Prions consist of aggregates of abnormal conformers of the cellular prion protein (PrPC). They propagate by recruiting host-encoded PrPC although the critical interacting proteins and the reasons for the differences in susceptibility of distinct cell lines and populations are unknown. We derived a lineage of cell lines with markedly differing susceptibilities, unexplained by PrPC expression differences, to identify such factors. Transcriptome analysis of prion-resistant revertants, isolated from highly susceptible cells, revealed a gene expression signature associated with susceptibility and modulated by differentiation. Several of these genes encode proteins with a role in extracellular matrix (ECM) remodelling, a compartment in which disease-related PrP is deposited. Silencing nine of these genes significantly increased susceptibility. Silencing of Papss2 led to undersulphated heparan sulphate and increased PrPC deposition at the ECM, concomitantly with increased prion propagation. Moreover, inhibition of fibronectin 1 binding to integrin α8 by RGD peptide inhibited metalloproteinases (MMP)-2/9 whilst increasing prion propagation. In summary, we have identified a gene regulatory network associated with prion propagation at the ECM and governed by the cellular differentiation state.

Vaccination with live attenuated simian immunodeficiency virus (SIVmacC8) confers potent, reproducible protection against homologous wild-type virus challenge (SIVmacJ5). The ability of SIVmacC8 to confer resistance to superinfection with an uncloned ex vivo derivative of SIVmac251 (SIVmac32H/L28) was investigated. In naïve, Mauritian-derived cynomolgus macaques (Macaca fascicularis), SIVmac32H/L28 replicated to high peak titres (.10 8 SIV RNA copies ml), persisted at high levels and induced distinctive pathology in lymphoid tissues. In cynomolgus macaques vaccinated with SIVmacC8, no evidence of detectable superinfection was observed in 3/8 vaccinates following challenge 3 or 20 weeks later with SIVmac32H/L28. Analyses after SIVmac32H/L28 challenge revealed a significant reduction in viral RNA (P,0.001) and DNA levels between 20 week vaccinates and challenge controls. Amongst 3 week vaccinates, less potent protection was observed. However, analysis of env from breakthrough virus indicated .99 % sequence similarity with the vaccine virus. Highly sensitive PCR assays that distinguish vaccine and challenge virus stocks demonstrated restimulation of replication of the vaccine virus SIVmacC8 in the face of potent protection against a vigorous, homologous challenge virus. Vaccine-induced antiviral neutralizing antibodies and anti-Nef CD8 + cytotoxic T cell responses did not correlate with the outcome of the challenge. Defining the mechanism of vaccine protection will need to account for the effective control of a genetically closely related challenge virus whilst remaining unable to suppress replication of the pre-existing vaccine virus. The role of innate and intrinsic anti-retroviral immunity in the protection conferred by live attenuated SIV vaccines warrants careful study.

Prions are transmissible agents that cause lethal neurodegeneration in humans and other mammals. Prions bind avidly to metal surfaces such as steel wires and, when surface-bound, can initiate infection of brain or cultured cells with remarkable efficiency. While investigating the properties of metal-bound prions by using the scrapie cell assay to measure infectivity, we observed, at low frequency, positive assay results in control groups in which metal wires had been coated with uninfected mouse brain homogenate. This phenomenon proved to be reproducible in rigorous and exhaustive control experiments designed to exclude prion contamination. The infectivity generated in cell culture could be readily transferred to mice and had strain characteristics distinct from the mouse-adapted prion strains used in the laboratory. The apparent "spontaneous generation" of prions from normal brain tissue could result if the metal surface, possibly with bound cofactors, catalyzed de novo formation of prions from normal cellular prion protein. Alternatively, if prions were naturally present in the brain at levels not detectable by conventional methods, metal surfaces might concentrate them to the extent that they become quantifiable by the scrapie cell assay.cell culture | scrapie | metal | Creutzfeldt-Jakob disease P rions, the transmissible agents that cause fatal neurodegenerative diseases such as Creutzfeldt-Jakob disease in humans, or bovine spongiform encephalopathy and scrapie in animals, occur in the form of various strains and are composed principally or entirely of aggregates of misfolded cellular prion protein (PrP C ), generally referred to as PrP Sc . Replication comes about by autocatalytic conversion of PrP C to the pathogenic isoform (1).In vitro and in vivo studies using model systems have suggested host-encoded cofactors are required to facilitate the propagation of prions (2-5). A variety of polyanionic compounds, lipids, proteoglycans from brain homogenate or purified protein have been shown to stimulate the conversion of PrP C to PrP Sc in vitro (6-10).Steel wire exposed to scrapie prion-infected brain or brain homogenate acquires an extraordinary infectious potential (11,12). Although the amount of protein bound was undetectable by conventional methods, the wires were as effective in eliciting disease as injection of approximately 10 6 LD 50 units in the form of brain homogenate. We have isolated an N2a neuroblastoma-derived cell line, N2aPK1 (i.e., "PK1") (13), that is highly susceptible to infection by a wide spectrum of murine strains, including RML and 22L, but not ME7 and 301C (13,14). When PK1 cells were grown on RML prion-coated wires, adherent cells became PrP Sc -positive, in contrast to neighboring cells, which remained negative, implying that adherence to the prion-coated surface is a prerequisite for infectivity transfer (15). We exploited these observations to detect prions in RML prion-infected brain homogenates diluted as much as 10 −10 , by adsorbing infectivity to steel wire surfaces and sub...