New Isolates of Pandoraviruses: Contribution to the Study of Replication Cycle Steps

Abstract

Giant viruses are complex members of the virosphere, exhibiting outstanding structural and genomic features. Among these viruses, the pandoraviruses are some of the most intriguing members, exhibiting giant particles and genomes presenting at up to 2.5 Mb, with many genes having no known function. In this work, we analyzed, by virological and microscopic methods, the replication cycle steps of three new pandoravirus isolates from samples collected in different regions of Brazil. Our data indicate that all analyzed pandoravirus isolates can deeply modify the Acanthamoeba cytoplasmic environment, recruiting mitochondria and membranes into and around the electron-lucent viral factories. We also observed that the viral factories start forming before the complete degradation of the cellular nucleus. Various patterns of pandoravirus particle morphogenesis were observed, and the assembly of the particles seemed to be started either by the apex or by the opposite side. On the basis of the counting of viral particles during the infection time course, we observed that pandoravirus particles could undergo exocytosis after their morphogenesis in a process that involved intense recruitment of membranes that wrapped the just-formed particles. The treatment of infected cells with brefeldin affected particle exocytosis in two of the three analyzed strains, indicating biological variability among isolates. Despite such particle exocytosis, the lysis of host cells also contributed to viral release. This work reinforces knowledge of and reveals important steps in the replication cycle of pandoraviruses.IMPORTANCE The emerging Pandoraviridae family is composed of some of the most complex viruses known to date. Only a few pandoravirus isolates have been described until now, and many aspects of their life cycle remain to be elucidated. A comprehensive description of the replication cycle is pivotal to a better understanding of the biology of the virus. For this report, we describe new pandoraviruses and used different methods to better characterize the steps of the replication cycle of this new group of viruses. Our results provide new information about the diversity and biology of these giant viruses.

Keywords: giant virus; pandoravirus; replication cycle; viral morphogenesis; viral release; virus diversity.

FIG 1

Sites of collection and electron microscopy and phylogenetic analysis of the pandoravirus isolated in this work. (A) Map of Brazil showing where the samples were collected for the isolation of pandoraviruses. (B to D) Representative pictures from the areas of collection: Bom Jesus Creek (B), Mergulhão Creek (C), and the city of Bonito (D). (E) P. tropicalis particles were analyzed using scanning electron microscopy at 24 h.p.i. and an MOI of 0.01. (F, G, and H) Transmission electron microscopy (24 h.p.i./MOI 0.01) for the viral particles corresponding to the isolates of P. pampulha, P. tropicalis, and P. kadiweu, respectively. (I) Alignment of the sequences, showing that P. kadiweu and P. tropicalis represent strains of pandoraviruses with many exclusive polymorphisms (highlighted in yellow), compared to the sequences of other isolated pandoraviruses. (J) Maximum likelihood tree constructed using predicted sequence of 251 amino acids of a DNA polymerase B subunit in different isolates of pandoraviruses. The giant viruses isolated in this work are highlighted in red.

FIG 2

Initial steps of the pandoravirus replication cycle inside the amoebal host. (A and B) Scanning electron microscopy (A) and transmission electron microscopy (B) images show pandoravirus particles entering Acanthamoeba castellanii cells, likely as a consequence of phagocytosis. (C) The amoebas project pseudopods involving the viral particles and internalize them into vesicle-like structures known as phagosomes. (D and E) The phagosome then fuses with another component resembling a lysosome-like structure that, upon releasing their combined content, stimulates the uncoating of the pandoravirus particles (F). Although we used representative images in this figure, all the described steps were observed for all three isolated pandoraviruses. L, lysosome-like organelles; panels A and B, Pandoravirus tropicalis; panels C to F, Pandoravirus kadiweu.

FIG 3

Characterization of pandoravirus viral factories. Viral factories of (A) Pandoravirus tropicalis, (B) P. pampulha, and (C) P. kadiweu were observed by transmission electron microscopy. The region of the viral factories is highlighted in red, the mitochondria present in the interior of the viral factories are highlighted in green, and the lysosomes are pointed out by orange arrowheads.

FIG 4

Membranes recruited inside pandoravirus viral factories. (A) Transmission electron microscopy of P. tropicalis viral factories. (B) Transmission electron microscopy of P. pampulha viral factories. (C) Transmission electron microscopy of P. kadiweu viral factories. The membranes recruited inside the viral factories are highlighted in blue.

FIG 5

The Acanthamoeba castellanii cell nucleus becomes disorganized and loses its natural shape during the course of pandoravirus infection. (A) Transmission electron microscopy image showing a noninfected Acanthamoeba castellanii cell and how its nucleus is normally organized in this situation; it occupies about 2/3 of the cellular area, and it is delimited by a double-membrane layer known as the nuclear envelope (digitally highlighted in orange). The image at lower left represents the same conditions but visualized on a light microscope with Hemacolor staining. The nucleolus is observed as a dark spot surrounded by a bright area that represents the nucleus. (B) Image representing the amoeba observed just after the first steps of the pandoravirus replication cycle, as the virus (red arrow) is still harboring inside the amoebal phagosome. The nucleus does not yet seem to have suffered any modification at this stage. (C) At between h 3 and h 6 of infection, it seems that the nucleolus starts to be absent, as shown by one of the several images of transmission electron microscopy analyzed in this work. At lower left, the Hemacolor staining also shows the beginning of the appearance of the early viral factory. (D) The later steps of viral replication lead to the formation of the mature viral factory, marked by a bright area, easily recognizable in the images with Hemacolor staining. N, nucleus; Nc, nucleolus; eVF, early viral factory; mVF, mature viral factory.

FIG 6

Morphogenesis of pandoravirus particles. Transmission electron microscopy images show stages of pandoravirus particle formation. (A to C) Crescent-like structures with different sizes, inside the viral factory, growing in thickness and complexity. (D to F) Particles being formed from the ostiolo-like apex. (G to I) Particles being formed from the end opposite the ostiolo-like apex. We used representative images of P. tropicalis, P. pampulha, and P. kadiweu in this panel; all the described steps were observed for the three isolates.

FIG 7

Transmission electron microscopy images showing pandoravirus particles being packaged into exosomes. (A) The late steps in pandoravirus replication are marked by intense membrane trafficking in the cytoplasm of the amoebal host (highlighted in red). This event is easily observed around the viral factory where the viral morphogenesis occurs. (B to D) Then, at around h 6 to h 9 postinfection, these double-membrane layers start to surround isolated or grouped viral particles, suggesting the beginning of exocytosis.

FIG 8

Transmission electron microscopy images demonstrating sequential steps of pandoravirus particle exocytosis. The images demonstrate that in late stages of infection the particles of pandoravirus start being packaged inside vesicles (A and B), becoming closer to the cytoplasmic membrane of the host cell and being released to the external milieu (C).

FIG 9

Analysis of the time course of infection for the pandoravirus isolates in Acanthamoeba castellanii cultures. The course of infection of P. tropicalis, P. pampulha, and P. kadiweu was established. (A to C) First, we observed the kinetics for the diminishing of the number of amoebas during the replication cycles of P. pampulha (A), P. kadiweu (B), and P. tropicalis (C) analyzed by cell counting. (D to F) Then, the number of viral particles present in the supernatant of these infected cells was also observed for P. pampulha (D), P. kadiweu (E), and P. tropicalis (F), at different time points. After setting a time point at which we observed an increase of more than 1 log of virus particles in the supernatant but without observing cellular lysis, the amoebal cultures were treated with an inhibitor of membrane trafficking (brefeldin A). (G to I) The cells were then infected with P. pampulha (G), P. kadiweu (H), and P. tropicalis (I) to check the influence of brefeldin A in the viral release. The number of exocyted viral particles in supernatant was counted. DMSO, dimethyl sulfoxide.