Inside the Lifestyle of the Virophage

Abstract

<i>Objective(s):</i> We sought to better characterize Sputnik, the first isolated virophage, and to analyze its parasitic lifestyle during co-infection with Marseillevirus (a new giant virus) in <i>Acanthamoeba castellanii</i>. <i>Methods:</i> A combination of electron microscopy, immunofluorescence microscopy, and real-time PCR was used to characterize the kinetics of the viral replication cycle. RT-PCR was performed to detect RNAs inside the Sputnik virions. <i>Results:</i> Sputnik is a new viral entity carrying an almost complete ready-to-use set of viral RNAs (20 out of 21). Sputnik does not replicate with Marseillevirus but delays its replication cycle. While Marseillevirus is successfully internalized by <i>A. castellanii</i> following co-infections with Mamavirus and Sputnik, it does not initiate a replication cycle. In contrast, both Marseillevirus and Mamavirus can replicate in the amoeba in case of co-infection, but the development of one is exclusive from the other inside a single amoeba cell. <i>Conclusions:</i> This work provides new insight into the Sputnik replication cycle with another giant virus and confirms that Sputnik is a virophage. It shows new dimensions of the interactions existing among giant viruses.

Mimivirus shows dramatic genome reduction after intraamoebal culture

“…Immunofluorescence performed 6 h p.i. confirmed these results and showed that the M1 viral factories produce sputnik virions (24). However, in M4, only the VF was visible, and no production of sputnik virions could be observed (Fig.…”

“…Sputnik is a small icosahedral virus that successfully parasitizes Mimiviridae (11). It was previously hypothesized that sputnik uses the Mimivirus fibers as a gateway to penetrate the amoeba (24). Another virophage (called sputnik 2) was recently found to be associated with a giant virus isolated from a liquid from a contact lens of a patient with keratitis (25).…”

“…Even though we cannot rule out that some of the genes deleted in M4 are required for intracellular replication of the virophage, we assume that the loss of fibers could also affect the association between mimiviruses and sputnik virophages. It is likely that the sputnik particles are phagocytosed by amoebae because of their attachment to the Mimivirus fibers (24). So far, all characterized isolates of Mimiviridae exhibited fibers in early cultures (25).…”

Structure of Sputnik, a virophage, at 3.5-Å resolution

“…The requirement of Mimivirus for the propagation of Sputnik gave rise to the term "virophage" (4). EM images suggest that Sputnik virus matures in viral factories together with Mimivirus itself (5). This observation is supported by in situ hybridization experiments (6), which showed that Sputnik replicates its genome in viral factories.…”

Virophages go nuclear in the marine alga Bigelowiella natans

“…Virophages appear to have evolved multiple strategies to track down their viral and cellular hosts. The Sputnik virophage can adhere to long fibers on the surface of the mimivirus capsid, and it is assumed that Sputnik hitches a ride when mimivirus is phagocytosed by the amoebal host cell (8). By contrast, the mavirus virophage enters the host cell independently of its giant virus CroV, which lacks an external fiber coat (9).…”