Background
Giant viruses belong to a complex group of viruses commonly referred to as nucleocytoplasmic large DNA viruses (NCLDVs). The group gained notoriety after the isolation of Acanthamoeba polyphaga mimivirus (APMV), which has large particles (~ 750 nm) capable of infecting amoebae of the genus
Acanthamoeba [
1]. The study and search for new giant viruses has been intensified, and these viruses were uncovered in different samples and environments, considerably expanding our knowledge about their diversity and ubiquity [
2,
3]. Most giant viruses such as Mimivirus, Marseillevirus, Pandoravirus and Cedratvirus are associated with free-living amoebae of the genus
Acanthamoeba; however, other giant viruses have been isolated in
Vermamoeba vermiformis, such as Faustovirus and Kaumoebavirus [
4,
5].
In 2018, a new virus, Orpheovirus IHUMI-LCC2 (hence forward called Orpheovirus), was described that is capable of infecting
V. vermiformis, which was isolated from rat faecal samples collected in France. These viruses have ovoid-shaped particles, as observed for Cedratvirus, Pandoravirus, and Pithovirus, ranging from 900 to 1100 nm in length and approximately 500 nm in diameter. These viruses are also marked by the presence of a circular double-stranded DNA genome of 1,473,573 bp, encoding 1512 predicted genes of which 57.5% are ORFans [
6]. Regarding the predicted genes, it was observed that the best hits were with Pithovirus sibericum, Pithovirus massiliensis and Cedratvirus A11. Furthermore, pan-genomic analysis and phylogenetic reconstructions based on different core genes demonstrated a distant relationship between Orpheovirus and pithoviruses and cedratviruses [
6].
Due to its large size, it was proposed that the replication cycle of Orpheovirus was initiated by phagocytosis of viral particles by
V. vermiformis cells. After particle entry, the genome would be released into the cell cytoplasm through an ostiole located at the apex of the virion. An eclipse phase is established, and then viral factories (VFs) are formed, where new viral particles are assembled. In the final steps of the cycle, the cell cytoplasm is completely filled by new synthesized particles, which are released from the host cell by lysis [
6]. Despite the information described in the first proposed model, many steps of the replication cycle and particles of this virus still need to be elucidated.
In the present work, we present an in-depth investigation of the steps of the replication cycle of Orpheovirus. Our data revealed that Orpheovirus induces profound changes in the morphology of V. vermiformis, including increased cell motility at some time points of infection. We also provide details about virus entry, VFs formation, organelle recruitment and particle morphogenesis. We also observe that viral particles are released from infected cells both by exocytosis and cell lysis. The data presented here reveal several peculiar characteristics of the life cycle, structure and host interaction of this new giant virus.
Methods
Cell culture, viral production, purification and titration
V. vermiformis (ATCC CDC19) were cultivated in Peptone Yeast Extract Glucose (PYG) medium supplemented with 0.14 mg/mL penicillin (Sigma-Aldrich, USA), 50 mg/mL gentamicin (Thermo Fisher Scientific, USA), and 2.5 mg/mL amphotericin (Bristol-Myers Squibb, New York, USA) at 32 °C. For Orpheovirus production and purification, ten T175 cm
2 flasks (Thermo Fisher Scientific, USA) containing 20 × 10
6 cells in PYG medium were infected with Orpheovirus at a multiplicity of infection (M.O.I.) of 0.01 and incubated for 4 days at 32 °C. The lysate was centrifuged at 1200 x
g to remove cell debris. Then, the supernatant was collected, added over a 40% sucrose (Merck, Germany) cushion and centrifuged at 36,000 x
g for 1 h. The pellet was re-suspended in PBS and stored at − 20 °C. Three aliquots of the virus stock were titrated to the 50% end-point and calculated by the Reed-Muench method [
7,
8].
Cytopathic effect, one-step growth curve assays and particle counts
To investigate the cytopathic effect (CPE) of Orpheovirus in V. vermiformis cells by optical microscopy, 25 cm2 cell culture flasks containing 3 × 106 V. vermiformis cells were infected with Orpheovirus at an M.O.I. of 10, incubated at 32 °C and observed at different hours post infection (h.p.i) (1, 3, 6, 9, 12 and 24 h.p.i) for 24 h. Uninfected V. vermiformis cells (control) were also observed. A one-step growth curve was constructed using 25 cm2 flasks in duplicate at an M.O.I of 10. At different time points (1, 3, 6, 9, 12, 24, 48 and 72 h.p.i), the infected V. vermiformis cells and supernatants were collected, titred and calculated using the end point method. We also performed a quantitative polymerase chain reaction (qPCR) assay to quantify the viral genome load targeting the DNA polymerase gene, using oligonucleotide primer sequences Forward 5′- ATGGCGAAATATGCGGAAGGG-3′ and Reverse 5′-TCTTGTGCTCCTAACGCACC-3′. The thermal cycling conditions used were: one cycle at 95 °C for 10 min and 40 cycles at 95 °C for 10 s and 60 °C for 40 s; a melting curve analysis at 95 °C for 15 s, 58 °C for 15 s and a final cycle at 95 °C for 15 s was completed.
To investigate if the particles were released from the host cell by exocytosis, 3 × 106 V. vermiformis cells were infected with Orpheovirus at an M.O.I. of 5 and analyses were carried out at the infection times of 3, 6, 9, 12 and 24 h.p.i. Thirty minutes after infection, the monolayer of cells was washed once with PBS and the flasks were filled with 4 mL of PYG medium. After each time point, we separated 12 μL of the supernatant to count the number of released Orpheovirus particles during infection. The particles were observed by light microscopy (OlympusBX41, Japan) under 1000x magnification using a cell counting chamber (Kcell Olen Kasvi, Brazil).
Transmission and scanning electron microscopy
For the transmission electron microscopy (TEM) assays, the cells were infected at an M.O.I. of 0.01, and when the CPE was observed, we centrifuged the flask content for 10 min at 800 x g. The pellet was washed twice with 0.1 M sodium phosphate buffer (pH 7.4) and fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer for at least 1 h at room temperature. The pellet was then washed twice with 0.1 M sodium phosphate buffer and suspended in the same solution. After, the amoebae were embedded in EPON resin using a standard method, as follows: 2 h of fixation in 2% osmium tetroxide, five washes in distilled water, overnight incubation in uranyl acetate 2% at 2–8 °C, two washes in distilled water, 10 min dehydration in increasing ethanol concentrations (35, 50, 70, 85, 95 and 100%), 20 min incubation in acetone and embedding in EPON resin. Ultrathin sections were subsequently analysed under TEM (Spirit Biotwin FEI-120 kV) at the Center of Microscopy of UFMG, Brazil.
For the scanning electron microscopy (SEM) assays, the cells infected at an M.O.I. of 0.01 were added to round glass blades covered by poly-L-lysine and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 1 h at room temperature. Samples were then washed three times with 0.1 M cacodylate buffer and post-fixed with 1.0% osmium tetroxide for 1 h at room temperature. After a second fixation, the samples were washed three times with 0.1 M cacodylate buffer and immersed in 0.1% tannic acid for 20 min. Samples were then washed in cacodylate buffer and dehydrated by serial passages in ethanol solutions with concentrations ranging from 35 to 100%. They were dried at the critical CO2 point, transferred onto stubs and metalized with a 5 nm gold layer. The analyses were completed with SEM (FEG Quanta 200 FEI) at the Center of Microscopy of UFMG, Brazil.
Immunofluorescence assays
For immunofluorescence (IF) analysis, 3 × 105 V. vermiformis cells were added to round glass blades and after incubation during 24 h at 32 °C, the cells were infected at an M.O.I. of 5. After 1, 3, 6, 8, 12 and 24 h.p.i., we fixed with acetone for 10 min at − 20 °C. After fixation, cells were stained with polyclonal anti-orpheovirus whole particle antibodies produced in mouse (1:400 diluted in PBS) for 1 h at 37 °C, followed by three rinses with PBS. Cells were then incubated with 3% bovine serum albumin (BSA)-PAS for 30 min, followed by three rinses with PBS. After a 1 h of incubation with anti-mouse secondary antibodies (1:400 diluted in PBS), followed by three rinses with PBS, the cells were also incubated with DAPI Sigma (1:1000 diluted in PBS) for 1 h at room temperature. After three rinses with PBS, the cells were incubated with rodamine-phalloidine (Invitrogen) (1:1000 diluted in PBS) for 1 h at room temperature. Uninfected cells (control) were also fixed and prepared as described above. Fluorescently labelled cells were observed using an Axio Imager Z2-Apotome 2 microscope (Zeiss). The Zen Lite software from Zeiss microscopy was used for image processing at the Center of Microscopy of UFMG, Brazil.
Membrane inhibitor assays
To evaluate the role of cell membranes in the viral replication cycle, 14 × 105 V. vermiformis cells were infected with Orpheovirus at an M.O.I. of 5. Thirty minutes post-infection, the amoebae were washed with PAS and then we added 15 mL of PYG medium maintained at 32 °C. After 1 h, Brefeldin A (BFA), an inhibitor of membrane traffic, was added at a final concentration of 10 μM, and at 8 h post-infection, the amoebae were collected for TEM analysis. Samples were prepared for microscopy as previously described.
Discussion
Giant viruses have notable differences in their replication cycles and structures of their virions. After the discovery of this group of viruses, studies have been conducted to increase the knowledge of their biology and host interaction through investigations related to the life cycle of these organisms [
11,
16,
17]. This study presents an in-depth description of the stages of the Orpheovirus life cycle, providing information about the entry, morphogenesis, release, structural characteristics and CPE triggered by this virus in its host cells. Other giant viruses are capable of inducing morphological changes in
V. vermiformis, triggering amoebae aggregates called bunches, as described for Tupanvirus [
10], and formation of plaque forming units evidenced for Faustovirus [
9], as well as rounding and cell lysis. The morphological changes triggered by the Orpheovirus are different from those previously described, presenting cells with fusiform shapes and some branched cells. It is known that most giant viruses trigger cell rounding, which prevents the formation of pseudopods, and consequently decrease the motility of amoebae. Interestingly, in addition to morphological changes, we observed an increase in
V. vermiformis cell motility never described before. Although speculative, one hypothesis is that this increased motility would be a strategy that would facilitate the infection of neighbouring cells, leading to an increased spread of viral particles.
The strategy of phagocytosis entry has often been described for other giant viruses, demonstrated through biological assays with pharmacological inhibitors for APMV and Cedratvirus getuliensis, which showed a considerable decrease in viral titres [
11,
12]. Considering the fact that Orpheovirus particles are large (900–1100 nm), it was previously proposed that particle penetration would occur by phagocytosis [
6]. After observing several SEM images, we noticed the formation of pseudopods in contact with Orpheovirus particles at the surface of host cells. Due to this, we reinforce the hypothesis that phagocytosis would be exploited as an entry strategy, although other entry mechanisms should not be discarded. Once viruses enter cells, the internal particle content is released into the cell cytoplasm through an ostiole located in its apical region. This mechanism is also described for other giant viruses that have particles with similar morphology and are phylogenetically related to Orpheovirus [
12,
14].
Unlike VFs described for mimiviruses, which present well-defined zones [
11], VFs visualized during Orpheovirus infections are large electron-lucent areas located in perinuclear regions, where the morphogenesis of new particles occurs. Different than that previously observed for pandoraviruses, the nucleus remains during viral infection [
3]. In addition, mitochondrial recruitment was observed in peripheral regions of VFs. Membrane recruitment was also observed inside VFs and is suggested to be important for the morphogenesis and vesicle formation phases for further particle exocytosis. Nevertheless, the origin of the recruited membranes needs to be investigated. Marseillevirus vesicles have recently been shown to consist of endoplasmic reticulum membranes, and after treatment with inhibitors, there was a decrease in formation of new particles and vesicles [
17]. Also for Orpheovirus, we observe that the treatment with brefeldin impacts VF formation and morphogenesis. The morphogenesis of Orpheovirus particles occurs within the VFs, beginning with the formation of electron-dense semicircular structures called crescents. The formation of these structures is described for other giant amoeba viruses as well as other viruses belonging to the NCLDV group, such as Poxvirus [
18]. These crescents become thicker, and throughout the cycle, they are filled with the inner particle content and become more electron-dense until completely closed.
Although Orpheovirus particles present an ovoid-shape, similar to pandoraviruses, cedratviruses, and pithoviruses, they have a distinct structure. They have a fibril layer that is smaller compared to other giant viruses, such as mimiviruses, that have fibrils, and despite scarce information about their constitution, they may be related to host cell adhesion, as demonstrated for mimiviruses [
13]. Unlike Mimivirus, no specific area in which fibrils could be acquired was observed in VFs during Orpheovirus infection, and thus we hypothesized that they would be acquired within VFs [
11].
Moreover, it was observed that, in addition to fibrils, the particles have an outer layer to the capsid layer and an inner membrane surrounding the core. The presence of one or more particles involved by membranes is described for Cedratvirus getiliensis and Pithovirus sibericum, suggesting that they would be released from the host cell by exocytosis [
12,
14]. Although cell lysis has been demonstrated by SEM images and is described as the main strategy for viral progeny release, it is believed that exocytosis is an alternative mechanism used by Orpheovirus. A significant increase in particle counts of the supernatant was noted at 12 h.p.i., with no decrease in cell counts. In addition, by IF microscopy images throughout the cycle, we visualized an increase in the number of particles outside the cells at 12 h.p.i. and 24 h.p.i., respectively.
One-step growth assays showed an increase in viral titres at 12 h.p.i. and increased viral genome amplification by qPCR at 9 h.p.i., reaching a plateau at 48 h.p.i. by both qPCR analysis and titration. These data corroborate those previously proposed [
6], which showed a replication cycle duration of approximately 30 h. Other giant viruses, such as Tupanvirus, Kaumoebavirus and Faustovirus, capable of infecting
V. vermiformis have also been shown to have a longer duration multiplication cycle [
4,
5,
10] compared to giant viruses associated with amoebae of the genus
Acanthamoeba. This suggests that the replication cycle duration profile of these viruses might be related to their host. Altogether, this work brings valuable information concerning the replication cycle, structure and CPE triggered by Orpheovirus, therefore contributing to enhance our understanding about this new giant virus. Future studies applying alternative methods will help to fill some gaps, especially concerning the virus-host interaction.
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