Formation of antiviral cytoplasmic granules during orthopoxvirus infection

Vaccinia virus (VV) mutants lacking the double-stranded RNA (dsRNA)-binding E3L protein (ΔE3L mutant VV) show restricted replication in most cell types, as dsRNA produced by VV activates protein kinase R (PKR), leading to eIF2α phosphorylation and impaired translation initiation. Here we show that cells infected with ΔE3L mutant VV assemble cytoplasmic granular structures which surround the VV replication factories at an early stage of the nonproductive infection. These structures contain the stress granule-associated proteins G3BP, TIA-1, and USP10, as well as poly(A)-containing RNA. These structures lack large ribosomal subunit proteins, suggesting that they are translationally inactive. Formation of these punctate structures correlates with restricted replication, as they occur in >80% of cells infected with ΔE3L mutant VV but in only 10% of cells infected with wild-type VV. We therefore refer to these structures as antiviral granules (AVGs). Formation of AVGs requires PKR and phosphorylated eIF2α, as mouse embryonic fibroblasts (MEFs) lacking PKR displayed reduced granule formation and MEFs lacking phosphorylatable eIF2α showed no granule formation. In both cases, these decreased levels of AVG formation correlated with increased ΔE3L mutant VV replication. Surprisingly, MEFs lacking the AVG component protein TIA-1 supported increased replication of ΔE3L mutant VV, despite increased eIF2α phosphorylation and the assembly of AVGs that lacked TIA-1. These data indicate that the effective PKR-mediated restriction of ΔE3L mutant VV replication requires AVG formation subsequent to eIF2α phosphorylation. This is a novel finding that supports the hypothesis that the formation of subcellular protein aggregates is an important component of the successful cellular antiviral response.     

TRIM5 alpha cytoplasmic bodies are highly dynamic structures

Tripartite motif (TRIM)5 alpha has recently been identified as a host restriction factor that has the ability to block infection by certain retroviruses in a species-dependent manner. One interesting feature of this protein is that it is localized in distinct cytoplasmic clusters designated as cytoplasmic bodies. The potential role of these cytoplasmic bodies in TRIM5 alpha function remains to be defined. By using fluorescent fusion proteins and live cell microscopy, we studied the localization and dynamics of TRIM5 alpha cytoplasmic bodies. This analysis reveals that cytoplasmic bodies are highly mobile, exhibiting both short saltatory movements and unidirectional long-distance movements along the microtubule network. The morphology of the cytoplasmic bodies is also dynamic. Finally, photobleaching and photoactivation analysis reveals that the TRIM5 alpha protein present in the cytoplasmic bodies is very dynamic, rapidly exchanging between cytoplasmic bodies and a more diffuse cytoplasmic population. Therefore, TRIM5 alpha cytoplasmic bodies are dynamic structures more consistent with a role in function or regulation rather than protein aggregates or inclusion bodies that represent dead-end static structures.     

Dynamic movements of Ro52 cytoplasmic bodies along microtubules

The RING-finger protein Ro52/TRIM21 is known as an autoantigen and is recognized by anti-Ro/SSA antibodies, which are commonly found in patients with Sjögren’s syndrome and systemic lupus erythematosus. Recently, Ro52 has been shown to localize to distinct structures called cytoplasmic bodies and function as an E3 ubiquitin ligase. However, the Ro52 cytoplasmic bodies have not been well characterized. In this study, we investigated the Ro52 cytoplasmic bodies using fluorescence microscopy. This analysis revealed that the Ro52 cytoplasmic bodies are diffusely located in the cytoplasm and exist independently of TRIM5α cytoplasmic bodies. Our results further showed that the Ro52 cytoplasmic bodies are not stained with MitoTracker dye and are not colocalized with the proteasome subunit Rpt5, the caveolae component caveolin-1, the endosome markers (EEA1, Rab5, and Rab7), and the lysosome marker LAMP2. These results indicate that the Ro52 cytoplasmic bodies are not mitochondria, proteasome-enriched structures, caveolae, endosomes, or lysosomes. Importantly, the Ro52 cytoplasmic bodies are highly motile and are located along the microtubule network. These results suggest that the Ro52 cytoplasmic bodies are unidentified structures that are transported along the microtubule network.     

Actin filaments connected with the microtubules of lipotubuloids, cytoplasmic domains rich in lipid bodies and microtubules

Summary. Lipotubuloids, i.e., cytoplasmic domains containing an agglomeration of lipid bodies surrounded by half-unit membrane, entwined and held together by a system of microtubules, have been found in the ovary epidermis of Ornithogalum umbellatum. Ultrastructural studies demonstrated thin filaments in lipotubuloids that are probably actin filaments arranged parallel to microtubules. It is suggested that interaction of actin filaments with the microtubules determines the driving force for the rotary motion characteristic of lipotubuloids, as this movement is sensitive to cytochalasin B. Correspondence: Department of Cytophysiology, University of Łódź, Pilarskiego 14, 90-231 Łódź, Poland.     

Virus particle packaging in baculovirus and cytoplasmic polyhedrosis virus inclusion bodies

The structure of the inclusion bodies (IBs) of three multiply enveloped nuclear polyhedrosis viruses (MNPVs), one singly enveloped NPV (SNPV), two granulosis viruses (GVs) and one cytoplasmic polyhedrosis virus (CPV) were compared. A method was devised to calculate the numbers of virus particles and nucleocapsids in IBs using data from light microscopy and thin sections. The three MNPVs, from Agrotis segetum (English and Polish virus isolates) and Mamestra brassicae had similar concentrations of virus particles ranging from 17.3 to 19.6 per μm³ of IB. Plusia gamma SNPV had a higher density of 59.6 virus particles per μm³ of IB, which partly compensated for its having smaller IBs (mean volume 0.65 μm³) than the MNPVs (2.60–9.71 μm³). The English A. segetum MNPV isolate had the most nucleocapsids in each virus particle (mean, 4.04) and the largest IBs (mean volume, 9.71 μm³), giving 674 nucleocapsids per IB on average. The GVs, from A. segetum and Pieris brassicae, mainly contained one nucleocapsid per IB. P. gamma CPV IBs had a much higher density of virus particles than the baculoviruses (260 per μm³ compared with 17–60 per μm³). These data are discussed in relation to the biological properties of these viruses, and possible adaptational advantages of alternative IB designs are considered.     

Huntingtin inclusion bodies are iron-dependent centers of oxidative events

Recently, we reported that the transient expression of huntingtin exon1 polypeptide containing polyglutamine tracts of various sizes (httEx1-polyQ) in cell models of Huntington disease generated an oxidative stress whose intensity was CAG repeat expansion-dependent. Here, we have analyzed the intracellular localization of the oxidative events generated by the httEx1-polyQ polypeptides. Analysis of live COS-7 cells as well as neuronal SK-N-SH and PC12 cells incubated with hydroethidine or dichlorofluorescein diacetate revealed oxidation of these probes at the level of the inclusion bodies formed by httEx1-polyQ polypeptides. The intensity and frequency of the oxidative events among the inclusions were CAG repeat expansion-dependent. Electron microscopic analysis of cell sections revealed the presence of oxidation-dependent morphologic alterations in the vicinity of httEx1-polyQ inclusion bodies. Moreover, a high level of oxidized proteins was recovered in partially purified inclusions. We also report that the iron chelator deferroxamine altered the structure, localization and oxidative potential of httEx1-polyQ inclusion bodies. Hence, despite the fact that the formation of inclusion bodies may represent a defense reaction of the cell to eliminate httEx1 mutant polypeptide, this phenomenon appears inherent to the generation of iron-dependent oxidative events that can be deleterious to the cell.     

Overexpression of bacterial hemoglobin causes incorporation of pre-beta-lactamase into cytoplasmic inclusion bodies.

The expression of Vitreoscilla hemoglobin (VHb) in Escherichia coli JM101 (pRED2) causes the incorporation of the TEM beta-lactamase precursor into cytoplasmic inclusion bodies (IBs). Less pre-beta-lactamase is translocated and processed to its mature, periplasmic form in the strain coexpressing VHb than in the control strain E. coli JM101(pUC19) not expressing VHb. When cells are grown in a special fed-batch procedure, the formation of cytoplasmic IBs consisting of pre-beta-lactamase is also inducible in the control strain. Comparative microscopic and compositional analyses of IBs generated in E. coli JM101(pUC19) and JM101(pRED2) under identical growth conditions strongly suggest that pre-beta-lactamase and VHb coaggregate into common IBs in E. coli JM101 (pRED2).     

Immunocytochemical demonstration of human proinsulin chimeric polypeptide within cytoplasmic inclusion bodies of Escherichia coli

Immunocytochemical techniques were used to identify human proinsulin chimeric protein in cytoplasmic inclusion bodies of genetically modified Escherichia coli. Antibodies to proinsulin chimeric protein (human proinsulin coupled at its amino-terminus to a portion of the E. coli tryptophan E gene product) were localized in E. coli using post-embedding staining with protein A-peroxidase labelling for transmission electron microscopy. The observable distribution of the labelled antibody was limited to that portion of the E. coli cytoplasm occupied by inclusion bodies. The localization of human peptides as insoluble masses within the bacterial cytoplasm has important implications in relation to the synthesis, recovery and purification of pharmacologically useful substances produced through the application of recombinant DNA technology.     

A role for plant microtubules in the formation of transmission-specific inclusion bodies of Cauliflower mosaic virus

Interactions between microtubules and viruses play important roles in viral infection. The best-characterized examples involve transport of animal viruses by microtubules to the nucleus or other intracellular destinations. In plant viruses, most work to date has focused on interaction between viral movement proteins and the cytoskeleton, which is thought to be involved in viral cell-to-cell spread. We show here, in Cauliflower mosaic virus (CaMV)-infected plant cells, that viral electron-lucent inclusion bodies (ELIBs), whose only known function is vector transmission, require intact microtubules for their efficient formation. The kinetics of the formation of CaMV-related inclusion bodies in transfected protoplasts showed that ELIBs represent newly emerging structures, appearing at late stages of the intracellular viral life cycle. Viral proteins P2 and P3 are first produced in multiple electron-dense inclusion bodies, and are later specifically exported to transiently co-localize with microtubules, before concentrating in a single, massive ELIB in each infected cell. Treatments with cytoskeleton-affecting drugs suggested that P2 and P3 might be actively transported on microtubules, by as yet unknown motors. In addition to providing information on the intracellular life cycle of CaMV, our results show that specific interactions between host cell and virus may be dedicated to a later role in vector transmission. More generally, they indicate a new unexpected function for plant cell microtubules in the virus life cycle, demonstrating that microtubules act not only on immediate intracellular or intra-host phenomena, but also on processes ultimately controlling inter-host transmission.     

Characterization of the cytoplasmic inclusion bodies of the spleens from patients with adult form Gaucher's disease

The twisted tubular cytoplasmic inclusion bodies were isolated from spleens of four patients with adult form Gaucher's disease. The chemical composition of the CIB (cytoplasmic inclusion bodies) was as follows: proteins, 10%, cholesterol, 10%, phospholipids, 10%, glycolipids, 70%. More than 90% of glycolipids from CIB were glucosylceramide. The structural protein profile of these bodies was examined by SDS-polyacrylamide disc gel electrophoresis on a semimicro-scale (s-PAGE). A similar protein composition which included two glycoproteins was found in all the four cases. The tubular structure of the bodies was changed to a small and round form by the treatment with 1 mM EDTA-Na2 which removed some structural proteins from the bodies. This indicated that some proteins might have an important role in maintaining the tubular structure of CIB.     

Reovirus core protein mu2 determines the filamentous morphology of viral inclusion bodies by interacting with and stabilizing microtubules

Cells infected with mammalian reoviruses often contain large perinuclear inclusion bodies, or "factories," where viral replication and assembly are thought to occur. Here, we report a viral strain difference in the morphology of these inclusions: filamentous inclusions formed in cells infected with reovirus type 1 Lang (T1L), whereas globular inclusions formed in cells infected with our laboratory's isolate of reovirus type 3 Dearing (T3D). Examination by immunofluorescence microscopy revealed the filamentous inclusions to be colinear with microtubules (MTs). The filamentous distribution was dependent on an intact MT network, as depolymerization of MTs early after infection caused globular inclusions to form. The inclusion phenotypes of T1L x T3D reassortant viruses identified the viral M1 genome segment as the primary genetic determinant of the strain difference in inclusion morphology. Filamentous inclusions were seen with 21 of 22 other reovirus strains, including an isolate of T3D obtained from another laboratory. When the mu2 proteins derived from T1L and the other laboratory's T3D isolate were expressed after transfection of their cloned M1 genes, they associated with filamentous structures that colocalized with MTs, whereas the mu2 protein derived from our laboratory's T3D isolate did not. MTs were stabilized in cells infected with the viruses that induced filamentous inclusions and after transfection with the M1 genes derived from those viruses. Evidence for MT stabilization included bundling and hyperacetylation of alpha-tubulin, changes characteristically seen when MT-associated proteins (MAPs) are overexpressed. Sequencing of the M1 segments from the different T1L and T3D isolates revealed that a single-amino-acid difference at position 208 correlated with the inclusion morphology. Two mutant forms of mu2 with the changes Pro-208 to Ser in a background of T1L mu2 and Ser-208 to Pro in a background of T3D mu2 had MT association phenotypes opposite to those of the respective wild-type proteins. We conclude that the mu2 protein of most reovirus strains is a viral MAP and that it plays a key role in the formation and structural organization of reovirus inclusion bodies.     

Formation of soluble inclusion bodies by hpv e6 oncoprotein fused to maltose-binding protein

Many polypeptides overexpressed in bacteria are produced misfolded and accumulate as solid structures called inclusion bodies. Inclusion-body-prone proteins have often been reported to escape precipitation when fused to maltose-binding protein (MBP). Here, we have examined the case of HPV 16 oncoprotein E6. The unfused sequence of E6 is overexpressed as inclusion bodies in bacteria. By contrast, fusions of E6 to the C-terminus of MBP are produced soluble. We have analyzed preparations of soluble MBP-E6 fusions by using three independent approaches: dynamic light scattering, lateral turbidimetry, and sandwich ELISA. All three methods showed that MBP-E6 preparations contain highly aggregated material. The behavior of these soluble aggregates under denaturating conditions suggests that they are formed by agglomeration of misfolded E6 moieties. However, precipitation is prevented by the presence of the folded and highly soluble MBP moieties, which maintain the aggregates in solution. Therefore, the fact that a protein or protein domain is produced soluble when fused to the C-terminus of a carrier protein does not guarantee that the protein of interest is properly folded and active. We suggest that aggregation of fusion proteins should be systematically assayed, especially when these fusions are to be used for binding measurements or activity tests.     

A 37-kilodalton glycoprotein from a baculovirus of Orgyia pseudotsugata is localized to cytoplasmic inclusion bodies.

The gene encoding a 37-kDa glycoprotein (gp37) of Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) was located and sequenced. gp37 of OpMNPV was found to have 62 and 37% amino acid sequence identity with gp37 of Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) and with a protein reported to be a component of occlusion bodies from Choristoneura biennis entomopoxvirus, respectively. The mRNA start site of the OpMNPV gp37 gene was mapped within a late promoter sequence (TTAAG). A TrpE fusion protein containing 55% of the OpMNPV gp37 gene amino acid sequence was used to generate a monospecific antiserum. Western immunoblot analysis of OpMNPV-infected Lymantria dispar cells detected gp37 beginning at 24 h postinfection. Immunoelectron microscopy indicated that the protein is concentrated in cytoplasmic inclusion bodies late in infection. In contrast to gp37 of AcMNPV which was present in the matrix of occlusion bodies, OpMNPV gp37 was not observed in this location. Neither OpMNPV nor AcMNPV gp37 was associated with the polyhedron envelope.