Motohiro Horiuchi

Specific binding of normal prion protein to the scrapie form via a localized domain initiates its conversion to the protease-resistant state.

In the transmissible spongiform encephalopathies, normal prion protein (PrP‐sen) is converted to a protease‐resistant isoform, PrP‐res, by an apparent self‐propagating activity of the latter. Here we describe new, more physiological cell‐free systems for analyzing the initial binding and subsequent conversion reactions between PrP‐sen and PrP‐res. These systems allowed the use of antibodies to map the sites of interaction between PrP‐sen and PrP‐res. Binding of antibodies (α219–232) to hamster PrP‐sen residues 219–232 inhibited the binding of PrP‐sen to PrP‐res and the subsequent generation of PK‐resistant PrP. However, antibodies to several other parts of PrP‐sen did not inhibit. The α219–232 epitope itself was not required for PrP‐res binding; thus, inhibition by α219–232 was likely due to steric blocking of a binding site that is close to, but does not include the epitope in the folded PrP‐sen structure. The selectivity of the binding reaction was tested by incubating PrP‐res with cell lysates or culture supernatants. Only PrP‐sen was observed to bind PrP‐res. This highly selective binding to PrP‐res and the localized nature of the binding site on PrP‐sen support the idea that PrP‐sen serves as a critical ligand and/or receptor for PrP‐res in the course of PrP‐res propagation and pathogenesis in vivo.

Sulfated glycans and elevated temperature stimulate PrPSc-dependent cell-free formation of protease-resistant prion protein

A conformational conversion of the normal, protease‐ sensitive prion protein (PrP‐sen or PrPC) to a protease‐resistant form (PrP‐res or PrPSc) is commonly thought to be required in transmissible spongiform encephalopathies (TSEs). Endogenous sulfated glycosaminoglycans are associated with PrP‐res deposits in vivo, suggesting that they may facilitate PrP‐res formation. On the other hand, certain exogenous sulfated glycans can profoundly inhibit PrP‐res accumulation and serve as prophylactic anti‐TSE compounds in vivo. To investigate the seemingly paradoxical effects of sulfated glycans on PrP‐res formation, we have assayed their direct effects on PrP conversion under physiologically compatible cell‐free conditions. Heparan sulfate and pentosan polysulfate stimulated PrP‐res formation. Conversion was stimulated further by increased temperature. Both elevated temperature and pentosan polysulfate promoted interspecies PrP conversion. Circular dichroism spectropolarimetry measurements showed that pentosan polysulfate induced a conformational change in PrP‐sen that may potentiate its PrP‐res‐induced conversion. These results show that certain sulfated glycosaminoglycans can directly affect the PrP conversion reaction. Therefore, depending upon the circumstances, sulfated glycans may be either cofactors or inhibitors of this apparently pathogenic process.

A CELLULAR FORM OF PRION PROTEIN (PRPC) EXISTS IN MANY NON-NEURONAL TISSUES OF SHEEP

A cellular form of the prion protein (PrPC) is thought to be a substrate for an abnormal isoform of th eprion protein (PrPSc) in scrapie. PrPC is abundant in tissues of the central nervous system, but little is known about the distribution of PrPC in non-neuronal tissues of sheep, the natural host of scrapie. This study investigated the tissue distribution of PrPC in sheep. Although PrPC was abundant in neuronal tissues, it was detected in non-neuronal tissues such as spleen, lymph node, lung, heart, kidney, skeletal muscle, uterus, adrenal gland, parotid gland, intestine, proventriculus, abomasum and mammary gland. Neither PrPC nor PrP mRNA was detected in the liver. The tissue distribution of PrPC appears to be inconsistent with the tissues which possess scrapie infectivity, suggesting that factor(s) specific to certain cell types may be required to support multiplication of the scrapie agent.

Amino acid polymorphisms of PrP with reference to onset of scrapie in Suffolk and Corriedale sheep in Japan

We investigated the relationships between amino acid polymorphisms of the prion protein (PrP), restriction fragment length polymorphisms (RFLP) of the PrP gene and the incidence of natural scrapie in Japan. Six variant alleles of the PrP gene were found in healthy sheep. Based on the substitutions at codons 112, 136, 154 and 171, these allelic variants were designated PrPMARQ, PrPTARQ, PrPMVRQ, PrPMAHQ, PrPMARR and PrPMARH. Each RFLP haplotype (e1h2, e1h2 or e3h1) consisted bo multiple alleles including PrPMARQ. Three of these variant alleles were found in scrapie-affected Suffolk sheep. PrPMARQ was associated with high disease incidence, PrPTARQ and PrPMARR were associated with low disease incidence. We found that one scrapie-affected Suffolk was homozygous for PrPMARR and four PrPSc-positive Suffolks carried PrPMVRQ. Both of two scrapie-affected Corriedales and two out of three scrapie-affected cross-breeds between Suffolk and Corriedale carried PrPMARH, suggesting that this allele associates with high incidence of scrapie in Corriedale and its cross-breeds.

Cellular Prion Protein Promotes Brucella Infection into Macrophages

The products of the Brucella abortus virB gene locus, which are highly similar to conjugative DNA transfer system, enable the bacterium to replicate within macrophage vacuoles. The replicative phagosome is thought to be established by the interaction of a substrate of the VirB complex with macrophages, although the substrate and its host cellular target have not yet been identified. We report here that Hsp60, a member of the GroEL family of chaperonins, of B. abortus is capable of interacting directly or indirectly with cellular prion protein (PrPC) on host cells. Aggregation of PrPC tail-like formation was observed during bacterial swimming internalization into macrophages and PrPC was selectively incorporated into macropinosomes containing B. abortus. Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex–associated process. Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC. Hsp60 of B. abortus, expressed on the surface of Lactococcus lactis, promoted the aggregation of PrPC but not PrPC tail formation on macrophages. The PrPC deficiency prevented swimming internalization and intracellular replication of B. abortus, with the result that phagosomes bearing the bacteria were targeted into the endocytic network. These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection.

Prion protein interconversions and the transmissible spongiform encephalopathies

Autocatalytic changes in the conformation and aggregation state of prion protein appear to be fundamental to transmissible spongiform encephalopathies or prion diseases. Here we review the considerable progress that has been made in describing the normal properties of prion protein and the changes that occur during these devastating neurodegenerative diseases.

Mapping of determinants of the host range for canine cells in the genome of canine parvovirus using canine parvovirus/mink enteritis virus chimeric viruses.

Feline panleukopenia virus (FPLV), mink enteritis virus (MEV) and canine parvovirus (CPV) are more than 98% similar in DNA and predicted amino acid sequences, but they show different host-cell specificities; CPV is able to replicate in canine cells in culture, whereas FPLV and MEV cannot or replicate only to a low titre. To map the genomic region responsible for the host range of CPV in vitro, CPV/MEV chimeric viruses were generated by transfecting infectious CPV/MEV chimeric plasmids into a cultured feline kidney cell line, and their host cell ranges were analysed. The 60 to 91 map units (m.u.) region of the CPV genome, which contains a part of the capsid protein (VP) gene encoding from amino acid 91 (in the VP2 sequence) to the carboxy terminus of VP protein, was required to impart the ability to replicate in canine cells to MEV, although the chimeric virus containing the 60 to 91 m.u. region of the CPV genome in the MEV background did not replicate in canine cells as efficiently as did CPV derived from the infectious plasmid of CPV. Not only the VP gene, but also a part of the NS gene of CPV were considered to participate in the full expression of the ability to replicate in canine cells. Within the 60 to 91 m.u. region, five of nine amino acid changes between MEV-Abashiri and CPV-Y1 were thought to be phylogenetically CPV-common; however, a recombinant virus containing all five amino acid changes of CPV in the MEV background replicated minimally in canine cells.

Photoinactivation of Virus Infectivity by Hypocrellin A

Abstract— We investigated the photoinactivation of virus infectivity by hypocrellin A and its mechanism. The titers of vesicular stomatitis virus (VSV) and human immunodeficiency virus type 1 (HIV‐1), both of which are enveloped viruses, were reduced upon illumination with hypocrellin A in a concentration‐dependent manner, whereas canine parvovirus, a nonenveloped virus, was not killed. The removal of oxygen or addition of sodium azide or bT‐carotene both inhibited VSV inactivation. Mannitol and superoxide dismutase had no effect on VSV inactivation. These results indicate that singlet oxygen was involved in the process of VSV inactivation. Of the three major VSV membrane proteins, peripheral membrane protein M was most damaged by the hypocrellin A phototreatment.

Cutting Edge: Brucella abortus Exploits a Cellular Prion Protein on Intestinal M Cells as an Invasive Receptor

Brucella abortus is a Gram-negative bacterium causing brucellosis. Although B. abortus is known to infect via the oral route, the entry site in the gastrointestinal tract has been unclear. We found that B. abortus was selectively internalized by microfold cells (M cells), a subset of epithelial cells specialized for mucosal Ag uptake. During this process, colocalization of cellular prion protein (PrPC) and B. abortus was evident on the apical surface as well as in subapical vacuolar structures in M cells. Internalization of B. abortus by M cells of PrPC-deficient (Prnp−/−) mice was greatly reduced compared with that in wild-type mice. Furthermore, an oral infection study revealed that translocation of B. abortus into the Peyer’s patch was significantly lower in Prnp−/− than in wild-type mice. These observations suggest that orally infected B. abortus invades the host through M cells by using PrPC on the apical surface of M cells as an uptake receptor.

Improvement of PrPSc-detection in mouse spleen early at the preclinical stage of scrapie with collagenase-completed tissue homogenization and Sarkosyl-NaCl extraction of PrPSc

SummaryScrapie in sheep has recently become again a target of control measures and eradication programs. Crucial for the effectiveness of these measures is the detection of infected sheep during the long and potentially hazardous incubation period. However, routine-diagnosis is mostly limited to clinical examinations when disease becomes apparent, and to postmortem investigations. Through the detection of the scrapie-specific isoform of the prion protein (PrPSc) by Western blot in the spleen and lymph nodes from scrapie-infected mice and sheep, we have shown previously that diagnosis during the preclinical stage is possible. We introduce here an improved method for the diagnosis of mouse scrapie shortly after infection. Through a homogenization procedure that includes a collagenase digestion step, and through extraction and salting-out of PrPSc by Sarkosyl and NaCl, respectively, we were able to detect PrPSc in spleen tissue of intraperitoneally infected mice seven days postinfection. Moreover, the new protocol makes sample-handling easier and reduces the hands-on time. We also successfully enriched PrPSc from spleen tissue through immobilized metal affinity chromatography (IMAC); however, for the diagnosis at the earliest stage of infection, extraction of PrPSc by Sarkosyl and NaCl was more effective.