Visualization of the Oncolytic Alphavirus M1 Life Cycle in Cancer Cells

Abstract

Oncolytic alphavirus M1 has been shown to selectively target and kill cancer cells, but cytopathic morphologies induced by M1 virus and the life cycle of the M1 strain in cancer cells remain unclear. Here, we study the key stages of M1 virus infection and replication in the M1 virus-sensitive HepG2 liver cancer cell line by transmission electron microscopy, specifically examining viral entry, assembly, maturation and release. We found that M1 virus induces vacuolization of cancer cells during infection and ultimately nuclear marginalization, a typical indicator of apoptosis. Specifically, our results suggest that the endoplasmic reticulum participates in the assembly of nucleocapsids. In the early and late stage of infection, three kinds of special cytopathic vacuoles are formed and appear to be involved in the replication, maturation and release of the virus. Taken together, our data displayed the process of M1 virus infection of tumor cells and provide the structural basis for the study of M1 virus-host interactions.

Keywords: Alphavirus; Electron microscopy; Life cycle of virus; M1; Oncolytic virus.

Fig. 1

M1 virus replicated and induced apoptosis in the HepG2 liver cancer cell line. A Control HepG2 cells uninfected with M1 virus. B ER distension. C ER became swollen. D Condensation and marginalization of nuclear chromatin are indicated by red arrows. E The decrease and vacuolization of pseudopodia are indicated by red arrows. F Vacuolization of mitochondria with damage to the membrane and ridges. ER: endoplasmic reticulum. M: mitochondria. Nu: nucleus. Time points: 6 h (B), 12 h (C) and 18 h (D–F). Bar: 2 μm (A, B) and 200 nm (C–F).

Fig. 2

The early stage of M1 virus infection of the HepG2 cancer cells. A A few virus particles aggregated near the pseudopodia and appeared in the invaginated space (the circled area) of the cell membrane. BSeveral M1 viruses gathered in the same vesicle inside the cell (circled with red), and nucleocapsids (with only a high electron density core) appeared in the cytoplasm matrix (circled with black). C Many viruses absorbed on the cellular pseudopodia. D M1 virus particles also gathered in pits in the cell membrane. Time points: 15 min (A), 2 h (B) and 6 h (C, D). MOI: 100 pfu per cell (A, B) and 3 pfu per cell (C, D). Bar: 200 nm.

Fig. 3

M1 virus precursors were scattered in the cytoplasmic matrix and inside the ER cavity. A “Spherules” were formed on the plasma membrane (indicated by black arrows). B Representative morphology of the CPV-I and “spherules” were formed inside it. Nucleocapsids were distributed in the cytoplasm (indicated by the red arrow). C and D Virus particles with a high electron density core were scattered inside the ER cavity. E The representative 2D classification of particles in the cytoplasmic matrix and inside the ER, with the number, average diameter and radial density curve shown. The contrast of the images was inverted because the software recognizes the density as white. The colored curves correspond to the radial density curves of the selected classifications. Time points: 6 h (A and C), 6 h (B) and 18 h (D). Bar: 200 nm.

Fig. 4

The maturation and release of M1 virus by budding on the plasma membrane. A M1 virus precursors stacked in the cytoplasm near the plasma membrane for budding. Red arrows indicate precursors, and black arrows indicate mature virions. B M1 virus precursors accumulated in the cellular pseudopodia. C The 2D classification of the extracellular particles. The number of particles in the classes, average diameters and radial density curves is indicated. The highlighted blue curve corresponds to the radial density of the selected majority classification. Time point: 18 h. Bar: 200 nm.

Fig. 5

M1 virus induced vacuolization of the HepG2 cancer cells. A–C M1 induced vacuolization of the HepG2 cells in the samples at 6 h, 12 h and 18 h p.i. With increasing infection time, both the number and size of the vacuoles gradually increased in the infected HepG2 cells. D Representative morphology of the CPV-II-1. E Representative morphology of the CPV-II-2. F Representative morphology of the CPV-0. Bar: 200 nm.

Fig. 6

The 2D classification of progeny M1 virus particles in different locations. Representative images, 2D map of the majority classes, weighted average diameters and radial density curves of the progeny M1 virus particles in different locations. Dw the average diameter. Bar: 100 nm.

Fig. 7

Schematic diagram of the M1 virus life cycle in HepG2 cells. According to our results and those of previous studies on alphaviruses, we have drawn a schematic diagram of the M1 virus life cycle. M1 viruses enter HepG2 cells via endocytosis and release their nucleocapsids in the cytoplasmic matrix for replication. Then, viral proteins and RNA are synthesized in CPV-I; capsid proteins and viral genomes are assembled into nucleocapsids in the ER and cytoplasmic matrix. There may be 3 ways that M1 virus matures: (1) envelope proteins are transported to the plasma membrane, and the M1 progeny mature through budding from the plasma membrane (red); (2) CPV-II-1 and CPV-II-2 form, envelope proteins are transported to the membrane of the CPV-IIs, nucleocapsids obtain envelopes through budding from the CPV-IIs (blue); and (3) nucleocapsids are assembled and the virus matures in the ER.