Acanthamoeba polyphaga Mimivirus Prevents Amoebal Encystment-Mediating Serine Proteinase Expression and Circumvents Cell Encystment

Abstract

Acanthamoeba is a genus of free-living amoebas distributed worldwide. Few studies have explored the interactions between these protozoa and their infecting giant virus, Acanthamoeba polyphaga mimivirus (APMV). Here we show that, once the amoebal encystment is triggered, trophozoites become significantly resistant to APMV. Otherwise, upon infection, APMV is able to interfere with the expression of a serine proteinase related to amoebal encystment and the encystment can no longer be triggered. Acanthamoeba is a genus of free-living amoebas found in a variety of environments and distributed worldwide (1-3). The life cycle of the amoebas involves two cellular forms: one that is metabolically active, known as the trophozoite form, and a dormant form, called the cyst, that is also responsible for promoting resistance in adverse environments (3). In natural environments, members of Acanthamoeba spp. are hosts and reservoirs for many microorganisms, including pathogenic bacteria and yeasts (4-7). Recently, the study of members of the Acanthamoeba genus has gained increased attention since the description of them as natural hosts for viruses of the Mimiviridae family, which are among the largest and most complex viruses described to date (8). Despite the recent interest in both organisms, there are few studies focusing on the interactions between Acanthamoeba spp. and the infecting mimivirus (9, 10). Thus, the objective of this study was to analyze the interactions between Acanthamoeba spp. and Acanthamoeba polyphaga mimivirus (APMV) in response to encystment stimulation that may commonly happen in the natural environment.First, we performed a one-step growth curve analysis to compare the levels of replication of APMV at the amoebal trophozoite and cyst stages. Acanthamoeba castellanii cells (ATCC 30234) were seeded on 24-well microplates (Corning Incorporated, Corning, NY) in phosphate-amoeba saline (PAS) at 10 5 cells per well. The cells (cysts or trophozoites) were then infected with APMV at a multiplicity of infection (MOI) of 10, collected at different time points, and titrated as described before (8). All experiments were performed three times in triplicate. APMV was unable to infect cysts of A. castellanii, given that final viral titers remained close to the initial inoculum titer, revealing no viral replication (Fig. 1A). On the other hand, when trophozoites were infected, the viral progeny titer increased about 2.5 logs (500-fold) 24 h postinfection and evident cytopathic effect was observed (Fig. 1A). These results were confirmed by electron microscopy (12 h postinfection). APMV morphogenesis, including the presence of mature virions, was observed in trophozoites (Fig. 1B), while no virions could be seen inside the cysts (Fig. 1C).Next, we investigated if the stimulation of encystment affects APMV infection of amoebas. Amoebal trophozoites were infected with APMV after being incubated in Neff saline solution (Neff) to trigger encystment. Trophozoites were transferred to 24-well microplates, at 10 5 cells per we...

Faustovirus-Like Asfarvirus in Hematophagous Biting Midges and Their Vertebrate Hosts

“…Similar results were obtained for the whole proteomic analysis, with slight differences in the proportions of identified peptides, except that four and seven peptides were obtained for Kroon virus and Courdo11 Mimiviridae proteins, respectively (Yoosuf et al, 2014 ; Boratto et al, 2015 ). In addition, Mimiviridae -related sequences were obtained in the metagenomes (data not shown).…”

Trapping the Enemy: Vermamoeba vermiformis Circumvents Faustovirus Mariensis Dissemination by Enclosing Viral Progeny inside Cysts

“…It has been also demonstrated that haploid cells (but not diploid cells) of Emiliania huxleyi, a free-living marine protist, are refractory to Emiliania huxleyi virus 86 infection in an evasion mechanism known as Cheshire Cat (8). Analogously, our team described the same phenomenon for free-living amoebas belonging to the genus Acanthamoeba, in which the cysts, but not the trophozoitic forms, are resistant to mimivirus infection (9,10). We have demonstrated that, once infected by mimivirus, Acanthamoeba trophozoites are no longer able to encyst because mimivirus blocks the expression of a serine proteinase gene, a canonical element involved in the encystment process (10).…”

“…Analogously, our team described the same phenomenon for free-living amoebas belonging to the genus Acanthamoeba, in which the cysts, but not the trophozoitic forms, are resistant to mimivirus infection (9,10). We have demonstrated that, once infected by mimivirus, Acanthamoeba trophozoites are no longer able to encyst because mimivirus blocks the expression of a serine proteinase gene, a canonical element involved in the encystment process (10). Although it has been described that Acanthamoeba trophozoites are able to encyst in the presence of some intracellular bacteria (11), this phenomenon has never been described during amoebal virus infections.…”

“…Faustovirus mariensis is unable to circumvent V. vermiformis encystment. Our team has previously shown that Mimivirus was capable of inhibiting the process of encystment in Acanthamoeba castellanii by downregulating the expression of a cellular serine protease gene, which is an essential enzyme responsible for triggering the encystment pathway in these cells (10). Once infected by Mimivirus, a given trophozoite of A. castellanii inevitably suffers from lysis due to viral replication, since the virus blocks encystment.…”

Persistent virus and addiction modules: an engine of symbiosis

“…This clearly resembles a viral cyst and identifies a viral life strategy in which persistence in the environment may be crucial. Interestingly, Mimivirus not only superimposes a new genetic program for producing this stable 'cyst-like' virus, it also prevent the cyst production of its host amoeba [74]. In a sense, the virus has displaced the host program with a new viral programming for a cryptic state of viral life.…”