Role of the CM2 protein in the influenza C virus replication cycle

Abstract

CM2 is the second membrane protein of influenza C virus. Although its biochemical characteristics, coding strategy, and properties as an ion channel have been extensively studied, the role(s) of CM2 in the virus replication cycle remains to be clarified. In order to elucidate this role, in the present study we generated CM2-deficient influenza C virus-like particles (VLPs) and examined the VLP-producing 293T cells, VLPs, and VLP-infected HMV-II cells. Quantification of viral RNA (vRNA) in the VLPs by real-time PCR revealed that the CM2-deficient VLPs contain approximately one-third of the vRNA found in wild-type VLPs although no significant differences were detected in the expression levels of viral components in VLP-producing cells or in the number and morphology of the generated VLPs. This finding suggests that CM2 is involved in the genome packaging process into VLPs. Furthermore, HMV-II cells infected with CM2-deficient VLPs exhibited significantly reduced reporter gene expression. Although CM2-deficient VLPs could be internalized into HMV-II cells as efficiently as wild-type VLPs, a smaller amount of vRNA was detected in the nuclear fraction of CM2-deficient VLP-infected cells than in that of wild-type VLP-infected cells, suggesting that the uncoating process of the CM2-deficient VLPs in the infected cells did not proceed in an appropriate manner. Taken together, the data obtained in the present study indicate that CM2 has a potential role in the genome packaging and uncoating processes of the virus replication cycle.

FIG. 1.

VLPs generated from 293T cells transfected with plasmid DNAs. VLPs generated from 293T cells transfected with the 10 (WT VLP) or nine (ΔCM2 VLP) plasmid DNAs were analyzed (see Materials and Methods for plasmid composition). (A) The supernatant of the 293T cells was negatively stained and observed using an electron microscope. Black triangles indicate hexagonal arrays of HEF (right panel). Bar, 200 nm. (B) Purified VLPs were lysed, electrophoresed by SDS-PAGE, and subjected to immunoblotting using a mixture of MAbs against HEF, NP, and M1 (left) and with antiserum against CM2 (right). Band intensities of HEF, NP, and M1 in the respective lanes were measured, and the ratios of HEF/M1, NP/M1, and NP/HEF are shown. (C) RNA was extracted from the purified VLPs, treated with DNase, reverse transcribed, and then subjected to real-time PCR for quantification of GFP-vRNA. Because the initial data were not normally distributed, they were log transformed for statistical analysis. The copy number of the GFP-vRNA in the WT VLPs was used for normalization. Each bar represents the mean ± standard errors of the means.

FIG. 2.

Expression of viral components and GFP in the 293T cells transfected with plasmid DNAs. 293T cells transfected with the 10 (WT) or 9 (ΔCM2) plasmid DNAs were analyzed (see Materials and Methods for plasmid composition). (A) The lysates of the 293T cells at 24 h p.t. were electrophoresed and subjected to immunoblotting using a mixture of MAbs against HEF, NP, and M1 (left) and with antisera against CM2, NS1, and NS2 (right). (B) The plasmid-transfected 293T cells were observed using a fluorescent microscope. The mock-transfected cells are shown in the left panel (magnification, ×50). (C) RNA was extracted from the 293T cells, treated with DNase, reverse transcribed, and then subjected to real-time PCR for quantification of GFP-vRNA. The relative copy number of the GFP-vRNA from 293T cells transfected with the 10 plasmids (WT) is expressed as 1.0. The result from mock-transfected cells was shown as the control. A representative result is shown.

FIG. 3.

Reporter gene expression in HMV-II cells infected with VLPs. (A) HMV-II cells infected with mock (Control), WT VLPs, or ΔCM2 VLPs, followed by superinfection with AA/50, were incubated for 48 h and observed by fluorescence microscopy (magnification, ×100). (B) WT VLPs or ΔCM2 VLPs containing luciferase-vRNA were used for infection, and luciferase activity detected in the infected HMV-II cells was measured at 24 h p.i. and is expressed as relative light units (RLU). The RLU value from the HMV-II cells infected with WT VLPs was expressed as 100 and used for normalization. Each bar represents the mean ± standard errors of the means. The supernatant from mock-transfected 293T cells was used as a control.

FIG. 4.

Flow cytometry of HMV-II cells infected with VLPs. HMV-II cells infected with WT VLPs or ΔCM2 VLPs were analyzed by flow cytometry using anti-HEF MAb J14. The VLP-infected cells were incubated at 4°C for 30 min and then incubated at 33°C for a further 180 min. The histogram from mock-infected cells is shown as a shaded area. Vertical and horizontal lines indicate the number of cells and fluorescence intensities, respectively.

FIG. 5.

VLP-mediated hemolysis. Hemolysis mediated by WT VLPs or ΔCM2 VLPs was measured using chicken erythrocytes. A total of 100 μl of VLP suspension in PBS was added to 0.5 ml of 2% (vol/vol) chicken erythrocytes in PBS at pH 7.0 and incubated on ice for 30 min. The mixture was centrifuged at 500 × g, and the pellet was suspended in 0.5 ml of saline buffered with 10 mM MES at various pH levels and incubated at 37°C for 60 min. The mixture was then centrifuged, and the supernatants were measured for the optical density at 540 nm (OD₅₄₀).

FIG. 6.

Fractionation and real-time PCR of HMV-II cells infected with VLPs. (A) HMV-II cells were fractionated as described in Materials and Methods, and the whole-cell lysates, the nuclear fraction, and the cytoplasmic fraction of the cells were analyzed by immunoblotting using anti-lamin B and anti-α-tubulin antibodies. (B) The HMV-II cells infected with WT VLPs or ΔCM2 VLPs were incubated at 4°C for 30 min and then transferred to 33°C and incubated for 60 min. The cells were divided into cytoplasmic and nuclear fractions, and the GFP-vRNA contained in the respective fractions was quantified by real-time PCR. The vertical line indicates the copy number of GFP-vRNA from 1.0 × 10⁶ HMV-II cells infected with VLPs. The data obtained from three independent experiments are shown as the means ± standard deviations. All comparisons between groups were statistically evaluated by using a paired t test. NS, not significant.