sequence homology to the papain and rhinovirus 3C proteases. The 3CL protease also is commonly known as the main protease (Mpro) because of the major role it plays in the proteolytic pathway, which makes it the most attractive pharmacological target for anti-CoV drug design. CoV Mpros have been intensively studied, and crystal structures have been determined for the Mpros from the following CoVs: HCoV strain 229E (HCoV-229E) (2), porcine TGEV (1), avian IBV (41), and SARS-CoV (44). These structures are representative of group 1 (HCoV-229E and TGEV), group 2B (SARS-CoV), and group 3 (IBV) CoVs. However, no structure of the Mpro from a group 2A CoV (MHV, HCoV-HKU1, and HCoV-OC43) has been determined to date. The absence of structural data presents a major obstacle for structure-aided drug optimization targeting group 2A CoVs. The Mpros from different CoV groups are homologous in both sequence and main-chain architecture. They share a similar substrate binding sequence, with a requirement for glutamine at the P1 position and a strong preference for leucine/methionine at P2. Based on this information, broad-spectrum lead compounds (43) with micromolar values have been designed that target CoV Mpros. However, structural data for Coluracetam the Mpros from classical group 2A CoVs still are not available, posing a problem for further optimization. Although CoV Mpros exhibit absolute specificity for glutamine in the P1 position, recent research (38) has shown that the Mpro from HCoV-HKU1 may possess an unusual.