Danielle Blondel

Interaction of the Rabies Virus P Protein with the LC8 Dynein Light Chain

ABSTRACT The rabies virus P protein is involved in viral transcription and replication but its precise function is not clear. We investigated the role of P (CVS strain) by searching for cellular partners by using a two-hybrid screening of a PC12 cDNA library. We isolated a cDNA encoding a 10-kDa dynein light chain (LC8). LC8 is a component of cytoplasmic dynein involved in the minus end-directed movement of organelles along microtubules. We confirmed that this molecule interacts with P by coimmunoprecipitation in infected cells and in cells transfected with a plasmid encoding P protein. LC8 was also detected in virus particles. Series of deletions from the N- and C-terminal ends of P protein were used to map the LC8-binding domain to the central part of P (residues 138 to 172). These results are relevant to speculate that dynein may be involved in the axonal transport of rabies virus along microtubules through neuron cells.

Low-affinity nerve-growth factor receptor (P75NTR) can serve as a receptor for rabies virus.

A random‐primed cDNA expression library constructed from the mRNA of neuroblastoma cells (NG108) was used to clone a specific rabies virus (RV) receptor. A soluble form of the RV glycoprotein (Gs) was utilized as a ligand to detect positive cells. We identified the murine low‐affinity nerve‐growth factor receptor, p75NTR. BSR cells stably expressing p75NTR were able to bind Gs and G‐expressing lepidopteran cells. The ability of the RV glycoprotein to bind p75NTR was dependent on the presence of a lysine and arginine in positions 330 and 333 respectively of antigenic site III, which is known to control virus penetration into motor and sensory neurons of adult mice. P75NTR‐expressing BSR cells were permissive for a non‐adapted fox RV isolate (street virus) and nerve growth factor (NGF) decreased this infection. In infected cells, p75NTR associates with the RV glycoprotein and could be precipitated with anti‐G monoclonal antibodies. Therefore, p75NTR is a receptor for street RV.

Rabies Virus P Protein Interacts with STAT1 and Inhibits Interferon Signal Transduction Pathways

ABSTRACT Rabies virus P protein is a cofactor of RNA polymerase. We investigated other potential roles of P (CVS strain) by searching for cellular partners using two-hybrid screening. We isolated a cDNA encoding the signal transducer and activator of transcription 1 (STAT1) that is a critical component of interferon type I (IFN-α/β) and type II (IFN-γ) signaling. We confirmed this interaction by glutathione S-transferase-pull-down assay. Deletion mutant analysis indicated that the carboxy-terminal part of P interacted with a region containing the DNA-binding domain and the coiled-coil domain of STAT1. The expression of P protein inhibits IFN-α- and IFN-γ-induced transcriptional responses, thus impairing the IFN-induced antiviral state. Mechanistic studies indicate that P protein does not induce STAT1 degradation and does not interfere with STAT1 phosphorylation but prevents IFN-induced STAT1 nuclear accumulation. These results indicate that rabies P protein overcomes the antiviral response of the infected cells.

Rabies virus P and small P products interact directly with PML and reorganize PML nuclear bodies.

The interferon-induced promyelocytic leukaemia (PML) protein localizes both in the nucleoplasm and in matrix-associated multi-protein complexes known as nuclear bodies (NBs). NBs are disorganized in acute promyelocytic leukaemia or during some viral infections, suggesting that PML NBs could be a part of cellular defense mechanism. Rabies virus, a member of the rhabdoviridae family, replicates in the cytoplasm. Rabies phosphoprotein P and four other amino-terminally truncated products (P2, P3, P4, P5) are all translated from P mRNA. P and P2 are located in the cytoplasm, whereas P3, P4 and P5 are found mostly in the nucleus. Infection with rabies virus reorganized PML NBs. PML NBs became larger and appeared as dense aggregates when analysed by confocal or electron microscopy, respectively. The expression of P sequesters PML in the cytoplasm where both proteins colocalize, whereas that of P3 results in an increase in PML body size, as observed in infected cells. The P and P3 interacted directly in vivo and in vitro with PML. The C-terminal domain of P and the PML RING finger seem to be involved in this binding. Moreover, PML−/− primary mouse embryonic fibroblasts expressed viral proteins at a higher level and produced 20 times more virus than wild-type cells, suggesting that the absence of all PML isoforms resulted in an increase in rabies virus replication.

Functional Characterization of Negri Bodies (NBs) in Rabies Virus-Infected Cells: Evidence that NBs Are Sites of Viral Transcription and Replication

ABSTRACT Rabies virus infection induces the formation of cytoplasmic inclusion bodies that resemble Negri bodies found in the cytoplasm of some infected nerve cells. We have studied the morphogenesis and the role of these Negri body-like structures (NBLs) during viral infection. The results indicate that these spherical structures (one or two per cell in the initial stage of infection), composed of the viral N and P proteins, grow during the virus cycle before appearing as smaller structures at late stages of infection. We have shown that the microtubule network is not necessary for the formation of these inclusion bodies but is involved in their dynamics. In contrast, the actin network does not play any detectable role in these processes. These inclusion bodies contain Hsp70 and ubiquitinylated proteins, but they are not misfolded protein aggregates. NBLs, in fact, appear to be functional structures involved in the viral life cycle. Specifically, using in situ fluorescent hybridization techniques, we show that all viral RNAs (genome, antigenome, and every mRNA) are located inside the inclusion bodies. Significantly, short-term RNA labeling in the presence of BrUTP strongly suggests that the NBLs are the sites where viral transcription and replication take place.

Herpesvirus Capsid Association with the Nuclear Pore Complex and Viral DNA Release Involve the Nucleoporin CAN/Nup214 and the Capsid Protein pUL25

ABSTRACT After penetrating the host cell, the herpesvirus capsid is transported to the nucleus along the microtubule network and docks to the nuclear pore complex before releasing the viral DNA into the nucleus. The viral and cellular interactions involved in the docking process are poorly characterized. However, the minor capsid protein pUL25 has recently been reported to be involved in viral DNA uncoating. Here we show that herpes simplex virus type 1 (HSV-1) capsids interact with the nucleoporin CAN/Nup214 in infected cells and that RNA silencing of CAN/Nup214 delays the onset of viral DNA replication in the nucleus. We also show that pUL25 interacts with CAN/Nup214 and another nucleoporin, hCG1, and binds to the pUL36 and pUL6 proteins, two other components of the herpesvirus particle that are known to be important for the initiation of infection and viral DNA release. These results identify CAN/Nup214 as being a nuclear receptor for the herpesvirus capsid and pUL25 as being an interface between incoming capsids and the nuclear pore complex and as being a triggering element for viral DNA release into the nucleus.

IN VIVO INTERACTION OF RABIES VIRUS PHOSPHOPROTEIN (P) AND NUCLEOPROTEIN (N) : EXISTENCE OF TWO N-BINDING SITES ON P PROTEIN

The rabies virus phosphoprotein (P) and nucleoprotein (N) are involved in transcription and replication of the viral genome. Interaction between N and P was studied in vivo in transfected cells expressing both proteins. Co-immunoprecipitation assays revealed that the N-P complex is present in cells expressing both proteins as well as in infected cells. Furthermore, immunostaining showed that coexpression of N and P was sufficient to induce the formation of cytoplasmic inclusions similar to those found in infected cells. In addition, deletion mutant analysis of P was performed to identify the regions of P interacting with N. The results indicate that at least two independent N-binding sites exist on P protein: one is located in the carboxy-terminal part of the protein and another between amino acids 69 and 177. The formation of cytoplasmic inclusions seems to require the presence of both N-binding sites on P protein.

The Nucleocytoplasmic Rabies Virus P Protein Counteracts Interferon Signaling by Inhibiting both Nuclear Accumulation and DNA Binding of STAT1

ABSTRACT Rabies virus P protein inhibits alpha interferon (IFN-α)- and IFN-γ-stimulated Jak-STAT signaling by retaining phosphorylated STAT1 in the cytoplasm. Here, we show that P also blocks an intranuclear step that is the STAT1 binding to the DNA promoter of IFN-responsive genes. As P is a nucleocytoplasmic shuttling protein, we first investigated the effect of the cellular distribution of P on the localization of STAT1 and consequently on IFN signaling. We show that the localization of STAT1 is correlated with the localization of P: in cells expressing a nuclear form of P (the short P3 isoform or the complete P in the presence of the export inhibitor leptomycin B), STAT1 is nuclear, whereas in cells expressing a cytoplasmic form of P, STAT1 is cytoplasmic. However, the expression of nuclear forms of P inhibits the signaling of both IFN-γ and IFN-α, demonstrating that the retention of STAT1 in the cytoplasm is not the only mechanism involved in the inhibition of IFN signaling. Electrophoretic mobility shift analysis indicates that P expression in the cell extracts of infected cells or in stable cell lines prevents IFN-induced DNA binding of STAT1. The loss of the DNA binding of STAT1 and ISGF3 was also observed when purified recombinant P or P3 was added to the extracts of IFN-γ- or IFN-α-treated cells, indicating that P directly affects the DNA binding activity of STAT1. Then products of the rabies virus P gene are able to counteract IFN signaling by creating both cytoplasmic and nuclear blocks for STAT1.

Structure of Recombinant Rabies Virus Nucleoprotein-RNA Complex and Identification of the Phosphoprotein Binding site

ABSTRACT Rabies virus nucleoprotein (N) was produced in insect cells, in which it forms nucleoprotein-RNA (N-RNA) complexes that are biochemically and biophysically indistinguishable from rabies virus N-RNA. We selected recombinant N-RNA complexes that were bound to short insect cellular RNAs which formed small rings containing 9 to 11 N monomers. We also produced recombinant N-RNA rings and viral N-RNA that were treated with trypsin and that had lost the C-terminal quarter of the nucleoprotein. Trypsin-treated N-RNA no longer bound to recombinant rabies virus phosphoprotein (the viral polymerase cofactor), so the presence of the C-terminal part of N is needed for binding of the phosphoprotein. Both intact and trypsin-treated recombinant N-RNA rings were analyzed with cryoelectron microscopy, and three-dimensional models were calculated from single-particle image analysis combined with back projection. Nucleoprotein has a bilobed shape, and each monomer has two sites of interaction with each neighbor. Trypsin treatment cuts off part of one of the lobes without shortening the protein or changing other structural parameters. Using negative-stain electron microscopy, we visualized phosphoprotein bound to the tips of the N-RNA rings, most likely at the site that can be removed by trypsin. Based on the shape of N determined here and on structural parameters derived from electron microscopy on free rabies virus N-RNA and from nucleocapsid in virus, we propose a low-resolution model for rabies virus N-RNA in the virus.

Characterization of rabies virus nucleocapsids and recombinant nucleocapsid-like structures.

Rabies virus nucleoprotein (N) was produced in insect cells using the baculovirus expression system described by Préhaud et al. (Virology 178, 486-497, 1990). The protein was either purified on a CsCl gradient, resulting in a mixture of nucleocapsid-like structures and beaded rings, as observed by electron microscopy, or on a glycerol gradient that resulted in a preparation of the rings only. The rings and nucleocapsid-like structures had the same morphological characteristics as viral nucleocapsids. N in these structures is an 84 A long and thin molecule that is spaced at around 34 A along the length of the nucleocapsid, identical in shape and spacing as the nucleoprotein in nucleocapsids of rabies virus and very similar to those of vesicular stomatitis virus. The recombinant nucleocapsids contained RNA with a stoichiometry similar to that found in viral nucleocapsids. The RNA bound in the beaded rings was a subset of the insect cellular RNA. One of the RNA species was partially sequenced and, although a positive identification could not be made, could correspond to a tRNA. With respect to sensitivity to trypsin and RNase digestion, the recombinant and viral nucleocapsids behaved similar. Trypsin cleaved a 17 kDa fragment from the carboxy terminus of N with only a very small effect on the morphology of the nucleocapsids. RNase A completely digested the resident RNA in both viral and recombinant nucleocapsids into fragments of 4-5 nt long, again with no effect on the morphology of the nucleocapsids. Thus, when the RNA is cleaved, the structure must be maintained by protein-protein contacts. Experiments to remove the resident RNA from viral and recombinant rabies virus nucleocapsids failed, whereas the same methods used to eliminate the RNA from vesicular stomatitis virus nucleocapsids was successful.