Rotavirus viroplasms are cytosolic, electron-dense inclusions corresponding to the viral machinery of replication responsible for viral template transcription, dsRNA genome segments replication and assembly of new viral cores. We have previously observed that, over time, those viroplasms increase in size and decrease in number. Therefore, we hypothesized that this process was dependent on the cellular microtubular network and its associated dynamic components. Here, we present evidence demonstrating that viroplasms are dynamic structures, which, in the course of an ongoing infection, move towards the perinuclear region of the cell, where they fuse among each other, thereby gaining considerably in size and, simultaneouly, explaining the decrease in numbers. On the viral side, this process seems to depend on VP2 for movement and on NSP2 for fusion. On the cellular side, both the temporal transition and the maintenance of the viroplasms are dependent on the microtubular network, its stabilization by acetylation, and, surprisingly, on a kinesin motor of the kinesin-5 family, Eg5. Thus, we provide for the first time deeper insights into the dynamics of rotavirus replication, which can explain the behavior of viroplasms in the infected cell.

The protozoan parasite Giardia intestinalis belongs to one of the earliest diverged eukaryotic lineages. This is also reflected in a simple intracellular organization, as Giardia lacks common subcellular compartments such as mitochondria, peroxisomes, and apparently also a Golgi apparatus. During encystation, developmentally regulated formation of large secretory compartments containing cyst wall material occurs. Despite the lack of any morphological similarities, these encystation-specific vesicles (ESVs) show several biochemical characteristics of maturing Golgi cisternae. Previous studies suggested that Golgi structure and function are induced only during encystation in Giardia, giving rise to the hypothesis that ESVs, as a Giardia Golgi equivalent, are generated de novo. Alternatively, ESV compartments could be built on the template structure of a cryptic Golgi in trophozoites in response to ER export of cyst wall material during encystation. We addressed this question by defining the molecular framework of the Giardia secretory apparatus using a comparative genomic approach. Analysis of the corresponding transcriptome during growth and encystation revealed surprisingly little stage-specific regulation. A panel of antibodies was generated against selected marker proteins to investigate the developmental dynamics of the endomembrane system. We show evidence that Giardia accommodates the export of large amounts of cyst wall material through re-organization of membrane compartment(s) in trophozoites with biochemical similarities to ESVs. This suggests that ESVs are selectively stabilized Golgi-like compartments in a unique and archetypical secretory system, which arise from a structural template in trophozoites rather than being generated de novo.

Dynamins are universally conserved large guanosine triphosphatases, which function as mechanoenzymes in membrane scission. The primitive protozoan Giardia lamblia has a single dynamin-related protein (GlDRP) with an unusual domain structure. Giardia lacks a Golgi apparatus but generates transient Golgi-like delay compartments dubbed encystation-specific vesicles (ESVs), which serve to accumulate and mature cyst wall proteins during differentiation to infectious cyst forms. Here, we analyze the function of GlDRP during growth and encystation and demonstrate that it relocalizes from peripheral endosomal-lysosomal compartments to nascent ESVs. We show that GlDRP is necessary for secretion of the cyst wall material and ESV homeostasis. Expression of a dominantnegative GlDRP variant does not interfere with ESV formation but blocks cyst formation completely prior to regulated exocytosis. GlDRP colocalizes with clathrin at the cell periphery and is necessary for endocytosis of surface proteins to endosomal-lysosomal organelles in trophozoites. Electron microscopy and live cell imaging reveal gross morphological changes as well as functional impairment of the endocytic system in cells expressing the dominantnegative GlDRP. Thus, giardial DRP plays a key role in two distinct trafficking pathways and in organelle homeostasis, both essential functions for the proliferation of the parasite in the gut and its transmission to a new host.

The highly reduced protozoan parasite Giardia lamblia has minimal machinery for cellular processes such as protein trafficking. Giardia trophozoites maintain diverse and regulated secretory pathways but lack an identifiable Golgi complex. During differentiation to cysts, however, they produce specialized compartments termed encystation-specific vesicles (ESVs). ESVs are hypothesized to be unique developmentally regulated Golgi-like organelles dedicated to maturation and export of pre-sorted cyst wall proteins. Here we present a functional analysis of this unusual compartment by direct interference with the functions of the small GTPases Sar1, Rab1 and Arf1. Conditional expression of dominant-negative variants revealed an essential role of Sar1 in early events of organelle neogenesis, whilst inhibition of Arf1 uncoupled morphological changes and cell cycle progression from extracellular matrix export. The latter led to development of `naked cysts', which lacked water resistance and thus infectivity. Time-lapse microscopy and photobleaching experiments showed that putative Golgi-like cisternae in Giardia develop into a network capable of exchanging soluble cargo at a high rate via dynamic, tubular connections, presumably to synchronize maturation. The minimized and naturally pulsed trafficking machinery for export of the cyst wall biopolymer in Giardia is a simple model for investigating basic principles of neogenesis and maturation of Golgi compartments.

Transmission of the protozoan parasite Giardia intestinalis to vertebrate hosts presupposes the encapsulation of trophozoites into an environmentally resistant and infectious cyst form. We have previously shown that cyst wall proteins were faithfully sorted to large encystation-specific vesicles (ESVs), despite the absence of a recognizable Golgi apparatus. Here, we demonstrate that sorting to a second constitutively active pathway transporting variant-specific surface proteins (VSPs) to the surface depended on the cytoplasmic VSP tail. Moreover, pulsed endoplasmic reticulum (ER) export of chimeric reporters containing functional signals for both pathways showed that protein sorting was done at or very soon after export from the ER. Correspondingly, we found that a limited number of novel transitional ER-like structures together with small transport intermediates were generated during encystation. Colocalization of transitional ER regions and early ESVs with coat protein (COP) II and of maturing ESVs with COPI and clathrin strongly suggested that ESVs form by fusion of ER-derived vesicles and subsequently undergo maturation by retrograde transport. Together, the data supported the hypothesis that in Giardia, a primordial secretory apparatus is in operation by which proteins are sorted in the early secretory pathway, and the developmentally induced ESVs carry out at least some Golgi functions.

Herpesvirus envelopment is assumed to follow an uneconomical pathway including primary envelopment at the inner nuclear membrane, de-envelopment at the outer nuclear membrane, and reenvelopment at the trans-Golgi network. In contrast to the hypothesis of de-envelopment by fusion of the primary envelope with the outer nuclear membrane, virions were demonstrated to be transported from the perinuclear space to rough endoplasmic reticulum (RER) cisternae. Here we show by high-resolution microscopy that herpes simplex virus 1 envelopment follows two diverse pathways. First, nuclear envelopment includes budding of capsids at the inner nuclear membrane into the perinuclear space whereby tegument and a thick electron dense envelope are acquired. The substance responsible for the dense envelope is speculated to enable intraluminal transportation of virions via RER into Golgi cisternae. Within Golgi cisternae, virions are packaged into transport vacuoles containing one or several virions. Second, for cytoplasmic envelopment, capsids gain direct access from the nucleus to the cytoplasm via impaired nuclear pores. Cytoplasmic capsids could bud at the outer nuclear membrane, at membranes of RER, Golgi cisternae, and large vacuoles, and at banana-shaped membranous entities that were found to continue into Golgi membranes. Envelopes originating by budding at the outer nuclear membrane and RER membrane also acquire a dense substance. Budding at Golgi stacks, designated wrapping, results in single virions within small vacuoles that contain electron-dense substances between envelope and vacuolar membranes.Much controversy has arisen about the pathway of herpesvirus capsids from their nuclear origin to the site of their release into the extracellular space (e.g., see references 4, 13, 19, 50, 52, 53, 55, and 56). The current widely accepted view suggests the formation of primary virions comprising capsid, primary tegument, and a primary envelope that originates by budding at the inner nuclear membrane into the perinuclear space (37). For de-envelopment, the primary envelope is assumed to be inserted into the outer nuclear membrane by fusion, releasing capsid and the primary tegument into the cytoplasm (14). In contrast to de-envelopment, many investigations clearly demonstrate that "primary" virions are transported from the perinuclear space into rough endoplasmic reticulum (RER) cisternae (12,15,45,51,52,58,62) and that "primary" wild-type virions can accumulate within the perinuclear space-RER compartment (55). Intraluminal accumulations of virions have also been explained as a failure in deenvelopment, e.g., due to the lack of US3 protein in pseudorabies virus (25). The de-envelopment theory also does not consider that membrane fusion is a fast but well-studied process starting by close apposition of the membranes to allow fusion followed by pore formation (24,27,32,36). Recognizing close apposition and pore formation is imperative to distinguishing fusion from budding and fission. To our knowledge, pore formation between "prim...

The family Arenaviridae comprises three genera, Mammarenavirus, Reptarenavirus and the most recently added Hartmanivirus. Arenaviruses have a bisegmented genome with ambisense coding strategy. For mammarenaviruses and reptarenaviruses the L segment encodes the Z protein (ZP) and the RNA-dependent RNA polymerase, and the S segment encodes the glycoprotein precursor and the nucleoprotein. Herein we report the full length genome and characterization of Haartman Institute snake virus-1 (HISV-1), the putative type species of hartmaniviruses. The L segment of HISV-1 lacks an open-reading frame for ZP, and our analysis of purified HISV-1 particles by SDS-PAGE and electron microscopy further support the lack of ZP. Since we originally identified HISV-1 in co-infection with a reptarenavirus, one could hypothesize that co-infecting reptarenavirus provides the ZP to complement HISV-1. However, we observed that co-infection does not markedly affect the amount of hartmanivirus or reptarenavirus RNA released from infected cells in vitro, indicating that HISV-1 does not benefit from reptarenavirus ZP. Furthermore, we succeeded in generating a pure HISV-1 isolate showing the virus to replicate without ZP. Immunofluorescence and ultrastructural studies demonstrate that, unlike reptarenaviruses, HISV-1 does not produce the intracellular inclusion bodies typical for the reptarenavirus-induced boid inclusion body disease (BIBD). While we observed HISV-1 to be slightly cytopathic for cultured boid cells, the histological and immunohistological investigation of HISV-positive snakes showed no evidence of a pathological effect. The histological analyses also revealed that hartmaniviruses, unlike reptarenaviruses, have a limited tissue tropism. By nucleic acid sequencing, de novo genome assembly, and phylogenetic analyses we identified additional four hartmanivirus species. Finally, we screened 71 individuals from a collection of snakes with BIBD by RT-PCR and found 44 to carry hartmaniviruses. These findings suggest that harmaniviruses are common in captive snake populations, but their relevance and pathogenic potential needs yet to be revealed.

Herpesvirus capsids originating in the nucleus overcome the nucleocytoplasmic barrier by budding at the inner nuclear membrane. The fate of the resulting virions is still under debate. The fact that capsids approach Golgi membranes from the cytoplasmic side led to the theory of fusion between the viral envelope and the outer nuclear membrane, resulting in the release of capsids into the cytoplasm. We recently discovered a continuum from the perinuclear space to the Golgi complex implying (i) intracisternal viral transportation from the perinuclear space directly into Golgi cisternae and (ii) the existence of an alternative pathway of capsids from the nucleus to the cytoplasm. Here, we analyzed the nuclear surface by high-resolution microscopy. Confocal microscopy of MDBK cells infected with recombinant bovine herpesvirus 1 expressing green fluorescent protein fused to VP26 (a minor capsid protein) revealed distortions of the nuclear surface in the course of viral multiplication. High-resolution scanning and transmission electron microscopy proved the distortions to be related to enlargement of nuclear pores through which nuclear content including capsids protrudes into the cytoplasm, suggesting that capsids use impaired nuclear pores as gateways to gain access to the cytoplasmic matrix. Close examination of Golgi membranes, rough endoplasmic reticulum, and outer nuclear membrane yielded capsid-membrane interaction of high identity to the budding process at the inner nuclear membrane. These observations signify the ability of capsids to induce budding at any cell membrane, provided the fusion machinery is present and/or budding is not suppressed by viral proteins.Herpesviruses comprise the capsid containing the viral genome, the viral envelope consisting of a lipid bilayer with embedded glycoproteins, and tegument proteins filling the space between capsid and envelope. DNA double strands formed during replication are packed into capsids built of proteins imported from the cytoplasm (32). Capsids are transported to the nuclear periphery. Their pathway through the nucleocytoplasmic barrier and the acquisition of tegument and envelope are yet not fully understood (18). Capsids bud through the inner nuclear membrane into the perinuclear space, concomitantly acquiring an envelope (15) and tegument proteins (43). It is assumed that the envelope derived from budding at the inner nuclear membrane fuses with the outer nuclear membrane, releasing both tegument and capsid into the cytoplasmic matrix (4, 6, 7, 12, 14-16, 20, 42, 46). Capsids are then transported to the trans-Golgi network, where they are wrapped by Golgi membranes leading to an enveloped virion within a transport vacuole. Alternatively, it is speculated that virions escape from the perinuclear space via vesicle formation at the outer nuclear membrane (5,7,11,16,31,37). These vesicles then pass the Golgi complex for final maturation of virions. Contradictory to both the fusion and vesicle formation theory is the fact that fully enveloped virions were found in...