Peptides based on heptad repeat (HR) domains of class I viral fusion proteins are considered promising antiviral drugs targeting virus cell entry. We have analyzed the evolution of the mouse hepatitis coronavirus during multiple passaging in the presence of an HR2-based fusion inhibitor. Drug-resistant variants emerged as a result of multiple substitutions in the spike fusion protein, notably within a 19-residue segment of the HR1 region. Strikingly, one mutation, an A1006V substitution, which consistently appeared first in four independently passaged viruses, was the main determinant of the resistance phenotype, suggesting that only limited options exist for escape from the inhibitory effect of the HR2 peptide.Coronaviruses are important pathogens for humans and animals, as illustrated by the severe acute respiratory syndrome (SARS) coronavirus, which caused the global SARS epidemic in [2002][2003]. Hence, there is a need for effective vaccines and antiviral therapeutics. While some progress has been made with the development of coronavirus vaccines (13), little success has been achieved in the search for antiviral therapies. For human immunodeficiency virus (HIV), the entry stage of the infection cycle has proven to be an attractive drug target (8). Entry of the enveloped coronaviruses is mediated by the viral spike (S) protein. The coronavirus S protein belongs to the class I viral fusion proteins (2), together with the fusion proteins of viruses of the orthomyxo-, retro-, paramyxo-, filo-, arena-, and baculoviridae. Class I fusion proteins mediate membrane fusion through extensive post-receptor-binding structural rearrangements in which two heptad repeat (HR) regions play a key role: HR1, positioned C-terminal of a hydrophobic fusion peptide, and HR2, located N-terminal of the transmembrane domain. It is hypothesized that upon anchoring of the fusion peptide in the target membrane, HR2 binds to the HR1 region in an antiparallel manner to form a stable six-helix bundle, with three HR1 fragments forming a central coiled coil surrounded by three coiled HR2 segments. The formation of this six-helix bundle is thought to mediate the juxtaposition of viral and cellular membranes, thereby facilitating the fusion process (for two recent reviews, see references 7 and 12). Peptides corresponding to the HR1 and HR2 regions were first shown for HIV to be potent virus inhibitors in vitro (6,16,17) and in vivo (8), most likely as a result of antagonistic binding to the HR regions within the fusion protein, thereby preventing hairpin formation and consequently membrane fusion. Similarly, we and others have shown that coronavirus entry can be effectively targeted by HR-derived peptides (1,2,9,10,18,19,21). Despite their effectiveness in vitro, the applicability of these HR homologues as therapeutics for treatment of coronavirus infections might be hampered by the emergence of drug-resistant viruses, as was illustrated by the development of clinical resistance of HIV against enfuvirtide, an HR2 peptide homologue (14).To analy...
