Replication of Mononegavirales occurs in viral factories which form inclusions in the host-cell cytoplasm. For rabies virus, those inclusions are called Negri bodies (NBs). We report that NBs have characteristics similar to those of liquid organelles: they are spherical, they fuse to form larger structures, and they disappear upon hypotonic shock. Their liquid phase is confirmed by FRAP experiments. Live-cell imaging indicates that viral nucleocapsids are ejected from NBs and transported along microtubules to form either new virions or secondary viral factories. Coexpression of rabies virus N and P proteins results in cytoplasmic inclusions recapitulating NBs properties. This minimal system reveals that an intrinsically disordered domain and the dimerization domain of P are essential for Negri bodies-like structures formation. We suggest that formation of liquid viral factories by phase separation is common among Mononegavirales and allows specific recruitment and concentration of viral proteins but also the escape to cellular antiviral response.

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.

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-␣/␤) and type II (IFN-␥) 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-␣-and IFN-␥-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.The interferon (IFN) response is one of the host response's primary defense mechanisms against viral infection. IFNs are classified as ␣, ␤, , and ␥ on the basis of their structures and antigenic properties: type I is composed of IFN-␣, -␤, and -and type II of only IFN-␥. IFN-␣/␤ is synthesized and secreted by cells in direct response to specifically viral products, including doublestranded RNA which triggers a cascade of kinase reactions and leads to the activation of specific cellular transcription factors. IFN-␣/␤ is produced by most cells as a direct response to viral infection, while IFN-␥ is synthesized almost exclusively by activated NK cells and activated T cells in response to virus-infected cells. Both type I and II IFNs achieve their antiviral effect by binding to their respective receptors (IFN-␣/␤ or IFN-␥ receptor), resulting in the activation of a distinct but related "Janus" tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway (17).Briefly, the interaction of IFN-␣/␤ with its receptors, which consist of IFNAR1 and IFNAR2 molecules, leads to the activation of the "Janus tyrosine kinases" Tyk 2 and Jak1, respectively, via tyrosine phosphorylation. Activated Tyk 2 phosphorylates IFNAR1, which then serves as a binding site for STAT2. STAT2 is then phosphorylated by Tyk 2 on tyrosine 689 and serves as a binding site for STAT1, which in turn is phosphorylated by Jak1 on tyrosine 701. The phosphorylated STATs heterodimerize; the heterodimers dissociate from the receptors and bind to the DNA-binding protein p48 (IFN regulatory factor 9 [IRF-9]) to form the complex IFN-stimulated growth factor 3 (ISGF3). The heterotrimer complexes translocate into the nucleus and bind to the IFN-stimulated response element (ISRE) to induce IFN-stimulated genes (ISGs). The binding of IFN-␥ to the IFN-␥ receptors IFNGR1 and IFNGR2 leads to the activation of the Janus kinases Jak1 and Jak2, respectively, via tyrosine phosphorylation, which in turn phosphorylate STAT1 on tyrosine. STAT1 homodimers ...

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.Rhabdoviruses have a single-stranded negative-sense RNA genome (11 to 15 kb) that is tightly encapsidated by the viral nucleoprotein (N) to form a ribonucleoprotein (RNP). This RNP serves as the template for viral transcription and replication. During transcription, a positive-strand leader RNA and five mRNAs are synthesized. The replication process yields nucleocapsids containing full-length antisense genome RNA which in turn serves as a template for the synthesis of sense genome RNA. The active virus-encoded RNA polymerase complex consists of the large protein (L) and its cofactor, the phosphoprotein (P) (13). The L protein is a multifunctional enzyme and acts as the RNA-dependent RNA polymerase. This polymerase complex carries out all the enzymatic steps of transcription, including the initiation and elongation of transcripts, and cotranscriptional modifications of RNAs, such as capping and polyadenylation (2). The functions of the P protein are not clear. Studies with vesicular stomatitis virus (VSV) have shown that the P protein works as a noncatalytic cofactor of the viral RNA polymerase and as a molecular chaperone helping the viral N protein to bind specifically and correctly to the nascent RNA chain during genome replication. VSV P protein has different phosphorylation states that are believed to bind to the RNP with different affinities and to have different transcription activities (3, 4, 17). The VSV P protein has also been shown to form oligomers, and oligomerization seems to be necessary for binding both to the L protein and to the template (16).By analogy with the VSV P protein, rabies virus P protein is also thought to act as a chaperone and to be a noncatalytic subunit of the viral RNA polymerase. In vitro and in vivo studies have shown that rabies virus P protein forms specific complexes with N and L proteins (10,11,14). We have previously demonstrated the existence of two N protein-binding sites on the P protein: one located between amino acids 69 and 138 and the other in the carboxy-terminal region comprising amino acids 268 to 297 (10). We have shown that the major L binding site re...

Interferons (IFNs) encode a family of secreted proteins that provide the front-line defense against viral infections. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. We have recently isolated a new human IFN-induced gene that we have termed ISG20, which codes for a 3 to 5 exonuclease with specificity for single-stranded RNA and, to a lesser extent, for DNA. In this report, we demonstrate that ISG20 is involved in the antiviral functions of IFN. In the absence of IFN treatment, ISG20-overexpressing HeLa cells showed resistance to infections by vesicular stomatitis virus (VSV), influenza virus, and encephalomyocarditis virus (three RNA genomic viruses) but not to the DNA genomic adenovirus. ISG20 specifically interfered with VSV mRNA synthesis and protein production while leaving the expression of cellular control genes unaffected. No antiviral effect was observed in cells overexpressing a mutated ISG20 protein defective in exonuclease activity, demonstrating that the antiviral effects were due to the exonuclease activity of ISG20. In addition, the inactive mutant ISG20 protein, which is able to inhibit ISG20 exonuclease activity in vitro, significantly reduced the ability of IFN to block VSV development. Taken together, these data suggested that the antiviral activity of IFN against VSV is partly mediated by ISG20. We thus show that, besides RNase L, ISG20 has an antiviral activity, supporting the idea that it might represent a novel antiviral pathway in the mechanism of IFN action. Interferons (IFNs)1 are a family of multifunctional secreted proteins characterized by their abilities to interfere with virus infection and replication (1, 2). IFNs can indirectly inhibit viral production by reducing the growth of target cells and by stimulating their susceptibility to apoptotic processes (3, 4) or by promoting the recognition and the cytotoxic killing of infected cells by the immune system (5, 6). IFNs also act directly at various steps of the viral multiplication cycle through the products of specific but usually overlapping sets of cellular genes induced in the target cells and involved in RNA and protein metabolism and signaling as well (7,8). Until now, three IFNregulated pathways have been considered to be involved in these processes: the double-stranded RNA-dependent protein kinase R (PKR) (9 -11), the 2-5A/RNase L system (12, 13), and the Mx proteins (14 -16). PKR is a serine/threonine kinase that, after binding to dsRNA, phosphorylates the protein synthesis initiation factor eIF2 and the inhibitor of nuclear factor B (IB), resulting in the inhibition of protein synthesis and specific transcription regulation (reviewed in . RNase L is a dormant cytosolic endoribonuclease that is activated by short oligoadenylates produced by the 2Ј-5Ј oligoadenylate synthetase after viral infection or IFN exposure (reviewed in Refs. 2 and 13). Degradation of viral RNAs and cleavage of cellular 18 S and 28 S rRNA...

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 (G s ) 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 G s 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.

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.Many nucleus-replicating viruses have evolved different strategies for delivering their genomes into the nucleus of their host cell through the nuclear pores, which provide the only route of transit across the physical barrier of the nuclear envelope. These strategies depend mainly on the nature of the capsid, which acts both as a protective element for the genome and as a delivery agent (for reviews, see references 21 and 60).Alphaherpesviruses are large, double-stranded DNA viruses. Their genomes are contained within a 125-nm-diameter capsid that is surrounded sequentially by a thick proteinaceous layer, called the tegument, and a lipid envelope. The herpes simplex virus type 1 (HSV-1) capsid structure has been extensively studied (66) and is a general model for other alphaherpesviruses. It has icosahedral symmetry with the major capsid protein VP5, forming hexamers and pentamers (termed hexons and pentons) at the faces and vertices, respectively, of the icosahedron. There are 150 hexons and 11 pentons per capsid. At one vertex, the penton is replaced by a portal, a structure common to tailed bacteriophages and herpesviruses, through which the viral DNA is encapsidated and released (7,8). In HSV-1, the portal is a dodecamer of the UL6 gene product, pUL6 (38, 57).The nuclear pore complex (NPC) is a multiprotein complex that selectively controls the passage of material through the nuclear envelope (for a review, see reference 28). The NPC has three structural components: the nuclear basket, the central framework, which is embedded in the nuclear envelope, and the cytoplasmic filaments. The diameter of the cytoplasmic face is ϳ125 nm, whereas the central channel is ϳ60 nm in diameter (3). Its component proteins, termed nucleoporins, perform various roles, being important both in forming a selective gate and in carrying out nucleocytoplasmic transport (41, 55). Several models have been proposed to explain the s...

Rabies virus P protein inhibits alpha interferon (IFN-␣)-and IFN-␥-stimulatedJak-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-␥ and IFN-␣, 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-␥-or IFN-␣-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.