Transmission characteristics of heterozygous cases of Creutzfeldt-Jakob disease with variable abnormal prion protein allotypes.

In the human prion disease Creutzfeldt-Jakob disease (CJD), different CJD neuropathological subtypes are defined by the presence in normal prion protein (PrPC) of a methionine or valine at residue 129, by the molecular mass of the infectious prion protein PrPSc, by the pattern of PrPSc deposition, and by the distribution of spongiform change in the brain. Heterozygous cases of CJD potentially add another layer of complexity to defining CJD subtypes since PrPSc can have either a methionine (PrPSc-M129) or valine (PrPSc-V129) at residue 129. We have recently demonstrated that the relative amount of PrPSc-M129 versus PrPSc-V129, i.e. the PrPSc allotype ratio, varies between heterozygous CJD cases. In order to determine if differences in PrPSc allotype correlated with different disease phenotypes, we have inoculated 10 cases of heterozygous CJD (7 sporadic and 3 iatrogenic) into two transgenic mouse lines overexpressing PrPC with a methionine at codon 129. In one case, brain-region specific differences in PrPSc allotype appeared to correlate with differences in prion disease transmission and phenotype. In the other 9 cases inoculated, the presence of PrPSc-V129 was associated with plaque formation but differences in PrPSc allotype did not consistently correlate with disease incubation time or neuropathology. Thus, while the PrPSc allotype ratio may contribute to diverse prion phenotypes within a single brain, it does not appear to be a primary determinative factor of disease phenotype.

Altered distribution, aggregation, and protease resistance of cellular prion protein following intracranial inoculation.

Prion protein (PrPC) is a protease-sensitive and soluble cell surface glycoprotein expressed in almost all mammalian cell types. PrPSc, a protease-resistant and insoluble form of PrPC, is the causative agent of prion diseases, fatal and transmissible neurogenerative diseases of mammals. Prion infection is initiated via either ingestion or inoculation of PrPSc or when host PrPC stochastically refolds into PrPSc. In either instance, the early events that occur during prion infection remain poorly understood. We have used transgenic mice expressing mouse PrPC tagged with a unique antibody epitope to monitor the response of host PrPC to prion inoculation. Following intracranial inoculation of either prion-infected or uninfected brain homogenate, we show that host PrPC can accumulate both intra-axonally and within the myelin membrane of axons suggesting that it may play a role in axonal loss following brain injury. Moreover, in response to the inoculation host PrPC exhibits an increased insolubility and protease resistance similar to that of PrPSc, even in the absence of infectious prions. Thus, our results raise the possibility that damage to the brain may be one trigger by which PrPC stochastically refolds into pathogenic PrPSc leading to productive prion infection.

Cellular prion protein is present in mitochondria of healthy mice.

Cellular prion protein (PrPC) is a mammalian glycoprotein which is usually found anchored to the plasma membrane via a glycophosphatidylinositol (GPI) anchor. PrPC misfolds to a pathogenic isoform PrPSc, the causative agent of neurodegenerative prion diseases. The precise function of PrPC remains elusive but may depend upon its cellular localization. Here we show that PrPC is present in brain mitochondria from 6-12 week old wild-type and transgenic mice in the absence of disease. Mitochondrial PrPC was fully processed with mature N-linked glycans and did not require the GPI anchor for localization. Protease treatment of purified mitochondria suggested that mitochondrial PrPC exists as a transmembrane isoform with the C-terminus facing the mitochondrial matrix and the N-terminus facing the intermembrane space. Taken together, our data suggest that PrPC can be found in mitochondria in the absence of disease, old age, mutation, or overexpression and that PrPC may affect mitochondrial function.

Efficient uptake and dissemination of scrapie prion protein by astrocytes and fibroblasts from adult hamster brain.

Prion infections target neurons and lead to neuronal loss. However, the role of non-neuronal cells in the initiation and spread of infection throughout the brain remains unclear despite the fact these cells can also propagate prion infectivity. To evaluate how different brain cells process scrapie prion protein (PrPres) during acute infection, we exposed neuron-enriched and non-neuronal cell cultures from adult hamster brain to fluorescently-labeled purified PrPres and followed the cultures by live cell confocal imaging over time. Non-neuronal cells present in both types of cultures, specifically astrocytes and fibroblasts, internalized PrPres more efficiently than neurons. PrPres was trafficked to late endosomal/lysosomal compartments and rapidly transported throughout the cell bodies and processes of all cell types, including contacts between astrocytes and neurons. These observations suggest that astrocytes and meningeal fibroblasts play an as yet unappreciated role in prion infections via efficient uptake and dissemination of PrPres.

Effects of FlAsH/tetracysteine (TC) Tag on PrP proteolysis and PrPres formation by TC-scanning.

Protein-protein interactions associated with proteolytic processing and aggregation are integral to normal and pathological aspects of prion protein (PrP) biology. Characterization of these interactions requires the identification of amino acid residues involved. The FlAsH/tetracysteine (FlAsH/TC) tag is a small fluorescent tag amenable to insertion at internal sites in proteins. In this study, we used serial FlAsH/TC insertions (TC-scanning) as a probe to characterize sites of protein-protein interaction between PrP and other molecules. To explore this application in the context of substrate-protease interactions, we analyzed the effect of FlAsH/TC insertions on proteolysis of cellular prion protein (PrPsen) in in vitro reactions and generation of the C1 metabolic fragment of PrPsen in live neuroblastoma cells. The influence of FlAsH/TC insertion was evaluated by TC-scanning across the cleavage sites of each protease. The results showed that FlAsH/TC inhibited protease cleavage only within limited ranges of the cleavage sites, which varied from about one to six residues in width, depending on the protease, providing an estimate of the PrP residues interacting with each protease. TC-scanning was also used to probe a different type of protein-protein interaction: the conformational conversion of FlAsH-PrPsen to the prion disease-associated isoform, PrPres. PrP constructs with FlAsH/TC insertions at residues 90-96 but not 97-101 were converted to FlAsH-PrPres, identifying a boundary separating loosely versus compactly folded regions of PrPres. Our observations demonstrate that TC-scanning with the FlAsH/TC tag can be a versatile method for probing protein-protein interactions and folding processes.

Isolation of novel synthetic prion strains by amplification in transgenic mice coexpressing wild-type and anchorless prion proteins.

Mammalian prions are thought to consist of misfolded aggregates (protease-resistant isoform of the prion protein [PrP(res)]) of the cellular prion protein (PrP(C)). Transmissible spongiform encephalopathy (TSE) can be induced in animals inoculated with recombinant PrP (rPrP) amyloid fibrils lacking mammalian posttranslational modifications, but this induction is inefficient in hamsters or transgenic mice overexpressing glycosylphosphatidylinositol (GPI)-anchored PrP(C). Here we show that TSE can be initiated by inoculation of misfolded rPrP into mice that express wild-type (wt) levels of PrP(C) and that synthetic prion strain propagation and selection can be affected by GPI anchoring of the host's PrP(C). To create prions de novo, we fibrillized mouse rPrP in the absence of molecular cofactors, generating fibrils with a PrP(res)-like protease-resistant banding profile. These fibrils induced the formation of PrP(res) deposits in transgenic mice coexpressing wt and GPI-anchorless PrP(C) (wt/GPI(-)) at a combined level comparable to that of PrP(C) expression in wt mice. Secondary passage into mice expressing wt, GPI(-), or wt plus GPI(-) PrP(C) induced TSE disease with novel clinical, histopathological, and biochemical phenotypes. Contrary to laboratory-adapted mouse scrapie strains, the synthetic prion agents exhibited a preference for conversion of GPI(-) PrP(C) and, in one case, caused disease only in GPI(-) mice. Our data show that novel TSE agents can be generated de novo solely from purified mouse rPrP after amplification in mice coexpressing normal levels of wt and anchorless PrP(C). These observations provide insight into the minimal elements required to create prions in vitro and suggest that the PrP(C) GPI anchor can modulate the propagation of synthetic TSE strains.

FluBlok, a next generation influenza vaccine manufactured in insect cells.

FluBlok, a recombinant trivalent hemagglutinin (rHA) vaccine produced in insect cell culture using the baculovirus expression system, provides an attractive alternative to the current egg-based trivalent inactivated influenza vaccine (TIV). Its manufacturing process presents the possibility for safe and expeditious vaccine production. FluBlok contains three times more HA than TIV and does not contain egg-protein or preservatives. The high purity of the antigen enables administration at higher doses without a significant increase in side-effects in human subjects. The insect cell-baculovirus production technology is particularly suitable for influenza where annual adjustment of the vaccine is required. The baculovirus-insect expression system is generally considered a safe production system, with limited growth potential for adventitious agents. Still regulators question and challenge the safety of this novel cell substrate as FluBlok continues to advance toward product approval. This review provides an overview of cell substrate characterization for expresSF cell line used for the manufacturing of FluBlok. In addition, this review includes an update on the clinical development of FluBlok. The highly purified protein vaccine, administered at three times higher antigen content than TIV, is well tolerated and results in stronger immunogenicity, a long lasting immune response and provides cross-protection against drift influenza viruses.

Molecular and phylogenetic analyses of bovine rhinovirus type 2 shows it is closely related to foot-and-mouth disease virus.

Bovine rhinovirus 2 (BRV2), a causative agent of respiratory disease in cattle, is tentatively assigned to the genus Rhinovirus in the family Picornaviridae. A nearly full-length cDNA of the BRV2 genome was cloned and the nucleotide sequence determined. BRV2 possesses a putative leader proteinase, a small 2A protein and a poly(C) tract, which are characteristic of aphthoviruses. Alignment of BRV-2 and FMDV polyproteins showed that 41% of amino acids were identical within the P1 region. Furthermore, 2A, 2C, 3B(3), 3C and 3D proteins are as much as 67%, 52%, 52%, 50%, and 64% identical, respectively. BRV2 leader protein is rapidly released from the viral polyprotein and cleaves eIF4G at a rate similar to FMDV leader proteinase, suggesting a functional relationship between the leader protein in these viruses. The results suggest that BRV2 is closely related to FMDV and should therefore be considered as a new species within the genus Aphthovirus.

Construction and characterization of new piggyBac vectors for constitutive or inducible expression of heterologous gene pairs and the identification of a previously unrecognized activator sequence in piggyBac.

BACKGROUND: We constructed and characterized several new piggyBac vectors to provide transposition of constitutively- or inducibly-expressible heterologous gene pairs. The dual constitutive control element consists of back-to-back copies of a baculovirus immediate early (ie1) promoter separated by a baculovirus enhancer (hr5). The dual inducible control element consists of back-to-back copies of a minimal cytomegalovirus (CMVmin) promoter separated by a synthetic operator (TetO7), which drives transcription in the presence of a mutant transcriptional repressor plus tetracycline. RESULTS: Characterization of these vectors revealed an unexpected position effect, in which heterologous genes adjacent to the 3'- terminal region ("rightward" genes) were consistently expressed at higher levels than those adjacent to the 5'-terminal region ("leftward" genes) of the piggyBac element. This position effect was observed with all six heterologous genes examined and with both transcriptional control elements. Further analysis demonstrated that this position effect resulted from stimulation of rightward gene expression by the internal domain sequence of the 3'-terminal region of piggyBac. Inserting a copy of this sequence into the 5'- terminal repeat region of our new piggyBac vectors in either orientation stimulated leftward gene expression. Representative piggyBac vectors designed for constitutive or inducible expression of heterologous gene pairs were shown to be functional as insect transformation vectors. CONCLUSION: This study is significant because (a) it demonstrates the utility of a strategy for the construction of piggyBac vectors that can provide constitutive or inducible heterologous gene pair expression and (b) it reveals the presence of a previously unrecognized transcriptional activator in piggyBac, which is an important and increasingly utilized transposable element.

Molecular cloning and functional characterization of beta-N-acetylglucosaminidase genes from Sf9 cells.

Sf9, a cell line derived from the lepidopteran insect, Spodoptera frugiperda, is widely used as a host for recombinant glycoprotein expression and purification by baculovirus vectors. Previous studies have shown that this cell line has one or more beta-N-acetylglucosaminidase activities that may be involved in the degradation and/or processing of N-glycoprotein glycans. However, these enzymes and their functions remain poorly characterized. Therefore, the goal of this study was to isolate beta-N-acetylglucosaminidase genes from Sf9 cells, over-express the gene products, and characterize their enzymatic activities. A degenerate PCR approach yielded three Sf9 cDNAs, which appeared to encode two distinct beta-N-acetylglucosaminidases, according to bioinformatic analyses. Baculovirus-mediated expression of these two cDNA products induced membrane-associated beta-N-acetylglucosaminidase activities in Sf9 cells, which cleaved terminal N-acetylglucosamine residues from the alpha-3 and -6 branches of a biantennary N-glycan substrate with acidic pH optima and completely hydrolyzed chitotriose to its constituent N-acetylglucosamine monomers. GFP-tagged forms of both enzymes exhibited punctate cytoplasmic fluorescence, which did not overlap with either lysosomal or Golgi-specific dyes. Together, these results indicated that the two new Sf9 genes identified in this study encode broad-spectrum beta-N-acetylglucosaminidases that appear to have unusual intracellular distributions. Their relative lack of substrate specificity and acidic pH optima are consistent with a functional role for these enzymes in glycoprotein glycan and chitin degradation, but not with a role in N-glycoprotein glycan processing.

A transgenic insect cell line engineered to produce CMP-sialic acid and sialylated glycoproteins.

We have previously engineered transgenic insect cell lines to express mammalian glycosyltransferases and showed that these cells can sialylate N-glycoproteins, despite the fact that they have little intracellular sialic acid and no detectable CMP-sialic acid. In the accompanying study, we presented evidence that these cell lines can salvage sialic acids for de novo glycoprotein sialylation from extracellular sialoglycoproteins, such as fetuin, found in fetal bovine serum. This finding led us to create a new transgenic insect cell line designed to synthesize its own sialic acid and CMP-sialic acid. SfSWT-1 cells, which encode five mammalian glycosyltransferases, were transformed with two additional mammalian genes that encode sialic acid synthase and CMP-sialic acid synthetase. The resulting cell line expressed all seven mammalian genes, produced CMP-sialic acid, and sialylated a recombinant glycoprotein when cultured in a serum-free growth medium supplemented with N-acetylmannosamine. Thus the addition of mammalian genes encoding two enzymes involved in CMP-sialic acid biosynthesis yielded a new transgenic insect cell line, SfSWT-3, that can sialylate recombinant glycoproteins in the absence of fetal bovine serum. This new cell line will be widely useful as an improved host for baculovirus-mediated recombinant glycoprotein production.