Characterisation of the differences between hepatitis C virus genotype 3 and 1 glycoproteins

Sequence variation in the envelope E1 and E2 glycoproteins of hepatitis C virus (HCV) could account for differences in disease pathogenesis in patients infected with different genotypes. A cDNA encoding the structural region of the hepatitis C polyprotein was constructed to match the majority sequence of viral RNA extracted from a patient infected with genotype 3a (designated strain HCV3a-Gla). The principal differences predicted between E2 of HCV3a-Gla and the corresponding H77c genotype 1a protein were that the former contained six more amino acids (361 vs. 355), but it had one fewer glycosylation site. Expression studies showed that, in common with the H77c glycoproteins, E1 and E2 from HCV3a-Gla localised to the endoplasmic reticulum (ER) membrane in both Huh-7 and BHK tissue culture cells and interacted to form native complexes. Analysis of the cross-reactivity of antibodies raised against glycoproteins of genotype 1a strains showed that three of five monoclonal antibodies that recognise linear epitopes were able to detect E2 from strain HCV3a-Gla. However, neither conformational E2 antibodies nor antibodies raised against E1 were able to detect the HCV3a-Gla glycoproteins. In receptor binding assays, E2 of HCV3a-Gla consistently failed to bind CD81, a putative cell receptor for HCV. Absence of binding to CD81 and lack of recognition by most antibodies raised to genotype 1a glycoproteins indicate important differences between these glycoproteins representative of genotypes 3a and 1a. These may be pertinent to the differences in response to interferon therapy and the prevalence of steatosis reported in patients infected with these genotypes.     

Expression of the murine CB2 cannabinoid receptor using a recombinant Semliki Forest virus

A recombinant Semliki Forest virus (SFV) RNA construct, SFV1-mCB(2) RNA, was employed for the high-level expression of the murine CB(2) (mCB(2)) cannabinoid receptor in baby hamster kidney cells. Biosynthetic radiolabel incorporation studies in concert with urea-sodium dodecylsulfate-polyacrylamide gel electrophoresis (urea-SDS-PAGE) and western immunoblotting revealed that two major proteins of approximately 26 and 40kDa were produced by the construct. The 40kDa product, but not the 26kDa product, was glycosylated as determined by 2-deoxy-D-glucose incorporation and peptide-N-glycosidase F digestion analysis. Assessment of [3H]CP55940 ([3H]-(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) binding data for membranes of cells transfected with SFV1-mCB(2) RNA indicated a K(d) of 0.35+/-0.04nM and a B(max) of 24.4+/-2.7pmol/mg. A rank order of binding affinities for cannabinoids, which paralleled that reported for native mCB(2) receptors, was observed. The CB(2) receptor-specific antagonist SR144528 (N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) blocked binding of [3H]CP55940, while the CB(1) receptor-specific antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] had a minimal effect. These results indicate that the recombinant receptor expressed from SFV1-mCB(2) RNA exhibits properties, including ligand binding features, that are consistent with those for the native mCB(2) receptor. However, the presence of both 26 and 40kDa receptor species is consistent with alternative translation from two AUG start sites using the SFV1-mCB(2) RNA expression system.     

Induction of EAE by T cells specific for alpha B-crystallin depends on prior viral infection in the CNS

While myelin-reactive T cells are widely believed to play a pathogenic role in multiple sclerosis (MS), no substantial differences appear to exist in T-cell responses to myelin antigens between MS patients and healthy subjects. As an example, indistinguishable peripheral T-cell responses and serum antibody levels have been found in MS patients and healthy controls to alpha B-crystallin, a dominant antigen in MS-affected brain myelin. This suggests that additional factors are relevant in allowing myelin-reactive T cells to become pathogenic. In this study, we examined whether the inflammatory state of the CNS is relevant to the pathogenicity of alpha B-crystallin-specific T cells in mice. In normal mice, T-cell responses against alpha B-crystallin are limited by robust immunological tolerance. Reactive T cells were therefore generated in alpha B-crystallin-deficient mice, and these T cells were transferred into C57BL/6 recipients. While such a transfer in itself never induced any clinical signs of experimental autoimmune encephalomyelitis (EAE) in healthy recipient mice, acute EAE could be induced in animals that had been infected 7 days before with the avirulent A7(74) strain of Semliki Forest virus (SFV). SFV infection alone did not induce clinical disease, nor did it alter the expression levels of the target antigen. Our findings indicate that at least in mice, alpha B-crystallin-specific T cells can trigger EAE but only when prior viral infection has induced an inflammatory state in the CNS that helps recruit and activate T cells.     

Infection of rat brain primary cell cultures with an avirulent A7 strain of Semliki Forest virus

The ability of A7 Semliki Forest Virus (SFV) to infect primary brain cell cultures has been examined using cultures prepared from 1-2-day neonatal rat cerebral hemispheres. These cultures, characterised immunocytochemically using cell-specified markers, contain mainly GFAP+ protoplasmic astrocytes and smaller multiprocessed A2B5+ cells, probably fibrous astrocytes. 10% of the cells are GC+ oligodendrocytes and some neurones are also present. These cultures support virus growth and a cytopathic effect was observed. Using double labelling techniques with the cell-specific markers and anti-SFV antibody A7 has been shown to readily infect cells which carry either the A2B5+ antigen or galactocerebroside marker. Protoplasmic astrocytes (GFAP+/A2B5-) are not readily infected under the conditions used. The protein labelling studies using [35S]methionine show that host cell protein synthesis is not completely shut off and continues in the astrocyte protein region. These results suggest that cells derived from a common A2B5+, GFAP-, GC- progenitor glial cell, i.e. GC+ oligodendrocytes and A2B5+/GFAP+ fibrous astrocytes, are more readily infected than other brain cell types including the protoplasmic astrocytes.     

In vitro evolution of high-titer,virus-like vesicles containing a single structural protein

Self-propagating, infectious, virus-like vesicles (VLVs) are generated when an alphavirus RNA replicon expresses the vesicular stomatitis virus glycoprotein (VSV G) as the only structural protein. The mechanism that generates these VLVs lacking a capsid protein has remained a mystery for over 20 years. We present evidence that VLVs arise from membrane-enveloped RNA replication factories (spherules) containing VSV G protein that are largely trapped on the cell surface. After extensive passaging, VLVs evolve to grow to high titers through acquisition of multiple point mutations in their nonstructural replicase proteins. We reconstituted these mutations into a plasmid-based system from which high-titer VLVs can be recovered. One of these mutations generates a late domain motif (PTAP) that is critical for high-titer VLV production. We propose a model in which the VLVs have evolved in vitro to exploit a cellular budding pathway that is hijacked by many enveloped viruses, allowing them to bud efficiently from the cell surface. Our results suggest a basic mechanism of propagation that may have been used by primitive RNA viruses lacking capsid proteins. Capsids may have evolved later to allow more efficient packaging of RNA, greater virus stability, and evasion of innate immunity.Enveloped RNA viruses have highly organized structures. One or more capsid proteins encase their RNA, matrix proteins often lie between the capsid and the membrane, and one or more transmembrane glycoproteins can interact with the matrix or capsid proteins to direct efficient particle assembly (1). Once the particles are released from cells, one or more glycoproteins in the viral envelope bind cellular receptors and catalyze membrane fusion to allow the viruses to enter new cells (2).Vesicular stomatitis virus (VSV) is a negative-strand RNA virus that encodes a single membrane glycoprotein (G), a matrix protein, and a nucleocapsid protein as well as two proteins that form the viral polymerase (3). Remarkably, when cells are transfected with an alphavirus RNA replicon encoding only the alphavirus nonstructural replicase proteins and the VSV G protein, infectious, self-propagating membrane-enveloped vesicles containing the VSV G protein are generated (4). These infectious, virus-like vesicles (VLVs) grow to only low titers of 10⁴ to 10⁵ infectious units (i.u.) per mL, but propagate like a virus in tissue culture cells. The vesicles contain the genomic RNA and VSV G protein, but unlike known enveloped RNA viruses, they lack a capsid protein encasing their RNA resulting in a low buoyant density (4). The mechanism by which these VLVs are generated has never been determined. A nonspecific packaging was postulated because the VLVs contained both genomic RNA and subgenomic mRNA (4) and the titers could be increased by sonication of the cells. Despite the low titers, VLVs expressing other proteins have proven useful as experimental vaccines (5, 6).The alphavirus replicon used in the studies described above was derived from Semliki Forest Virus (SFV), a positive-strand, membrane-enveloped RNA virus that encodes four nonstructural proteins called nsP 1–4 and three structural proteins: capsid, and the E1 and E2 transmembrane glycoproteins (7, 8). The nsP 1–4 proteins are translated from the first two-thirds of the genomic RNA. These proteins form a complex that directs replication of the genomic RNA to form antigenomic RNA, which is then copied to form full-length positive strand RNA and a subgenomic mRNA that encodes the structural proteins. The capsid protein encases the genomic RNA in the cytoplasm and then buds from the cell surface in a membrane containing the SFV glycoproteins. Alphavirus RNA replication occurs inside light-bulb shaped, membrane-bound compartments called spherules that initially form on the cell surface and are then endocytosed to form cytopathic vacuoles containing multiple spherules (9). The replicase proteins appear localized near the cytoplasmic side of the spherules (10). Positive-strand genomic RNA produced in the spherules is packaged into nucleocapsids before SFV budding. Alphavirus RNA replicons lacking structural protein genes can replicate efficiently inside a cell, but they are incapable of propagating beyond the cell.We have been interested in developing VLVs as a vaccine platform (5, 6). However, the relatively low titers generated were a major limitation of the system. We undertook the extensive serial passaging studies described here to determine whether VLVs could evolve in culture to grow to high titers. We succeeded in generating VLVs that grow to at least 1,000-fold higher titers. In the process of studying these VLVs, we generated data suggesting the mechanism of VLV formation. In addition, our data suggest that the high-titer VLVs have evolved through passaging to use a cellular budding machinery that is exploited by many enveloped viruses to drive efficient budding.Many enveloped RNA viruses use components of a cellular vesicular budding machinery to drive efficient budding from the cell surface (11–13). Short sequence motifs called late domains in their structural proteins recruit cellular protein complexes called ESCRT (endosomal sorting complex required for transport). The ESCRT complexes are normally involved in budding of vesicles into cellular multivesicular bodies (MVB) as well as other cellular processes (12). Retroviruses, paramyxoviruses, filoviruses, and rhabdoviruses have been shown to use components of the ESCRT pathway to drive budding (11–13). However, other enveloped RNA viruses including SFV and influenza virus do not use the ESCRT pathway in budding (14–16).     

An ultrastructural study of nerve profiles in the myenteric plexus of the rabbit colon

The ultrastructure of the myenteric plexus from the rabbit colon was examined in both conventionally fixed tissue and also material fixed with the chromaffin method. Montages of the ganglia were analysed semi-quantitatively. Six main types of axon profile are described and classified on a morphological consideration of the vesicle population. Most axon types formed synapses with myenteric neurons. Two kinds of chromaffin-positive nerve fibre were seen, one containing a predominance of small granular vesicles, the other containing many flattened vesicles. The difficulties in relating axon profile types to putative transmitters are discussed.     

Regulated sarcolemmal localization of the muscle-specific ClC-1 chloride channel

The skeletal muscle-specific ClC-1 is a voltage-gated chloride channel protein. Specific antibodies against ClC-1 revealed in muscle sections a sarcolemmal staining that was absent in the myotonic arrested development of righting response (ADR) mouse muscle. The intensity of the sarcolemmal staining varied from one type of muscle to another and in lateral sections showed a typical mosaic pattern that colocalized with beta-dystroglycan and left the transverse tubule openings clear. Surprisingly, in isolated myofibers, the ClC-1 protein was absent from the sarcolemma. Instead, it localized to intracellular I band areas as soon as the myofibers were isolated. When the isolated myofibers were incubated with the kinase inhibitor staurosporine, the ClC-1 protein shifted back to the sarcolemma. Electric stimulation of the cultivated fibers had a similar effect. Also, myofibers infected with a recombinant Semliki Forest virus (SFV) expressing myc-tagged ClC-1 showed intracellular localization of the protein. The virally expressed mycClC-1 reached the Golgi apparatus but sarcolemmal staining remained nondetectable, and addition of staurosporine into the growth medium recruited the mycClC-1 to the sarcolemma. These data indicate that sarcolemmal targeting of the ClC-1 requires specific signals that are provided by the physiological environment.