Peter Mastrangelo

Identification of nucleolin as a cellular receptor for human respiratory syncytial virus

Human respiratory syncytial virus (RSV) causes a large burden of disease worldwide. There is no effective vaccine or therapy, and the use of passive immunoprophylaxis with RSV-specific antibodies is limited to high-risk patients. The cellular receptor (or receptors) required for viral entry and replication has yet to be described; its identification will improve understanding of the pathogenesis of infection and provide a target for the development of novel antiviral interventions. Here we show that RSV interacts with host-cell nucleolin via the viral fusion envelope glycoprotein and binds specifically to nucleolin at the apical cell surface in vitro. We observed decreased RSV infection in vitro in neutralization experiments using nucleolin-specific antibodies before viral inoculation, in competition experiments in which virus was incubated with soluble nucleolin before inoculation of cells, and upon RNA interference (RNAi) to silence cellular nucleolin expression. Transfection of nonpermissive Spodoptera frugiperda Sf9 insect cells with human nucleolin conferred susceptibility to RSV infection. RNAi-mediated knockdown of lung nucleolin was associated with a significant reduction in RSV infection in mice (P = 0.0004), confirming that nucleolin is a functional RSV receptor in vivo.

Doppel-induced cerebellar degeneration in transgenic mice

Doppel (Dpl) is a paralog of the mammalian prion protein (PrP); it is abundant in testes but expressed at low levels in the adult central nervous system. In two Prnp-deficient (Prnp0/0) mouse lines (Ngsk and Rcm0), Dpl overexpression correlated with ataxia and death of cerebellar neurons. To determine whether Dpl overexpression, rather than the dysregulation of genes neighboring the Prn gene complex, was responsible for the ataxic syndrome, we placed the mouse Dpl coding sequence under the control of the Prnp promoter and produced transgenic (Tg) mice on the Prnp0/0-ZrchI background (hereafter referred to as ZrchI). ZrchI mice exhibit neither Dpl overexpression nor cerebellar degeneration. In contrast, Tg(Dpl)ZrchI mice showed cerebellar granule and Purkinje cell loss; the age of onset of ataxia was inversely proportional to the levels of Dpl protein. Crosses of Tg mice overexpressing wild-type PrP with two lines of Tg(Dpl)ZrchI mice resulted in a phenotypic rescue of the ataxic syndrome, while Dpl overexpression was unchanged. Restoration of PrP expression also rendered the Tg(Dpl) mice susceptible to prion infection, with incubation times indistinguishable from non-Tg controls. Whereas the rescue of Dpl-induced neurotoxicity by coexpression of PrP argues for an interaction between the PrP and Dpl proteins in vivo, the unaltered incubation times in Tg mice overexpressing Dpl in the central nervous system suggest that Dpl is unlikely to be involved in prion formation.

The CNS glycoprotein Shadoo has PrPC-like protective properties and displays reduced levels in prion infections

The cellular prion protein, PrPC, is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS‐expressed Doppel or internally deleted PrP (‘ΔPrP’). This paradigm has facilitated mapping of activity determinants in PrPC and implicated a cryptic PrPC‐like protein, ‘π’. Shadoo (Sho) is a hypothetical GPI‐anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N‐terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrPC, but is low in cerebellar granular neurons (CGNs) containing PrPC and high in PrPC‐deficient dendritic processes. In Prnp0/0 CGNs, Sho transgenes were PrPC‐like in their ability to counteract neurotoxic effects of either Doppel or ΔPrP. Additionally, prion‐infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for π, and since it engenders a PrPC‐like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology.

The prion gene complex encoding PrPC and Doppel: insights from mutational analysis

The prion protein gene, Prnp, encodes PrP(Sc), the major structural component of prions, infectious pathogens causing a number of disorders including scrapie and bovine spongiform encephalopathy (or BSE). Missense mutations in the human Prnp gene cause inherited prion diseases such as familial Creutzfeldt-Jakob disease. In uninfected animals Prnp encodes a glycophosphatidylinositol (GPI)-anchored protein denoted PrP(C) and in prion infections PrP(C) is converted to PrP(Sc) by templated refolding. Though Prnp is conserved in mammalian species, attempts to verify interactions of putative PrP binding proteins by genetic means have proven frustrating and the ZrchI and Npu lines of Prnp gene-ablated mice (Prnp(0/0) mice) lacking PrP(C) remain healthy throughout development. This indicates that PrP(C) serves a function that is not apparent in a laboratory setting or that other molecules have overlapping functions. Current possibilities involve shuttling or sequestration of synaptic Cu(II) via binding to N-terminal octapeptide residues and/or signal transduction involving the fyn kinase. A new point of entry into the issue of prion protein function has emerged from identification of a paralogue, Prnd, with 24% coding sequence identity to Prnp. Prnd lies downstream of Prnp and encodes the doppel (Dpl) protein. Like PrP(C), Dpl is presented on the cell surface via a GPI anchor and has three alpha-helices: however, it lacks the conformationally plastic and octapeptide repeat domains present in its well-known relative. Interestingly, Dpl is overexpressed in the Ngsk and Rcm0 lines of Prnp(0/0) mice via intergenic splicing events. These lines of Prnp(0/0) mice exhibit ataxia and apoptosis of cerebellar cells, indicating that ectopic synthesis of Dpl protein is toxic to central nervous system neurons: this inference has now been confirmed by the construction of transgenic mice expressing Dpl under the direct control of the PrP promoter. Remarkably, Dpl-programmed ataxia is rescued by wild-type Prnp transgenes. The interaction between the Prnp and Prnd genes in mouse cerebellar neurons may have a physical correlate in competition between Dpl and PrP(C) within a common biochemical pathway that when mis-regulated leads to apoptosis.

RSV Fusion: Time for a New Model

In this review we propose a partially hypothetical model of respiratory syncytial virus (RSV) binding and entry to the cell that includes the recently discovered RSV receptor nucleolin, in an attempt to stimulate further inquiry in this research area. RSV binding and entry is likely to be a two-step process, the first involving the attachment of the virus to the cell membrane, which may be enhanced by electrostatic interactions with cellular glycoproteins/heparin and the viral G protein, and the second involving fusion to the cell membrane mediated by the viral F protein and a specific cellular fusion receptor. With our recent discovery of nucleolin as a functional fusion receptor for RSV, comes the possibility of a number of new approaches to the development of novel strategies for RSV prophylaxis and therapy, as well as raising some new questions concerning the pathobiology of RSV infection and tropism.

Respiratory syncytial virus receptor expression in the mouse and viral tropism

Human respiratory syncytial virus (RSV) infects airway epithelium and can cause serious illnesses such as bronchiolitis and pneumonia. With the discovery of cell-surface nucleolin as a fusion receptor for RSV, the question arose as to whether nucleolin could explain RSV tropism in vivo. Here, we report the distribution of cell-surface nucleolin expression in tissues of normal mice and how this distribution of expression relates to what is known about RSV tropism and its clinical manifestations. Our results show evidence of cell-surface nucleolin expression in the respiratory tract. In addition, cell-surface nucleolin is expressed in tissues outside of the respiratory tract, many of which correspond to previous reports of tissue-specific RSV infection, and others that may allude to additional potential sites for RSV infection in vivo. Furthermore, our work provides a foundation for the investigation of nucleolins physiological function in various healthy mammalian tissues.

The RSV fusion receptor: not what everyone expected it to be.

This article reviews current knowledge about respiratory syncytial virus (RSV) binding and entry into cells. The recent discovery of Nucleolin as a fusion receptor for RSV opens new avenues for developing interventions, while raising questions concerning RSV pathobiology and tropism. We also discuss characteristics of a good RSV drug target.

A cluster of familial Creutzfeldt-Jakob disease mutations recapitulate conserved residues in Doppel: a case of molecular mimicry?

Intrachromosomal deletions linking Dpl expression to the PrP promoter produce cerebellar degeneration that can be abrogated by the introduction of wild‐type PrP transgenes. Since Dpl‐like truncated forms of PrP are neuropathogenic in mice and likewise counterbalanced by expression of PrPC we asked whether naturally occurring mutant forms of human PrP have Dpl‐like attributes. Five PRNP missense mutations causing familial Creutzfeldt–Jakob disease (F‐CJD) map to a helical region found in both PrPC and Dpl and result in amino acids identical to conserved residues in Dpl. These F‐CJD alleles may cause mutant PrP to become a weak mimetic of Dpl structure and/or function.

Host and Viral Factors in Respiratory Syncytial Virus Infection

Respiratory syncytial virus (RSV) is a major worldwide pathogen for which there is still no effective vaccine or antiviral treatment available, and immunoprophylaxis with RSV-specific antibodies (e.g., palivizumab) is used in limited clinical settings. In this review, we discuss virus–host interactions relevant to RSV pathobiology and how advances in cell and systems biology have accelerated knowledge in this area. We also highlight recent advances in understanding the relationship between RSV bronchiolitis and sequelae of recurrent wheezing and asthma, new findings into an intriguing interaction between RSV and air pollution, and exciting developments toward the goal of realizing a safe and effective RSV vaccine.

Targeting Host Cell Surface Nucleolin for RSV Therapy: Challenges and Opportunities

Nucleolin (NCL) has been reported as a cellular receptor for the human respiratory syncytial virus (RSV). We studied the effects of re-purposing AS1411, an anti-cancer compound that binds cell surface NCL, as a possible novel strategy for RSV therapy in vitro and in vivo. AS1411 was administered to RSV-infected cultures of non-polarized (HEp-2) and polarized (MDCK) epithelial cells and to virus-infected mice and cotton rats. Results of in vitro experiments showed that AS1411, used in micromolar concentrations, was associated with decreases in the number of virus-positive cells. Intranasal administration of AS1411 (50 mg/kg) to RSV-infected mice and cotton rats was associated with partial reductions in lung viral titers, decreased virus-associated airway inflammation, and decreased IL-4/IFN-γ ratios when compared to untreated, infected animals. In conclusion, our findings indicate that therapeutic use of AS1411 has modest effects on RSV replication and host response. While the results underscore the challenges of targeting cell surface NCL as a potential novel strategy for RSV therapy, they also highlight the potential of cell surface NCL as a therapeutic target.