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Structural Insight into the Interaction of Sendai Virus C Protein with Alix To Stimulate Viral Budding

Human parainfluenza virus type I (hPIV1) is a respiratory pathogen affecting young children, immunocompromised patients, and the elderly, with no available vaccines or antiviral drugs. Sendai virus (SeV), a murine counterpart of hPIV1, has been studied extensively to determine the molecular and biological properties of hPIV1. ABSTRACT Sendai virus (SeV), belonging to the Respirovirus genus of the family Paramyxoviridae, harbors an accessory protein, named C protein, which facilitates viral pathogenicity in mice. In addition, the C protein is known to stimulate the budding of virus-like particles by binding to the host ALG-2 interacting protein X (Alix), a component of the endosomal sorting complexes required for transport (ESCRT) machinery. However, small interfering RNA (siRNA)-mediated gene knockdown studies suggested that neither Alix nor C protein is related to SeV budding. In the present study, we determined the crystal structure of a complex comprising the C-terminal half of the C protein (Y3) and the Bro1 domain of Alix at a resolution of 2.2 Å to investigate the role of the complex in SeV budding. The structure revealed that a novel consensus sequence, LXXW, which is conserved among Respirovirus C proteins, is important for Alix binding. SeV possessing a mutated C protein with reduced Alix-binding affinity showed impaired virus production, which correlated with the binding affinity. Infectivity analysis showed a 160-fold reduction at 12 h postinfection compared with nonmutated virus, while C protein competes with CHMP4, one subunit of the ESCRT-III complex, for binding to Alix. All together, these results highlight the critical role of C protein in SeV budding. IMPORTANCE Human parainfluenza virus type I (hPIV1) is a respiratory pathogen affecting young children, immunocompromised patients, and the elderly, with no available vaccines or antiviral drugs. Sendai virus (SeV), a murine counterpart of hPIV1, has been studied extensively to determine the molecular and biological properties of hPIV1. These viruses possess a multifunctional accessory protein, C protein, which is essential for stimulating viral reproduction, but its role in budding remains controversial. In the present study, the crystal structure of the C-terminal half of the SeV C protein associated with the Bro1 domain of Alix, a component of cell membrane modulating machinery ESCRT, was elucidated. Based on the structure, we designed mutant C proteins with different binding affinities to Alix and showed that the interaction between C and Alix is vital for viral budding. These findings provide new insights into the development of new antiviral drugs against hPIV1.