The Endocytic Recycling Compartment Serves as a Viral Factory for Hepatitis E Virus

Background & Aims: Although Hepatitis E virus (HEV) is the major leading cause of enterically transmitted viral hepatitis worldwide, many gaps remain in the understanding of the HEV lifecycle. Notably, viral factories induced by HEV have not been documented yet and it is currently unknown whether HEV infection leads to cellular membrane modelling as many positive-strand RNA viruses. HEV genome encodes three proteins, the ORF1 replicase, the ORF2 capsid protein and the ORF3 protein involved in virion egress. Previously, we demonstrated that HEV produces different ORF2 isoforms including the virion-associated ORF2i form. Here, we aimed to probe infectious particles and viral factories in HEV-producing cells, using antibodies directed against the different ORF2 isoforms. Methods: We generated monoclonal antibodies that specifically recognize the particle-associated ORF2i form, and antibodies that recognize the different ORF2 isoforms. We used them in confocal and electron microscopy approaches to probe viral factories in HEV-producing cells. We performed an extensive colocalization study of viral proteins with subcellular markers. We analyzed the impact of silencing Rab11, a central player of the endocytic recycling compartment (ERC). Results: One of the antibodies, named P1H1 and targeting the N-terminus of ORF2i, recognized delipidated HEV particles. Confocal and ultrastructural microscopy analyses of HEV-producing cells revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. These subcellular structures were enriched in ORF2 and ORF3 proteins, and were dependent on the ORF3 expression and ORF2i capsid protein assembly. Colocalization and silencing analyses revealed that these structures are derived from the ERC. Conclusions: Our study reveals that HEV hijacks the ERC and forms a membrane network of vesicular and tubular structures that might be the hallmark of HEV infection.

Virophage: The hijacker of my hijacker is my friend

The discovery of virophage carries along the proof of existence of a new bio controlling agent in the entire biosystem. The virophage is a parasite to a giant virus and works by hijacking “the giant virus” viral factory, an essential machinery for the giant virus’s replication, leading to a sharp incline of the virophage viral load inside the host cell. Success of the host cell survival against the invading giant virus is shown by the decline of the destroyed cell during lytic stage after virophage co-infection to the giant virus. Virophage has a similar role to the bacteriophage but instead of targeting a bacterium, it targets specifically on virus. Hitherto, the existence of human-borne virophage and interactions of virophage to human microbiome remain elusive, thus future studies are required. This short review will highlight the discovery, types and recent known method of virophage replication. We also added some biological perspectives of the connections and interactions between the virophage and its host to exploit the virophage main role as a biocontrolling agent to pathogenic viruses that are potentially benevolent for human life.

Rotavirus viroplasm biogenesis involves microtubule-based dynein transport mediated by an interaction between NSP2 and dynein intermediate chain

Rotaviruses are the causative agents of severe and dehydrating gastroenteritis in children, piglets, and many other young animals. They replicate their genomes and assemble double-layered particles in cytoplasmic electron-dense inclusion bodies called ‘viroplasms’. The formation of viroplasms is reportedly associated with the stability of microtubules. Although material transport is an important function of microtubules, whether and how microtubule-based transport influences the formation of viroplasms is still unclear. Here, we demonstrate that the small viroplasms move and fuse in living cells. We show that microtubule-based dynein transport affects rotavirus infection, viroplasm formation, and the assembly of transient enveloped particles (TEPs) and triple-layered particles (TLPs). The dynein intermediate chain (DIC) is shown to localize in the viroplasm and to interact directly with non-structural protein 2 (NSP2), indicating that DIC is responsible for connecting the viroplasm to dynein. The WD40 repeat domain of DIC regulates the interaction between DIC and NSP2, and the knockdown of DIC inhibited rotaviral infection, viroplasm formation, and the assembly of TEPs and TLPs. Our findings show that rotavirus viroplasms hijack dynein transport for fusion events, required for maximal assembly of infectious viral progeny. This study provides novel insights into the intracellular transport of viroplasms, which is involved in their biogenesis. Importance Because the viroplasm is the viral factory for rotavirus replication, viroplasm formation undoubtedly determines the effective production of progeny rotavirus. Therefore, understanding the virus–host interactions involved in the biogenesis of the viroplasm is critical for the future development of prophylactic and therapeutic strategies. Previous studies have reported that the formation of viroplasms is associated with the stability of microtubules, whereas little is known about its specific mechanism. Here, we demonstrate that rotavirus viroplasm formation takes advantage of microtubule-based dynein transport mediated by an interaction between NSP2 and DIC. These findings provide new insight into the intracellular transport of viroplasms.

Quantitative conversion of biomass in giant DNA virus infection

Bioconversion of organic materials is the foundation of many applications in chemical engineering, microbiology and biochemistry. Herein, we introduce a new methodology to quantitatively determine conversion of biomass in viral infections while simultaneously imaging morphological changes of the host cell. As proof of concept, the viral replication of an unidentified giant DNA virus and the cellular response of an amoebal host are studied using soft X-ray microscopy, titration dilution measurements and thermal gravimetric analysis. We find that virions produced inside the cell are visible from 18 h post infection and their numbers increase gradually to a burst size of 280–660 virions. Due to the large size of the virion and its strong X-ray absorption contrast, we estimate that the burst size corresponds to a conversion of 6–12% of carbonaceous biomass from amoebal host to virus. The occurrence of virion production correlates with the appearance of a possible viral factory and morphological changes in the phagosomes and contractile vacuole complex of the amoeba, whereas the nucleus and nucleolus appear unaffected throughout most of the replication cycle.

In-depth analysis of the replication cycle of Orpheovirus

Background After the isolation of Acanthamoeba polyphaga mimivirus (APMV), the study and search for new giant viruses has been intensified. Most giant viruses are associated with free-living amoebae of the genus Acanthamoeba; however other giant viruses have been isolated in Vermamoeba vermiformis, such as Faustovirus, Kaumoebavirus and Orpheovirus. These studies have considerably expanded our knowledge about the diversity, structure, genomics, and evolution of giant viruses. Until now, there has been only one Orpheovirus isolate, and many aspects of its life cycle remain to be elucidated. Methods In this study, we performed an in-depth characterization of the replication cycle and particles of Orpheovirus by transmission and scanning electron microscopy, optical microscopy and IF assays. Results We observed, through optical and IF microscopy, morphological changes in V. vermiformis cells during Orpheovirus infection, as well as increased motility at 12 h post infection (h.p.i.). The viral factory formation and viral particle morphogenesis were analysed by transmission electron microscopy, revealing mitochondria and membrane recruitment into and around the electron-lucent viral factories. Membrane traffic inhibitor (Brefeldin A) negatively impacted particle morphogenesis. The first structure observed during particle morphogenesis was crescent-shaped bodies, which extend and are filled by the internal content until the formation of multi-layered mature particles. We also observed the formation of defective particles with different shapes and sizes. Virological assays revealed that viruses are released from the host by exocytosis at 12 h.p.i., which is associated with an increase of particle counts in the supernatant. Conclusions The results presented here contribute to a better understanding of the biology, structures and important steps in the replication cycle of Orpheovirus.

Evidence supporting a viral origin of the eukaryotic nucleus

The defining feature of the eukaryotic cell is the possession of a nucleus that uncouples transcription from translation. This uncoupling of transcription from translation depends on a complex process employing hundreds of eukaryotic specific genes acting in concert and requires the 7-methylguanylate (m7G) cap to prime eukaryotic mRNA for splicing, nuclear export, and cytoplasmic translation. The origin of this complex system is currently a paradox since it is not found or needed in prokaryotic cells which lack nuclei, yet it was apparently present and fully functional in the Last Eukaryotic Common Ancestor (LECA). According to the Viral Eukaryogenesis (VE) hypothesis the abrupt appearance of the nucleus in the eukaryotic lineage occurred because the nucleus descends from the viral factory of a DNA phage that infected the archaeal ancestor of the eukaryotes. Consequently, the system for uncoupling of transcription from translation in eukaryotes is predicted by the VE hypothesis to be viral in origin. In support of this hypothesis it is shown here that m7G capping apparatus that primes the uncoupling of transcription from translation in eukaryotes is present in viruses of the Mimiviridae but absent from bona-fide archaeal relatives of the eukaryotes such as Lokiarchaeota. Furthermore, phylogenetic analysis of the m7G capping pathway indicates that eukaryotic nuclei and Mimiviridae obtained this pathway from a common ancestral source that predated the origin of LECA. These results support the VE hypothesis and suggest the eukaryotic nucleus and the Mimiviridae descend from a common First Eukaryotic Nuclear Ancestor (FENA).

Evaluation of Viral Genome Copies Within Viral Factories on Different DNA Viruses

Summary This article describes a simple method of measuring the number of viral genomes within viral factories. For this purpose, we use three DNA viruses replicating in the cytoplasm of the infected cells: wild-type African swine fever virus (ASFV)-Georgia 2007, culture-adapted type ASFV-BA71V, and Vaccinia virus (VV). The measurements are conducted in three steps. In the first step, after DNA staining, we evaluate Integrated Optical Density (IOD) of total DNA for each viral factory. The second step involves the calculations of the mass of DNA in the viral factories in picograms (pg). And, in the third step, by dividing the mass of DNA within viral factory by the weight of a single viral genome, we obtain the number of viral genomes within the factory.

Filling Knowledge Gaps for Mimivirus Entry, Uncoating, and Morphogenesis

ABSTRACT 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.