Role of an FNIP Repeat Domain-Containing Protein Encoded by Megavirus Baoshan during Viral Infection.

FNIP repeat domain-containing protein (FNIP protein) is a little-studied atypical leucine-rich repeat domain-containing protein found in social amoebae and mimiviruses. Here, a recently reported mimivirus of lineage C, Megavirus baoshan, was analyzed for FNIP protein genes. A total of 82 FNIP protein genes were identified, each containing up to 26 copies of the FNIP repeat, and mostly having an F-box domain at the N terminus. Both nucleotide and amino acid sequences of FNIP repeat were highly conserved. Most of the FNIP protein genes clustered together tandemly in groups of two to 14 genes. Nearly all FNIP protein genes shared similar expression patterns and were expressed 4 to 9 h postinfection. A typical viral FNIP protein, Mb0983, was selected for functional analysis. Protein interactome analysis identified two small GTPases, Rap1B and Rab7A, that interacted with Mb0983 in cytoplasm. The overexpression of Mb0983 in Acanthamoeba castellanii accelerated the degradation of Rap1B and Rab7A during viral infection. Mb0983 also interacted with host SKP1 and cullin-1, which were conserved components of the SKP1-cullin-1-F-box protein (SCF)-type ubiquitin E3 ligase complex. Deletion of the F-box domain of Mb0983 not only abolished its interaction with SKP1 and cullin-1 but also returned the speed of Rap1B and Rab7A degradation to normal in infected A. castellanii. These results suggested that Mb0983 is a part of the SCF-type ubiquitin E3 ligase complex and plays a role in the degradation of Rap1B and Rab7A. They also implied that other viral F-box-containing FNIP proteins might have similar effects on various host proteins. IMPORTANCE Megavirus baoshan encodes 82 FNIP proteins, more than any other reported mimiviruses. Their genetic and transcriptional features suggest that they are important for virus infection and adaption. Since most mimiviral FNIP proteins have the F-box domain, they were predicted to be involved in protein ubiquitylation. FNIP protein Mb0983 interacted with host SKP1 and cullin-1 through the F-box domain, supporting the idea that it is a part of the SCF-type ubiquitin E3 ligase complex. The substrates of Mb0983 for degradation were identified as the host small GTPases Rap1B and Rab7A. Combining the facts of the presence of a large number of FNIP genes in megavirus genomes, the extremely high expression level of the viral ubiquitin gene, and the reported observation that 35% of megavirus-infected amoeba cells died without productive infection, it is likely that megavirus actively explores the host ubiquitin-proteasome pathway in infection and that viral FNIP proteins play roles in the process.

Kinetic Analysis of Acanthamoeba castellanii Infected with Giant Viruses Quantitatively Revealed Process of Morphological and Behavioral Changes in Host Cells.

Most virus-infected cells show morphological and behavioral changes, which are called cytopathic effects. Acanthamoeba castellanii, an abundant, free-living protozoan, serves as a laboratory host for some viruses of the phylum Nucleocytoviricota-the giant viruses. Many of these viruses cause cell rounding in the later stages of infection in the host cells. Here, we show the changes that lead to cell rounding in the host cells through time-lapse microscopy and image analysis. Time-lapse movies of A. castellanii cells infected with Mimivirus shirakomae, kyotovirus, medusavirus, or Pandoravirus japonicus were generated using a phase-contrast microscope. We updated our phase-contrast-based kinetic analysis algorithm for amoebae (PKA3) and used it to analyze these time-lapse movies. Image analysis revealed that the process leading to cell rounding varies among the giant viruses; for example, M. shirakomae infection did not cause changes for some time after the infection, kyotovirus infection caused an early decrease in the number of cells with typical morphologies, and medusavirus and P. japonicus infection frequently led to the formation of intercellular bridges and rotational behavior of host cells. These results suggest that in the case of giant viruses, the putative reactions of host cells against infection and the putative strategies of virus spread are diverse. IMPORTANCE Quantitative analysis of the infection process is important for a better understanding of viral infection strategies and virus-host interactions. Here, an image analysis of the phase-contrast time-lapse movies displayed quantitative differences in the process of cytopathic effects due to the four giant viruses in Acanthamoeba castellanii, which were previously unclear. It was revealed that medusavirus and Pandoravirus japonicus infection led to the formation of a significant number of elongated particles related to intercellular bridges, emphasizing the importance of research on the interaction of viruses with host cell nuclear function. Mimivirus shirakomae infection did not cause any changes in the host cells initially, so it is thought that the infected cells can actively move and spread over a wider area, emphasizing the importance of observation in a wider area and analysis of infection efficiency. These results suggest that a kinetic analysis using the phase-contrast-based kinetic analysis algorithm for amoebae (PKA3) reveals the infection strategies of each giant virus.

Structure and physiology of giant DNA viruses.

Although giant viruses have existed for millennia and possibly exerted great evolutionary influence in their environment. Their presence has only been noticed by virologists recently with the discovery of Acanthamoeba polyphaga mimivirus in 2003. Its virion with a diameter of 500 nm and its genome larger than 1 Mpb shattered preconceived standards of what a virus is and triggered world-wide prospection studies. Thanks to these investigations many giant virus families were discovered, each with its own morphological peculiarities and genomes ranging from 0.4 to 2.5 Mpb that possibly encode more than 400 viral proteins. This review aims to present the morphological diversity, the different aspects observed in host-virus interactions during replication, as well as the techniques utilized during their investigation.

A virophage cross-species infection through mutant selection represses giant virus propagation, promoting host cell survival.

Virus adaptation to new hosts is a major cause of infectious disease emergence. This mechanism has been intensively studied in the context of zoonotic virus spillover, due to its impact on global health. However, it remains unclear for virophages, parasites of giant viruses and potential regulators of microbial communities. Here, we present, for the first time to our knowledge, evidence of cross-species infection of a virophage. We demonstrated that challenging the native population of Guarani virophage with two previously unidentified giant viruses, previously nonpermissive to this virophage, allows the selection of a mutant genotype able to infect these giant viruses. We were able to characterize the potential genetic determinant (deletion) carried by the virophage with the expanded-host range. Our study also highlights the relevant biological impact of this host adaptation by demonstrating that coinfection with the mixture containing the mutant virophage abolishes giant virus production and rescues the host cell population from lysis.

Complex Membrane Remodeling during Virion Assembly of the 30,000-Year-Old Mollivirus Sibericum.

Cellular membranes ensure functional compartmentalization by dynamic fusion-fission remodeling and are often targeted by viruses during entry, replication, assembly, and egress. Nucleocytoplasmic large DNA viruses (NCLDVs) can recruit host-derived open membrane precursors to form their inner viral membrane. Using complementary three-dimensional (3D)-electron microscopy techniques, including focused-ion beam scanning electron microscopy and electron tomography, we show that the giant Mollivirus sibericum utilizes the same strategy but also displays unique features. Indeed, assembly is specifically triggered by an open cisterna with a flat pole in its center and open curling ends that grow by recruitment of vesicles never reported for NCLDVs. These vesicles, abundant in the viral factory (VF), are initially closed but open once in close proximity to the open curling ends of the growing viral membrane. The flat pole appears to play a central role during the entire virus assembly process. While additional capsid layers are assembled from it, it also shapes the growing cisterna into immature crescent-like virions and is located opposite to the membrane elongation and closure sites, thereby providing virions with a polarity. In the VF, DNA-associated filaments are abundant, and DNA is packed within virions prior to particle closure. Altogether, our results highlight the complexity of the interaction between giant viruses and their host. Mollivirus assembly relies on the general strategy of vesicle recruitment, opening, and shaping by capsid layers similar to all NCLDVs studied until now. However, the specific features of its assembly suggest that the molecular mechanisms for cellular membrane remodeling and persistence are unique.IMPORTANCE Since the first giant virus Mimivirus was identified, other giant representatives are isolated regularly around the world and appear to be unique in several aspects. They belong to at least four viral families, and the ways they interact with their hosts remain poorly understood. We focused on Mollivirus sibericum, the sole representative of "Molliviridae," which was isolated from a 30,000-year-old permafrost sample and exhibits spherical virions of complex composition. In particular, we show that (i) assembly is initiated by a unique structure containing a flat pole positioned at the center of an open cisterna, (ii) core packing involves another cisterna-like element seemingly pushing core proteins into particles being assembled, and (iii) specific filamentous structures contain the viral genome before packaging. Altogether, our findings increase our understanding of how complex giant viruses interact with their host and provide the foundation for future studies to elucidate the molecular mechanisms of Mollivirus assembly.

Filling Knowledge Gaps for Mimivirus Entry, Uncoating, and Morphogenesis.

Since the discovery of mimivirus, its unusual structural and genomic features have raised great interest in the study of its biology; however, many aspects concerning its replication cycle remain uncertain. In this study, extensive analyses of electron microscope images, as well as biological assay results, shed light on unclear points concerning the mimivirus replication cycle. We found that treatment with cytochalasin, a phagocytosis inhibitor, negatively impacted the incorporation of mimivirus particles by Acanthamoeba castellanii, causing a negative effect on viral growth in amoeba monolayers. Treatment of amoebas with bafilomicin significantly impacted mimivirus uncoating and replication. In conjunction with microscopic analyses, these data suggest that mimiviruses indeed depend on phagocytosis for entry into amoebas, and particle uncoating (and stargate opening) appears to be dependent on phagosome acidification. In-depth analyses of particle morphogenesis suggest that the mimivirus capsids are assembled from growing lamellar structures. Despite proposals from previous studies that genome acquisition occurs before the acquisition of fibrils, our results clearly demonstrate that the genome and fibrils can be acquired simultaneously. Our data suggest the existence of a specific area surrounding the core of the viral factory where particles acquire the surface fibrils. Furthermore, we reinforce the concept that defective particles can be formed even in the absence of virophages. Our work provides new information about unexplored steps in the life cycle of mimivirus.IMPORTANCE Investigating the viral life cycle is essential to a better understanding of virus biology. The combination of biological assays and microscopic images allows a clear view of the biological features of viruses. Since the discovery of mimivirus, many studies have been conducted to characterize its replication cycle, but many knowledge gaps remain to be filled. In this study, we conducted a new examination of the replication cycle of mimivirus and provide new evidence concerning some stages of the cycle which were previously unclear, mainly entry, uncoating, and morphogenesis. Furthermore, we demonstrate that atypical virion morphologies can occur even in the absence of virophages. Our results, along with previous data, allow us to present an ultimate model for the mimivirus replication cycle.

Cedratvirus, a Double-Cork Structured Giant Virus, is a Distant Relative of Pithoviruses.

Most viruses are known for the ability to cause symptomatic diseases in humans and other animals. The discovery of Acanthamoeba polyphaga mimivirus and other giant amoebal viruses revealed a considerable and previously unknown area of uncharacterized viral particles. Giant viruses have been isolated from various environmental samples collected from very distant geographic places, revealing a ubiquitous distribution. Their morphological and genomic features are fundamental elements for classifying them. Herein, we report the isolation and draft genome of Cedratvirus, a new amoebal giant virus isolated in Acanthamoeba castellanii, from an Algerian environmental sample. The viral particles are ovoid-shaped, resembling Pithovirus sibericum, but differing notably in the presence of two corks at each extremity of the virion. The draft genome of Cedratvirus-589,068 base pairs in length-is a close relative of the two previously described pithoviruses, sharing 104 and 113 genes with P. sibericum and Pithovirus massiliensis genomes, respectively. Interestingly, analysis of these viruses' core genome reveals that only 21% of Cedratvirus genes are involved in best reciprocal hits with the two pithoviruses. Phylogeny reconstructions and comparative genomics indicate that Cedratvirus is most closely related to pithoviruses, and questions their membership in an enlarged putative Pithoviridae family.

Identification of an L-rhamnose synthetic pathway in two nucleocytoplasmic large DNA viruses.

Nucleocytoplasmic large DNA viruses (NCLDVs) are characterized by large genomes that often encode proteins not commonly found in viruses. Two species in this group are Acanthocystis turfacea chlorella virus 1 (ATCV-1) (family Phycodnaviridae, genus Chlorovirus) and Acanthamoeba polyphaga mimivirus (family Mimiviridae), commonly known as mimivirus. ATCV-1 and other chlorovirus members encode enzymes involved in the synthesis and glycosylation of their structural proteins. In this study, we identified and characterized three enzymes responsible for the synthesis of the sugar L-rhamnose: two UDP-D-glucose 4,6-dehydratases (UGDs) encoded by ATCV-1 and mimivirus and a bifunctional UDP-4-keto-6-deoxy-D-glucose epimerase/reductase (UGER) from mimivirus. Phylogenetic analysis indicated that ATCV-1 probably acquired its UGD gene via a recent horizontal gene transfer (HGT) from a green algal host, while an earlier HGT event involving the complete pathway (UGD and UGER) probably occurred between a protozoan ancestor and mimivirus. While ATCV-1 lacks an epimerase/reductase gene, its Chlorella host may encode this enzyme. Both UGDs and UGER are expressed as late genes, which is consistent with their role in posttranslational modification of capsid proteins. The data in this study provide additional support for the hypothesis that chloroviruses, and maybe mimivirus, encode most, if not all, of the glycosylation machinery involved in the synthesis of specific glycan structures essential for virus replication and infection.

Acanthamoeba castellanii promotion of in vitro survival and transmission of coxsackie b3 viruses.

This work was undertaken to determine whether Acanthamoeba could play a role in the survival and transmission of coxsackieviruses and focused on in vitro interactions between Acanthamoeba castellanii and coxsackie B3 viruses (CVB-3). Residual virus titer evaluations and immunofluorescence experiments revealed a remarkable CVB-3 adsorption on amoeba surfaces and accumulation inside cells. The survival of viruses was independent of the dynamics of amoeba replication and encystment. In addition, our results indicated that virus-infected amoebas can release infectious viruses during interaction with human macrophages. On the basis of these data, Acanthamoeba appears to be a potential promoter of the survival of coxsackieviruses and their transmission to human hosts.