Interaction of the coronavirus infectious bronchitis virus membrane protein with beta-actin and its implication in virion assembly and budding

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 beta-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.

Figure 1. Co-Purification of actin with IBV virions.

Confluent monolayers of Vero cells were infected with IBV at a multiplicity of infection of approximately 2 PFU/cell. At 18 hours post-infection, the supernatants were collected, centrifuged at 4,000 rpm for 20 minutes to remove cell debris. The virus was pelleted by ultracentrifugation through a 2 ml surcrose cushion (20%) at 175,000×g for 3 hours and further purified using a linear gradient of 10–50% sucrose at 175,000×g for 18 hours. Eleven fractions from top to bottom were collected and separated by SDS-PAGE. Polypeptides were analyzed by Western blot with anti-N antibodies (upper panel) and anti-actin antibodies (lower panel). Total lysates prepared from confluent monolayers of Vero cells were ultra-centrifuged at 175,000×g for 18 hours on a linear gradient of 10–50% sucrose. Eleven fractions from top to bottom were collected and separated by SDS-PAGE. Polypeptides were analyzed by Western blot with anti-actin antibodies.

Figure 2. Interaction of the IBV M protein with human β-actin.

HeLa cells were transfected with the Myc-tagged actin alone (lanes 1, 4, 7 and 10), IBV M alone (lanes 2, 5, 8 and 11) or cotransfected with actin and IBV M (lanes 3, 6, 9 and 12). Cells were harvested at 24 hours post-transfection and lysates prepared. Polypeptides were either analyzed directly by Western blot with anti-Myc (lanes 1–3) and anti-IBV M (lanes 4–6) antibodies, or subjected to immunoprecipitation with anti-Myc antibodies. The precipitates were analyzed by Western blot with anti-Myc (lanes 7–9) and anti-IBV M (lanes 10–12) antibodies. Numbers on the left indicate molecular masses in kilodaltons.

Figure 3. Mapping the region on M protein responsible for interaction with β-actin.

a. Diagram showing the wild type IBV M and six deletion and mutation constructs (MΔ1, MΔ2, MΔ3, MΔ4, MΔ5 and Mm1). The two N-linked glycosylation sites (N-gly), three transmembrane domains (Tm1-3) and the C-terminal cytoplasmic domain of the M protein are highlighted. Also shown are the amino acid positions of the deleted and mutated region in each construct. b. The wild type IBV M and the six deletion and mutation constructs, respectively, were transformed into yeast cells with pACT-actin. The growth of the yeast cells on SD-Trp/-Leu/-His/-Ade- plate is shown. Also shown are the positive and negative controls. c. Co- immunoprecipitation of wild type M (the C-terminal region from amino acids 104–225) but not Mm1 with with human β-actin. HeLa cells were transfected with the Myc-tagged wild type M (Myc-M) (lane1), Myc-Mm1 (lane 2), empty vector (lane 3) or Myc-M+actin (lane 4). Cells were harvested at 24 hours post-transfection and lysates prepared. Polypeptides were either analyzed directly by Western blot with anti-Myc (upper panel), or subjected to immunoprecipitation with anti-actin antibodies. The precipitates were analyzed by Western blot with anti-Myc antibody (lower panel). LC-light chain.

Figure 4. Colocalizaion of M and actin proteins in cells.

The plasmids containing M gene were transfected into H1299 cells. At 4 hours post-transfection, the cells were treated by cytochalasin D (12.5 µg/ml) and permeabilized with 0.2% Triton X-100 and stained with anti-M antibodies at 48 hours post-transfection. The same cells were also stained with actin dye Alexa Fluor 488 Phalloidin (green).

Figure 5. Effects of A159-K160 deletion and mutation on replication and infectivity of IBV.

a. In vitro transcripts derived from wild type, MΔ5 and Mm1 full-length IBV clones were electroporated into Vero cells. At 24 and 72 hours post-electroporation, total RNA were extracted from the cells and specific primers were used to detect the minus RNA (upper panel) and subgenomic mRNA 4 (lower panel) by RT-PCR. b. H1299 cells expressing wild type IBV M and MΔ5 constructs with a vaccinia virus system were infected with IBV at a multiplicity of infection of approximately 0.5 PFU/cell. Cells were collected at 24 hours post-infection and polypeptides were analyzed by Western blot with anti-IBV M (second panel), anti-IBV N (third panel) and anti-actin (bottom panel) antibodies. H1299 cells expressing wild type IBV M and MΔ5 constructs with a vaccinia virus system without IBV infection harvested at 40 hours post-transfection were also analyzed by Western blot with anti-IBV M antibodies (top panel). c. Interaction of Mm1 mutant M protein with IBV E protein. H1299 cells expressing the E (lane 1), M (lane 2) and E+M (lane 3) and E+Mm1 were harvested at 24 hours post-transfection and lysed. The total cell lysates were either detected directly by Western blot with anti-E antibodies (top panel) or immunoprecipitated with anti-M antibodies. The precipitates were analyzed by Western blot with anti-M (middle panel) and anti-E (bottom panel) antibodies, respectively.

Figure 6. Effects of disruption of actin filaments by cytochalasin D on IBV replication.

Vero cells plated in six-well plates were infected with IBV at a multiplicity of infection of approximately 3 PFU/cell. Cytochalasin D (12.5 µg/ml) and DMSO were added to the infected cells at indicated time. Total cells and the culture media were separately collected at 24 hours post-infection. Cell lysates were prepared and analyzed by Western blot with anti-N (top panel) and anti-actin (middle panel) antibodies. Western blot analysis of the IBV N protein in the culture media with anti-N antibodies (bottom panel) was also shown.