Antibodies to the Core Proteins of Nairobi Sheep Disease Virus/Ganjam Virus Reveal Details of the Distribution of the Proteins in Infected Cells and Tissues

Nairobi sheep disease virus (NSDV; also called Ganjam virus in India) is a bunyavirus of the genus Nairovirus. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV). Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N) and polymerase (L) and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain) it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus in vivo. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN), where NSDV appeared to target monocytes and/or macrophages.     

Morphogenesis of Pestiviruses: New Insights from Ultrastructural Studies of Strain Giraffe-1

Knowledge on the morphogenesis of pestiviruses is limited due to low virus production in infected cells. In order to localize virion morphogenesis and replication sites of pestiviruses and to examine intracellular virion transport, a cell culture model was established to facilitate ultrastructural studies. Based on results of virus growth kinetic analysis and quantification of viral RNA, pestivirus strain Giraffe-1 turned out to be a suitable candidate for studies on virion generation and export from culture cells. Using conventional transmission electron microscopy and single-tilt electron tomography, we found virions located predominately in the lumen of the endoplasmic reticulum (ER) in infected cells and were able to depict the budding process of virions at ER membranes. Colocalization of the viral core protein and the envelope glycoprotein E2 with the ER marker protein disulfide isomerase (PDI) was demonstrated by immunogold labeling of cryosections. Moreover, pestivirions could be shown in transport vesicles and the Golgi complex and during exocytosis. Interestingly, viral capsid protein and double-stranded RNA (dsRNA) were detected in multivesicular bodies (MVBs), which implies that the endosomal compartment plays a role in pestiviral replication. Significant cellular membrane alterations such as those described for members of the Flavivirus and Hepacivirus genera were not found. Based on the gained morphological data, we present a consistent model of pestivirus morphogenesis.     

Virus-neuron interactions in the mouse brain infected with Japanese encephalitis virus

The virus-host interactions between Japanese encephalitis (JE) virus and mouse brain neurons were analyzed by electron microscopy. JE virus replicated exclusively in the rough endoplasmic reticulum (RER) of neurons. In the early phase of infection, the perikaryon of infected neurons had relatively normal-looking lamellar RER whose cisternae showed focal dilations containing progeny virions and characteristic endoplasmic reticulum (ER) vesicles. The reticular RER, consisted of rows of ribosomes surrounding irregular-shaped, membrane-unbounded cisternae and resembled that observed in JE-virus-infected PC12 cells, were also seen adjacent to the lamellar RER. The appearance of the reticular RER indicated that RER morphogenesis occurred in infected neurons in association with the viral replication. The fine network of Golgi apparatus was extensively obliterated by fragmentation and dissolution of the Golgi membranes and their replacement by the electron-lucent material. As the infection progressed, the lamellar RER was increasingly replaced by the hypertrophic RER which had diffusely dilated cisternae containing multiple progeny virions and ER vesicles. The Golgi apparatus, at this stage, was seen as coarse, localized Golgi complexes near the hypertrophic RER. In the later phase of infection, RER of infected neurons showed a degenerative change, with the cystically dilated cisternae being filled with ER vesicles and virions. Small, localized Golgi complexes frequently showed vesiculation, vacuolation, and dispersion. The present study, therefore, indicated that during the viral replication the normal lamellar RER which synthesized neuronal secretory and membrane proteins was replaced by the hypertrophic RER which synthesized the viral proteins. The hypertrophic RER eventually degenerated into cystic RER whose cisternae were filled with viral products. The constant degenerative change which occurred in the Golgi apparatus during the viral replication suggested that some of the viral proteins transported from RER to the Golgi apparatus were harmful to the Golgi apparatus and that increasing damage to the Golgi apparatus during the viral replication played the principal role in the pathogenesis of JE-virus-infected neurons in the central nervous system.     

Replication complex of human parechovirus 1

The parechoviruses differ in many biological properties from other picornaviruses, and their replication strategy is largely unknown. In order to identify the viral RNA replication complex in human parechovirus type 1 (HPEV-1)-infected cells, we located viral protein and RNA in correlation to virus-induced membrane alterations. Structural changes in the infected cells included a disintegrated Golgi apparatus and disorganized, dilated endoplasmic reticulum (ER) which had lost its ribosomes. Viral plus-strand RNA, located by electron microscopic (EM) in situ hybridization, and the viral protein 2C, located by EM immunocytochemistry were found on clusters of small vesicles. Nascent viral RNA, visualized by 5-bromo-UTP incorporation, localized to compartments which were immunocytochemically found to contain the viral protein 2C and the trans-Golgi marker 1,4-galactosyltransferase. Protein 2C was immunodetected additionally on altered ER membranes which displayed a complex network-like structure devoid of cytoskeletal elements and with no apparent involvement in viral RNA replication. This protein also exhibited membrane binding properties in an in vitro assay. Our data suggest that the HPEV-1 replication complex is built up from vesicles carrying a Golgi marker and forming a structure different from that of replication complexes induced by other picornaviruses.     

Intracellular Localization of the Peanut Clump Virus Replication Complex in Tobacco BY-2 Protoplasts Containing Green Fluorescent Protein-Labeled Endoplasmic Reticulum or Golgi Apparatus

RNA-1 of Peanut clump virus (PCV) encodes the proteins P131 and P191, containing the signature motifs of replication proteins, and P15, which regulates viral RNA accumulation. In PCV-infected protoplasts both P131 and P191 were immunodetected in the perinuclear region. Laser scanning confocal microscopy (LSCM) showed that P131 and P191 colocalized with neosynthesized 5-bromouridine 5'-triphosphate-labeled RNA and double-stranded RNA, demonstrating that they belong to the replication complex. On the contrary, the P15 fused to the enhanced green fluorescent protein (EGFP) never colocalized with the two proteins. In endoplasmic reticulum (ER)-GFP transgenic BY-2 protoplasts, the distribution of the green fluorescent-labeled ER was strongly modified by PCV infection. LSCM showed that both P131 and P191 colocalized with ER green fluorescent bodies accumulating around the nucleus during infection. The replication process was not inhibited by cerulenin and brefeldin A, suggesting that PCV replication does not depend on de novo-synthesized membrane and does not require transport through the Golgi apparatus. Electron microscopy of ultrathin sections of infected protoplasts showed aggregates of broken ER but also visualized vesicles, some of which resembled modified peroxisomes. The results suggest that accumulation of PCV during infection is accompanied by specific association of PCV RNA-1-encoded proteins with membranes of the ER and other organelles. The concomitant extensive rearrangement of these membranous structures leads to the formation of intracellular compartments in which synthesis and accumulation of the viral RNA occur in defined areas.     

Dengue Virus Uses a Non‐Canonical Function of the Host GBF1‐Arf‐COPI System for Capsid Protein Accumulation on Lipid Droplets

Dengue viruses cause the most important human viral disease transmitted by mosquitoes. In recent years, a great deal has been learned about molecular details of dengue virus genome replication; however, little is known about genome encapsidation and the functions of the viral capsid protein. During infection, dengue virus capsid progressively accumulates around lipid droplets (LDs) by an unknown mechanism. Here, we examined the process by which the viral capsid is transported from the endoplasmic reticulum (ER) membrane, where the protein is synthesized, to LDs. Using different methods of intervention, we found that the GBF1‐Arf1/Arf4‐COPI pathway is necessary for capsid transport to LDs, while the process is independent of both COPII components and Golgi integrity. The transport was sensitive to Brefeldin A, while a drug resistant form of GBF1 was sufficient to restore capsid subcellular distribution in infected cells. The mechanism by which LDs gain or lose proteins is still an open question. Our results support a model in which the virus uses a non‐canonical function of the COPI system for capsid accumulation on LDs, providing new ideas for antiviral strategies.      

Acid Phosphatase Activity in Mouse Brain Infected with Venezuelan Equine Encephalomyelitis Virus

The mode of development of Venezuelan equine encephalomyelitis virus and the activity of acid phosphatase in the central nervous system of newborn mice were investigated. Precursor particles appeared to be formed in masses of viroplasm, migrating to the membrane of the Golgi cisterns and vacuoles or to the plasma membrane and being transformed into mature viral particles by budding. Mature viral particles were also found in the lumen of the blood vessels and around the myelin sheath of axons. Increased number of Golgi complexes and depletion of polysomes were the main ultrastructural alterations of the nerve cells. Acid phosphatase activity was found to be increased in the Golgi cisterns, vacuoles, and lysosomes of nerve cells. The presence of acid phosphatase activity in the rough endoplasmic reticulum and perinuclear cisterns suggests increased production of the enzyme in the nerve cells infected with Venezuelan equine encephalomyelitis virus.     

Micromorphological Aspects of the Development of Rubella Virus in BHK-21 Cells

Sequential effects of rubella virus infection in BHK-21 cells were studied by electron microscopy of thin sections of control and infected cells, 2 to 7 days after infection. Vacuolization of cytoplasm in Golgi areas apparently preceded budding of virions from vacuole membranes and involvement of the endoplasmic reticulum. Newly formed endoplasmic reticulum cisternae encircled and segregated virionforming vacuoles together with other cellular elements. Large vacuolar complexes with numerous virus particles developed, and virus release from these areas occurred with disruption at the cell periphery. The viral particles, with a mean diameter of about 56 nm, consisted of an electron-dense core surrounded by a less dense capsid, enveloped by a typical unit membrane derived from the vacuole membrane.     

Routing of the G Protein During Maturation of Festuca Leaf Streak Rhabdovirus in its Plant Host

By immunogold labelling the location of Festuca leaf streak virus glycoprotein (FLSV-G) was investigated in developing phloem and mature leaf parenchyma of Festuca gigantea infected with Festuca leaf streak virus (FLSV: Rhabdotiridae). In developing phloem cells, FLSV-G was detected in endoplasmic reticulum (ER). at perinuclear membranes, and in assembled virions, but neither in Golgi stacks and Golgi vesicles nor at the plasma membrane of infected cells. These results indicate that FLSV-G stays in the ER after transmembrane synthesis, and is not routed through the secretory pathway in F. gigantea. The membranous inclusions, present in infected mature leaf parenchyma cells were found to contain FLSV-G. It is suggested that the, virus-induced membranous inclusions have developed from FLSV-G-containing ER. The residence of FLSV-G in ER (present study) is in contrast to results with vesicular stomatitis virus (VSV; vertebrate rhabdovirus). Here the G protein is known to be routed to the plasma membrane through the secretory pathway.     

Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Hepatitis C Virus Replication

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.     

Vaccinia virus L2 protein associates with the endoplasmic reticulum near the growing edge of crescent precursors of immature virions and stabilizes a subset of viral membrane proteins

The initial step in poxvirus morphogenesis, the formation of crescent membranes, occurs within cytoplasmic factories. L2 is one of several vaccinia virus proteins known to be necessary for formation of crescents and the only one synthesized early in infection. Virus replication was unaffected when the L2R open reading frame was replaced by L2R containing an N-terminal epitope tag while retaining the original promoter. L2 colocalized with the endoplasmic reticulum (ER) protein calnexin throughout the cytoplasm of infected and transfected cells. Topological studies indicated that the N terminus of L2 is exposed to the cytoplasm with the hydrophobic C terminus anchored in the ER. Using immunogold labeling and electron microscopy, L2 was detected in tubular membranes outside factories and inside factories near crescents and close to the edge or rim of crescents; a similar labeling pattern was found for the ER luminal protein disulfide isomerase (PDI). The phenotype of L2 conditional lethal mutants and the localization of L2 suggest that it participates in elongation of crescents by the addition of ER membrane to the growing edge. Small amounts of L2 and PDI were detected within immature and mature virions, perhaps trapped during assembly. The repression of L2, as well as A11 and A17, two other proteins that are required for viral crescent formation, profoundly decreased the stability of a subset of viral membrane proteins including those comprising the entry-fusion complex. To avoid degradation, these unstable membrane proteins may need to directly insert into the viral membrane or be rapidly shunted there from the ER.     

Requirements for ER-Arrest and Sequential Exit to the Golgi of Tomato Spotted Wilt Virus Glycoproteins

The envelope glycoproteins Gn and Gc are major determinants in the assembly of Tomato spotted wilt virus (TSWV) particles at the Golgi complex. In this article, the ER-arrest of singly expressed Gc and the transport of both glycoproteins to the Golgi upon co-expression have been analyzed. While preliminary results suggest that the arrest of Gc at the ER (endoplasmic reticulum) did not appear to result from improper folding, transient expression of chimeric Gc, in which the transmembrane domain (TMD) and/or cytoplasmic tail (CT) were swapped for those from Gn, showed that the TMD of Gn was sufficient to allow ER-exit and transport to the Golgi. Expression of both glycoproteins in the presence of overexpressed Sar1p-specific guanosine nucleotide exchange factor Sec 12p, resulted in ER-retention demonstrating that the viral glycoproteins are transported to the Golgi in a COPII (coat protein II)-dependent manner. Inhibition of ER-Golgi transport by brefeldin A (BFA) had a similar effect on the localization of Gn. However, inhibition of ER (endoplasmic reticulum) to Golgi transport of co-expressed Gc and Gn by overexpression of Sec 12p or by BFA revealed distinct localization patterns, i.e. diffuse ER localization versus concentration at specific spots.     

Hepatitis C virus, ER stress, and oxidative stress

Hepatitis C virus (HCV) replication is associated with the endoplasmic reticulum (ER), where the virus causes stress. Cells cope with ER stress by activating an adaptive program called the unfolded protein response (UPR), which alleviates this stress by stimulating protein folding and degradation in the ER and down-regulating overall protein synthesis. Recent work suggests that HCV also alters ER calcium homeostasis, inducing oxidative stress. Future progress in understanding the control that HCV exerts over the ER will provide insight into viral strategies for pathogenesis and persistence in chronically infected patients.     

Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein.

The mechanisms that direct positive-stranded RNA virus replication complexes to plant and animal cellular membranes are poorly understood. We describe a specific interaction between a replication protein of an RNA plant virus and membranes in vitro and in live cells. The tobacco etch virus (TEV) 6 kDa protein associated with membranes as an integral protein via a central 19 amino acid hydrophobic domain. In the presence or absence of other viral proteins, fluorescent fusion proteins containing the 6 kDa protein associated with large vesicular compartments derived from the endoplasmic reticulum (ER). Infection by TEV was associated with a collapse of the ER network into a series of discrete aggregated structures. Viral RNA replication complexes from infected cells were also associated with ER-like membranes. Targeting of TEV RNA replication complexes to membranous sites of replication is proposed to involve post-translational interactions between the 6 kDa protein and the ER.     

Influenza A viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (IRE1) stress pathway

Known therapies for influenza A virus infection are complicated by the frequent emergence of resistance. A therapeutic strategy that may escape viral resistance is targeting host cellular mechanisms involved in viral replication and pathogenesis. The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a primitive, evolutionary conserved molecular signaling cascade that has been implicated in multiple biological phenomena including innate immunity and the pathogenesis of certain viral infections. We investigated the effect of influenza A viral infection on ER stress pathways in lung epithelial cells. Influenza A virus induced ER stress in a pathway-specific manner. We showed that the virus activates the IRE1 pathway with little or no concomitant activation of the PERK and the ATF6 pathways. When we examined the effects of modulating the ER stress response on the virus, we found that the molecular chaperone tauroursodeoxycholic acid (TUDCA) significantly inhibits influenza A viral replication. In addition, a specific inhibitor of the IRE1 pathway also blocked viral replication. Our findings constitute the first evidence that ER stress plays a role in the pathogenesis of influenza A viral infection. Decreasing viral replication by modulating the host ER stress response is a novel strategy that has important therapeutic implications.     

Brome mosaic virus RNA replication proteins 1a and 2a colocalize and 1a independently localizes on the yeast endoplasmic reticulum

The universal membrane association of positive-strand RNA virus RNA replication complexes is implicated in their function, but the intracellular membranes used vary among viruses. Brome mosaic virus (BMV) encodes two mutually interacting RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and the polymerase-like 2a protein. In cells from the natural plant hosts of BMV, 1a and 2a colocalize on the endoplasmic reticulum (ER). 1a and 2a also direct BMV RNA replication and subgenomic mRNA synthesis in the yeast Saccharomyces cerevisiae, but whether the distribution of 1a, 2a, and active replication complexes in yeast duplicates that in plant cells has not been determined. For yeast expressing 1a and 2a and replicating BMV genomic RNA3, we used double-label confocal immunofluorescence to define the localization of 1a, 2a, and viral RNA and to explore the determinants of replication complex targeting. As in plant cells, 1a and 2a colocalized on and were retained on the yeast ER, with no detectable accumulation in the Golgi apparatus. 1a and 2a were distributed over most of the ER surface, with strongest accumulation on the perinuclear ER. In vivo labeling with bromo-UTP showed that the sites of 1a and 2a accumulation were the sites of nascent viral RNA synthesis. In situ hybridization showed that completed viral RNA products accumulated predominantly in the immediate vicinity of replication complexes but that some, possibly more mature cells also accumulated substantial viral RNA in the surrounding cytoplasm distal to replication complexes. Additionally, we find that 1a localizes to the ER when expressed in the absence of other viral factors. These results show that BMV RNA replication in yeast duplicates the normal localization of replication complexes, reveal the intracellular distribution of RNA replication products, and show that 1a is at least partly responsible for the ER localization and retention of the RNA replication complex.     

Distribution of the endoplasmic reticulum and its relationship with the sarcoplasmic reticulum in skeletal myofibers

We have analyzed the distribution of the endoplasmic reticulum (ER) within isolated rat skeletal muscle flexor digitorum brevis myofibers. Studies with confocal microscopy indicated that the resident ER proteins displayed a perinuclear and cross-striated distribution that extended over the I band areas. Interestingly, two discrete distribution patterns were observed when different receptor or viral marker proteins were blocked in the ER. Accordingly, the vesicular stomatitis virus G protein that lost its efficient export through the Golgi apparatus during myogenesis preferentially marked the A-I junctional areas. The proteins that retained their Golgi processing after myogenesis, on the contrary, concentrated around the myonuclei and over the Z lines. Furthermore, the ER exit site marker sec23 located to Z lines but not to A-I junctions. To analyze the ultrastructural organization of the ER, we infected myofibers with recombinant virus expressing KDEL-tagged peroxidase that is translocated into the ER. With transmission electron microscopy, peroxidase activity was found in perinuclear and Z line-flanking tubular structures, but also within the terminal cisternae of the sarcoplasmic reticulum. The translocon-associated protein exhibited a similar localization. Taken together, the terminal cisternae contained unevenly distributed rough ER structures apparently lacking the export function. The exporting ER comprised perinuclear and Z line-flanking structures.     

Cytopathology of PC12 cells infected with Japanese encephalitis virus.

Infection of a clonal rat pheochromocytoma cell line, PC12, with Japanese encephalitis (JE) virus produced successively higher titers of virus in the culture fluid during the 72-h experimental period. In electron microscopical observation, JE virus entered PC12 cells by direct penetration through the plasma membrane at 2 min postinoculation (p.i.) and caused marked cellular hypertrophy and extensive proliferation of the cellular secretory system including rough endoplasmic reticulum (RER) and Golgi complexes starting 24 h p.i. The proliferating RER of the virally infected cells contained progeny virions and characteristic endoplasmic reticulum vesicles in its cisternae, and the proliferating Golgi complexes contained virions in their saccules. These findings indicated that the proliferation of the cellular secretory system occurred in association with viral replication and maturation in the system. Seventy-two hours p.i., the cellular secretory system of infected PC12 cells showed degenerative changes with vesiculation, disorganization, and dispersion of the Golgi complexes and fragmentation, focal cystic dilation, and dissolution of the RER in the same manner as those seen in the secretory system of JE-virus-infected neurons in the mouse brain. Thus, JE-virus-infected PC12 cells seem to be a suitable neurogenic cell line for the study of the pathogenic mechanism of JE virus. At the same time, the virally infected cells seem to offer an interesting cell model for the study of the morphogenesis of the cellular secretory system.     

Retention of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein in the Endoplasmic Reticulum Does Not Redirect Virus Assembly from the Plasma Membrane

The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) has been shown to redirect the site of virus assembly in polarized epithelial cells. To test whether localization of the glycoprotein exclusively to the endoplasmic reticulum (ER) could redirect virus assembly to that organelle in nonpolarized cells, an ER -retrieval signal was engineered into an epitope-tagged variant of Env. The epitope tag, attached to the C terminus of Env, did not affect the normal maturation and transport of the glycoprotein or the incorporation of Env into virions. The epitope-tagged Env was also capable of mediating syncytium formation and virus entry with a similar efficiency to that of wild-type Env. When the epitope was modified to contain a consensus K(X)KXX ER retrieval signal, however, the glycoprotein was no longer proteolytically processed into its surface and transmembrane subunits and Env could not be detected at the cell surface by biotinylation. Endoglycosidase H analysis revealed that the modified Env was not transported to the Golgi apparatus. Immunofluorescent staining patterns were also consistent with the exclusion of Env from the Golgi. As expected, cells expressing the modified Env failed to form syncytia with CD4⁺ permissive cells. Despite this tight localization of Env to the ER, when the modified Env was expressed in the context of virus, virions continued to be produced efficiently from the plasma membrane of transfected cells. However, these virions contained no detectable glycoprotein and were noninfectious. Electron microscopy revealed virus budding from the plasma membrane of these cells, but no virus was seen assembling at the ER membrane and no assembled virions were found within the cell. These results suggest that the accumulation of Env in an intracellular compartment is not sufficient to redirect the assembly of HIV Gag in nonpolarized cells.