Giant viruses inhibit superinfection by downregulating phagocytosis in Acanthamoeba

Abstract

In the context of the virosphere, viral particles can compete for host cells. In this scenario, some viruses block the entry of exogenous virions upon infecting a cell, a phenomenon known as superinfection inhibition. The molecular mechanisms associated with superinfection inhibition vary depending on the viral species and the host, but generally, blocking superinfection ensures the genetic supremacy of the virus's progeny that first infects the cell. Giant amoeba-infecting viruses have attracted the scientific community's attention due to the complexity of their particles and genomes. However, there are no studies on the occurrence of superinfection and its inhibition induced by giant viruses. This study shows that mimivirus, moumouvirus, and megavirus, exhibit different strategies related to the infection of Acanthamoeba. For the first time, we have reported that mimivirus and moumouvirus induce superinfection inhibition in amoebas. Interestingly, megaviruses do not exhibit this ability, allowing continuous entry of exogenous virions into infected amoebas. Our investigation into the mechanisms behind superinfection blockage reveals that mimivirus and moumouvirus inhibit amoebic phagocytosis, leading to significant changes in the morphology and activity of the host cells. In contrast, megavirus-infected amoebas continue incorporating newly formed virions, negatively affecting the available viral progeny. This effect, however, is reversible with chemical inhibition of phagocytosis. This work contributes to the understanding of superinfection and its inhibition in mimivirus, moumouvirus, and megavirus, demonstrating that despite their evolutionary relatedness, these viruses exhibit profound differences in their interactions with their hosts.IMPORTANCESome viruses block the entry of new virions upon infecting a cell, a phenomenon known as superinfection inhibition. Superinfection inhibition in giant viruses has yet to be studied. This study reveals that even closely related viruses, such as mimivirus, moumouvirus, and megavirus, have different infection strategies for Acanthamoeba. For the first time, we have reported that mimivirus and moumouvirus induce superinfection inhibition in amoebas. In contrast, megaviruses do not exhibit this ability, allowing continuous entry of exogenous virions into infected amoebas. Our investigation shows that mimivirus and moumouvirus inhibit amoebic phagocytosis, causing significant changes in host cell morphology and activity. Megavirus-infected amoebas, however, continue incorporating newly formed viruses, affecting viral progeny. This research enhances our understanding of superinfection inhibition in these viruses, highlighting their differences in host interactions.

Keywords: Acanthamoeba; giant virus; phagocytosis; superinfection; virus-host relationship.

Fig 1

Particle, viral factory, and progeny features of mimi-, moumou-, and megaviruses observed by transmission electron microscopy. This panel shows that mimi-, moumou-, and megaviruses particles exhibit differences in the organization and abundance of surface fibrils, while their viral factories appear very similar. Notably, at 6 h post-infection, some megavirus particles are observed inside vesicles (black arrowheads).

Fig 2

The occurrence of megavirus particles within cytoplasmic vesicles. The replication cycles of megavirus caiporensis and other isolates were analyzed using transmission electron microscopy. All images were captured after the mature viral factory appearance, approximately 4–6 h post-infection. Vesicles contain from one to several megavirus particles.

Fig 3

Mimi-, moumou-, and megaviruses are not released via exocytosis in Acanthamoeba-infected cells. One-step growth curves of the supernatant from Acanthamoeba infected with (A) mimivirus, (B) moumouvirus, (C) megavirus, and (D) cedratvirus. The left Y-axis plots viral titers at different times post-infection. The right Y-axis plots data on viable cells at different times post-infection. The blue box in (D) highlights the period when cedratvirus is primarily released in the supernatant via exocytosis (the number of viable, non-lysed cells remains stable). Solid lines with circles represent virus titers, while dashed lines with squares represent viable cell counts. Panels (E–G) depict cedratvirus particles being released inside exosomes, 8 hpi.

Fig 4

Megavirus-infected cells phagocytose exogenous particles. Amoebas were infected by mimi-, moumou-, and megaviruses, and 2 h later were exposed to a “bait,” the Orpheovirus particles. At 6 hpi, cells and particles were analyzed by immunofluorescence microscopy. Orpheovirus particles, probed with a mouse primary antibody, appear green, while the amoeba cytoskeleton stained by blue Evans appears red. (A) Cells infected by mimi-, moumou-, or megavirus and exposed to Orpheovirus (bait). Only Orpheovirus particles colocalizing with amoeba vacuoles (black cytoplasm regions) were considered phagocytized (highlighted with dotted lines inside cells). (B) This panel shows several individual Acanthamoeba cells with phagocytized Orpheovirus particles.

Fig 5

The consequences of superinfection and its inhibition in mimi-, moumou-, and megavirus-infected cells. (A) Megavirus-infected cells phagocytized significantly more exogenous particles than mimi- and moumouvirus-infected cells. This graph was obtained after observing 50 cells infected by mimi-, moumou-, or megavirus and analyzed by immunofluorescence microscopy. The average of three independent replicates is presented. (B) Number of cells versus Orpheovirus-incorporated baits. The graph depicts all analyzed cells belonging to three independent replicates. (C) Titration of the residual Orpheovirus input in Vermoameba vermiformis cells. Mimi-, moumou-, and megavirus-infected cells were inoculated with Orpheovirus baits to verify phagocytosis activity. After 4 h (6 hpi), the supernatants were collected and titered in Vermoameba vermiformis, the laboratory host of Orpheovirus. This graph shows that almost the complete input of Orpheovirus baits was recovered from the supernatant of mimi- and moumouvirus-infected cells, suggesting a significant reduction in phagocytosis activity compared to megavirus-infected cells. (D) Transmission electron microscopy of megavirus-infected cells exposed to Orpheovirus baits. Asterisks denote megavirus particles; VF represents megavirus virus factory; red dashed circles indicate phagosomes containing Orpheovirus particles. (E) Evaluation of superinfection inhibition by mimi-, moumou-, and megaviruses. Acanthamoeba cells were infected by mimi-, moumou-, or megavirus at an MOI of 10. At times 30 min, 1 h, 2 h, 4 h, 6 h, and 12 h, cells were exposed to Orpheovirus baits. Four hours after this exposure, culture supernatants were collected and titered in Vermoameba vermiformis. The graph shows the almost complete recovery of Orpheovirus inputs from mimi- and moumouvirus-infected cultures from 2 hpi. This result indicates that mimi- and moumouvirus start to block phagocytosis of exogenous particles at early times post-infection. ***: P < 0.001, ****: P < 0.0001 (ANOVA, one-way).

Fig 6

Mimi- and moumouvirus cause compaction of the cellular cytoplasm and reduction in the formation of pseudopods and intracellular vacuoles. Acanthamoeba cells were infected with mimi-, moumou-, or megavirus at an MOI of 10. At 6 hpi, cells were analyzed by immunofluorescence microscopy. The amoeba cytoskeleton stained with blue Evans is represented in red. (A) Cells infected with mimi- and moumouvirus appear rounded and compacted compared to megavirus-infected and uninfected cells. Cell dimensions were measured (B), and vacuole counting (C) was performed by analyzing 50 cells randomly in three independent replicates. (D) Scanning electron microscopy of amoebas infected with mimi-, moumou-, or megavirus, 6 hpi. This panel shows that cells infected with mimi- and moumouvirus are smaller and exhibit fewer pseudopods compared to cells infected with megavirus or uninfected cells. ****: P < 0.0001 (ANOVA, one-way). Some of the images presented here (panel (A)) were also shown in Fig. 4A and B, as they were obtained from the same experiment.

Fig 7

Exogenous particles incorporated by 12-h megavirus-infected cells appear to be undergoing the uncoating process or are already empty. Amoebas were infected with megavirus at an MOI of 10. Twelve hours post-infection, infected cells were analyzed by transmission electron microscopy. (A–C) Megavirus exogenous particles (inside vesicles) undergo the uncoating process. In the images, it is possible to visualize the particle’s inner membrane fused with the phagosome membrane. (D and E) Empty exogenous particles inside vesicles (indicated by black arrowheads).

Fig 8

Cytochalasin D reverses the superinfection promoted by megavirus and increases the number of viral particles in the system. Acanthamoeba cells were infected with mimi-, moumou-, or megavirus at an MOI of 10. Two hours post-infection, amoebas were treated with different inhibitors of endocytic and phagocytic pathways: 2 µM of cytochalasin, 100 µM of chloroquine, or 1 µM of EIPA. Twenty-four hours post-infection, the culture supernatants were titrated. No significant changes in viral titers were observed in cells infected by mimi- (A) or moumouvirus (B) and treated with any of the mentioned inhibitors. However, cells infected with megavirus (C) and treated with cytochalasin produced significantly increased titers. ****: P < 0.0001 (ANOVA, one way).