Interaction of the Coronavirus Infectious Bronchitis Virus Membrane Protein with β-Actin and Its Implication in Virion Assembly and Budding

Coronavirus M protein is an essential component of virion and plays pivotal roles in virion assembly, budding and maturation. The M protein is integrated into the viral envelope with three transmembrane domains flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. In this study, we showed co-purification of the M protein from coronavirus infectious bronchitis virus (IBV) with actin. To understand the cellular factors that may be involved in virion assembly, budding and maturation processes, IBV M was used as the bait in a yeast two-hybrid screen, resulting in the identification of β-actin as a potentially interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation and immunofluorescence microscopy in mammalian cells, and mutation of amino acids A159 and K160 on the M protein abolished the interaction. Introduction of the A159-K160 mutation into an infectious IBV clone system blocks the infectivity of the clone, although viral RNA replication and subgenomic mRNA transcription were actively detected. Disruption of actin filaments with cell-permeable agent cytochalasin D at early stages of the infection cycle led to the detection of viral protein synthesis in infected cells but not release of virus particles to the cultured media. However, the same treatment at late stages of the infection cycle did not affect the release of virus particles to the media, suggesting that disruption of the actin filaments might block virion assembly and budding, but not release of the virus particles. This study reveals an essential function of actin in the replication cycle of coronavirus

Targeting non-human coronaviruses to human cancer cells using a bispecific single-chain antibody

To explore the potential of using non-human coronaviruses for cancer therapy, we first established their ability to kill human tumor cells. We found that the feline infectious peritonitis virus (FIPV) and a felinized murine hepatitis virus (fMHV), both normally incapable of infecting human cells, could rapidly and effectively kill human cancer cells artificially expressing the feline coronavirus receptor aminopeptidase N. Also 3-D multilayer tumor spheroids established from such cells were effectively eradicated. Next, we investigated whether FIPV and fMHV could be targeted to human cancer cells by constructing a bispecific single-chain antibody directed on the one hand against the feline coronavirus spike protein--responsible for receptor binding and subsequent cell entry through virus-cell membrane fusion--and on the other hand against the human epidermal growth factor receptor (EGFR). The targeting antibody mediated specific infection of EGFR-expressing human cancer cells by both coronaviruses. Furthermore, in the presence of the targeting antibody, infected cancer cells formed syncytia typical of productive coronavirus infection. By their potent cytotoxicity, the selective targeting of non-human coronaviruses to human cancer cells provides a rationale for further investigations into the use of these viruses as anticancer agents