Classic osteogenesis imperfecta, an autosomal dominant disorder associated with osteoporosis and bone fragility, is caused by mutations in the genes for type I collagen. A recessive form of the disorder has long been suspected. Since the loss of cartilage-associated protein (CRTAP), which is required for post-translational prolyl 3-hydroxylation of collagen, causes severe osteoporosis in mice, we investigated whether CRTAP deficiency is associated with recessive osteogenesis imperfecta. Three of 10 children with lethal or severe osteogenesis imperfecta, who did not have a primary collagen defect yet had excess post-translational modification of collagen, were found to have a recessive condition resulting in CRTAP deficiency, suggesting that prolyl 3-hydroxylation of type I collagen is important for bone formation.

The human polyomavirus JC virus (JCV) is the etiologic agent of a fatal central nervous system (CNS) demyelinating disease known as progressive multifocal leukoencephalopathy (PML). PML occurs predominantly in immunosuppressed patients and has increased dramatically as a result of the AIDS pandemic. The major target cell of JCV infection and lytic replication in the CNS is the oligodendrocyte. The mechanisms by which JCV initiates and establishes infection of these glial cells are not understood. The initial interaction between JCV and glial cells involves virus binding to N-linked glycoproteins containing terminal ␣(2-6)-linked sialic acids. The subsequent steps of entry and targeting of the viral genome to the nucleus have not been described. In this report, we compare the kinetics and mechanisms of infectious entry of JCV into human glial cells with that of the related polyomavirus, simian virus 40 (SV40). We demonstrate that JCV, unlike SV40, enters glial cells by receptor-mediated clathrin-dependent endocytosis.JC virus (JCV) is a small, nonenveloped, double-stranded DNA containing virus belonging to the family Papovaviridae and the subfamily Polyomavirinae (23, 26). In vivo, JCV infection is restricted to oligodendrocytes, astrocytes, and B lymphocytes (17,20). This highly restricted cell type specificity is also seen in vitro, as JCV infects primary cultures of human glial cells, human glial cell lines, and to a limited extent, primary human B cells and some B-cell lines (4,17,20,28). The life cycle of JCV begins with virus attachment to a cell surface glycoprotein receptor containing ␣-(2-6)-linked sialic acid (15). Following attachment, the JCV virion must penetrate the plasma membrane and target its genome to the nucleus. Very little is known about the mechanisms of polyomavirus entry and nuclear targeting. Early work with the mouse polyomavirus and simian virus 40 (SV40) demonstrated that these virions were internalized into monopinocytotic vesicles which then accumulated at the nuclear membrane (12, 16). In some studies, viral particles were also seen in the nucleus, suggesting that the nucleus was the site of uncoating (7,19,21). More recent studies have shown that SV40 enters cells by receptor-mediated endocytosis into uncoated membrane-bound invaginations known as caveolae (1,2,24). An interaction between SV40 and major histocompatibility complex-encoded class I proteins induces the clustering of virus-receptor complexes into caveolin-rich membrane domains (5,8,24). Intracellular signals induced by SV40 binding to cells result in increased caveola-dependent endocytosis of the virus and delivery of the virions to the endoplasmic reticulum (9, 24). It is unclear how the viral genome is then targeted from the endoplasmic reticulum to the nucleus.In this report, we studied the kinetics of JCV and SV40 infectious entry into human glial cells. Our results demonstrate that JCV rapidly enters glial cells and is completely internalized into a neutralizing antibody-resistant compartment within 30 min. SV40 ...

Prion diseases are fatal neurodegenerative disorders caused by aberrant metabolism of the cellular prion protein (PrPC). In genetic forms of these diseases, mutations in the globular C-terminal domain are hypothesized to favor the spontaneous generation of misfolded PrP conformers (including the transmissible PrPSc form) that trigger downstream pathways leading to neuronal death. A mechanistic understanding of these diseases therefore requires knowledge of the quality control pathways that recognize and degrade aberrant PrPs. Here, we present comparative analyses of the biosynthesis, trafficking, and metabolism of a panel of genetic disease-causing prion protein mutants in the C-terminal domain. Using quantitative imaging and biochemistry, we identify a misfolded subpopulation of each mutant PrP characterized by relative detergent insolubility, inaccessibility to the cell surface, and incomplete glycan modifications. The misfolded populations of mutant PrPs were neither recognized by ER quality control pathways nor routed to ER-associated degradation despite demonstrable misfolding in the ER. Instead, mutant PrPs trafficked to the Golgi, from where the misfolded subpopulation was selectively trafficked for degradation in acidic compartments. Surprisingly, selective re-routing was dependent not only on a mutant globular domain, but on an additional lysine-based motif in the highly conserved unstructured N-terminus. These results define a specific trafficking and degradation pathway shared by many disease-causing PrP mutants. As the acidic lysosomal environment has been implicated in facilitating the conversion of PrPC to PrPSc, our identification of a mutant-selective trafficking pathway to this compartment may provide a cell biological basis for spontaneous generation of PrPSc in familial prion disease.