Abstract
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.
Highlights
Enveloped viruses acquire their envelope by budding from the host cell
11 fractions were collected from top to bottom, and the presence of viral proteins was checked by Western blot with anti-N antibodies, showing that the infectious bronchitis virus (IBV) N protein was detected in fractions 7–11 with majority of the protein located in fractions 8–10 (Fig. 1, top panel)
It is proposed that the formation of coronavirus envelope is dominated by lateral interaction between M molecules that form a two-dimensional lattice in intracellular membranes
Summary
Enveloped viruses acquire their envelope by budding from the host cell. In this process, viral envelope proteins gather at a special membranous structure and cooperate with other viral components to induce budding [1]. Some viruses including human immunodeficiency virus bud from the plasma membrane and release the virion from host cells by pinching-off. The detailed mechanisms of viral assembly and budding, especially the host factors that are involved in these processes, are yet to be revealed for many viruses. We report that interaction between coronavirus membrane protein (M) and actin with functional implication in facilitating virion assembly and budding
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