Identification of a role for nucleolin in rabies virus infection.

Rabies virus replicates in the cytoplasm of host cells, but rabies virus phosphoprotein (P-protein) undergoes active nucleocytoplasmic trafficking. Here we show that the largely nuclear P-protein isoform P3 can localize to nucleoli and forms specific interactions with nucleolin. Importantly, depletion of nucleolin expression inhibits viral protein expression and infectious virus production by infected cells. This provides the first evidence that lyssaviruses interact with nucleolin and that nucleolin is important to lyssavirus infection.

Conservation of a unique mechanism of immune evasion across the Lyssavirus genus.

The evasion of host innate immunity by Rabies virus, the prototype of the genus Lyssavirus, depends on a unique mechanism of selective targeting of interferon-activated STAT proteins by the viral phosphoprotein (P-protein). However, the immune evasion strategies of other lyssaviruses, including several lethal human pathogens, are unresolved. Here, we show that this mechanism is conserved between the most distantly related members of the genus, providing important insights into the pathogenesis and potential therapeutic targeting of lyssaviruses.

Golgi staining-like retrograde labeling of brain circuits using rabies virus: Focus onto the striatonigral neurons.

BACKGROUND: The introduction of viral transneuronal tracers in the toolbox of neural tract-tracing methods has been an important addition in the field of connectomics for deciphering circuit-level architecture of the nervous system. One of the added values of viral compared to conventional retrograde tracers, in particular of rabies virus, is to provide a Golgi staining-like view of the infected neurons, revealing the thin dendritic arborizations and the spines that are major post-synaptic seats of neuronal connections. NEWMETHOD: Here, we comparatively illustrate the characteristics of the labeling obtained in the same model system, the basal ganglia circuitry, by different retrograde viral tracing approaches, using the Bartha strain of pseudorabies virus, the SAD and CVS strains of rabies virus and by the conventional retrograde tracer cholera toxin B. To best contrast the differences in the capacity of these tracers to reveal the dendritic morphology in details, we focused on one population of first-order infected neurons in the striatum, which exhibit high spine density, after tracer injection in the substantia nigra. RESULTS AND CONCLUSION: None of the viruses tested allowed to detect as many neurons as with cholera toxin B, but the SAD and CVS strains of rabies virus had the advantage of enabling detailed Golgi-like visualisation of the dendritic trees, the best numerical detection being offered by the transneuronal rCVS-N2c-P-mCherry while poor labeling was provided by rCVS-N2c-M-GFP. Results also suggest that, besides different viral properties, technical issues about constructs and detection methods contribute to apparently different efficiencies among the viral approaches.

[The virus rabies protein: a multifonctional protein at the interface between the virus and its host]. / La protéine P du virus rabique : une protéine multifonctionnelle à l'interface entre le virus et son hôte.

Rhabdoviruses P protein plays a central role in the network of protein-protein interactions by providing a bridge at the interface between the polymerase L, N-RNAtemplate and cellulars factors. The P protein contains two independent binding sites : a N-terminal domain interacting with the L protein and a C-terminal domain which binds to the N-RNA. The P protein has two roles: it stabilizes the RNA polymerase L to the N-RNA template and binds to the soluble No preventing N aggregation and keeping N in a suitable form for specific encapsidation of viral RNA. The two cellular partners of rabies virus P protein identified until now do not seem to be involved in transcription and replication processes indicating that P may have others functions in the virus cycle. Interaction of P with the dynein light chain LC8 suggests that P could mediate the transport of viral nucleocapsids in the nervous central system. The interaction of P with the protein PML that is induced by interferon suggests that P may overcome the immune response of the infected cells. The multifonctionality of P is probably linked to the polymorphism of the protein which is characterized by the expression of shorter P forms in different cellular compartments and by the existence of various phosphorylated and oligomeric forms. The results are not sufficient to establish the involvement of this polymorphism on the various fonctions of P.

Ultrasonically induced gas bubble production in agar based gels: Part I. Experimental investigation.

Macroscopically visible gas bubbles can be produced in an agar based gel by irradiation with either continuous or pulsed ultrasound at frequencies from 0.75 to 3.0 MHz. The variation in the number of bubbles formed with frequency, acoustic pressure, pulse length, duty cycle, and temperature closely resembles that seen in vivo. Furthermore, the acoustic pressure required to initiate bubble formation is also close to that required in vivo. It has been observed that alterations in the concentration and pH of the gels can have a profound effect on the nature and quantity of bubbles. This suggests that not only is this gel model suitable for the representation of the macroscopic features of bubble formation in vivo, but can be used to gain information about the preexisting bubble nuclei. Based on the experimental results obtained it can be suggested that for peak negative acoustic pressures of up 1 MPa (equivalent, for a plane travelling sinusoidal wave, to a time averaged intensity of 30 W/cm2) bubble formation can be avoided by the use of high frequencies, short pulse lengths and long duty cycles.

Vesicular stomatitis virus growth in Drosophila melanogaster cells. II. Modifications of viral protein phosphorylation.

The phosphoproteins of vesicular stomatitis virus released from infected Drosophila melanogaster cells were examined. The membrane (M) protein was more phosphorylated than after multiplication in chicken embryo cells, even in Drosophila cell cytoplasm before its association with cellular membranes. Analysis of phosphopeptides generated after partial proteolysis and of phosphoamino acids obtained after complete acid hydrolysis showed that M phosphorylation was quantitatively and qualitatively changed, while NS protein phosphorylation was only slightly modified.

Interaction between tubulin and the viral matrix protein of vesicular stomatitis virus: possible implications in the viral cytopathic effect.

The matrix (M) protein of vesicular stomatitis virus has been shown to induce the rounding of cells. Experiments were performed in order to define the mechanism by which M protein could cause this cytopathic effect (CPE). Immunofluorescence experiments performed on infected cells indicate that cellular rounding coincides with the disruption of the microtubular network. Immunoprecipitation of M protein or tubulin in infected cell extract demonstrates an association of these two proteins in vivo. We show that M protein is capable of interacting in vitro with tubulin in both its polymerized and nonassembled forms. Studies using proteolytically cleaved proteins indicate that this interaction occurs via the highly basic N-terminal domain of M protein and the highly acidic C-terminal region of tubulin. Furthermore, a thermosensitive mutant (tsG33) containing a mutation in the matrix protein gene which is unable to induce CPE at nonpermissive temperature interacts with tubulin with a lower affinity. These results demonstrate that M protein interacts with tubulin in vivo and in vitro and strongly suggest that CPE is caused by this interaction.

Interaction of the rabies virus P protein with the LC8 dynein light chain.

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.

Translation initiation at alternate in-frame AUG codons in the rabies virus phosphoprotein mRNA is mediated by a ribosomal leaky scanning mechanism.

The phosphoprotein of rabies virus is a 297-amino-acid polypeptide encoded by the longest open reading frame of the P gene. Immunoprecipitation experiments using a monoclonal antiserum directed against the P protein detected the P protein and at least four additional shorter products in infected cells, cells transfected with a plasmid encoding the wild-type P protein, and purified virus (CVS strain). By means of deletion analyses, these proteins were shown to be translated from secondary downstream in-frame AUG initiation codons. Immunofluorescence experiments indicated that all these P products were found in the cytoplasm of transfected cells; however, the proteins initiated from the third, fourth, and fifth AUG codons were found mostly in the nucleus. Changes in the 5'-terminal region of the P mRNA (including site-specific mutations, deletions, and insertions) demonstrated that a leaky scanning mechanism is responsible for translation initiation of the P gene at several sites.

Restricted expression of viral glycoprotein in vesicular stomatitis virus-infected Drosophila melanogaster cells.

Vesicular stomatitis virus (VSV) establishes a non-cytopathic persistent infection in Drosophila melanogaster cells. The synthesis of the viral glycoprotein G was specifically inhibited during a post-transcriptional step, whereas the synthesis and turnover of its mRNA were not modified compared with the other viral mRNAs. Another viral glycoprotein, migrating slightly faster than G protein on an SDS-polyacrylamide gel, was detected in infected Drosophila cells. This protein showed most of the characteristics of the intracellular Gs protein found in infected vertebrate cells. The amounts of G protein integrated into mature virions and of soluble Gs protein secreted into the culture medium were reduced greatly during VSV infection in Drosophila cells.

Mapping the interacting domains between the rabies virus polymerase and phosphoprotein.

The RNA polymerase of rabies virus consists of two subunits, the large (L) protein and the phosphoprotein (P), with 2,127 and 297 amino acids, respectively. When these proteins were coexpressed via the vaccinia virus-T7 RNA polymerase recombinant in mammalian cells, they formed a complex as detected by coimmunoprecipitation. Analysis of P and L deletion mutants was performed to identify the regions of both proteins involved in complex formation. The interaction of P with L was not disrupted by large deletions removing the carboxy-terminal half of the P protein. On the contrary, P proteins containing a deletion in the amino terminus were defective in complex formation with L. Moreover, fusion proteins containing the 19 or the 52 first residues of P in frame with green fluorescent protein (GFP) still bound to L. These results indicate that the major L binding site resides within the 19 first residues of the P protein. We also mapped the region of L involved in the interaction with P. Mutant L proteins consisting of the carboxy-terminal 1,656, 956, 690, and 566 amino acids all bound to the P protein, whereas deletion of 789 residues within the terminal region eliminated binding to P protein. This result demonstrates that the carboxy-terminal domain of L is required for the interaction with P.

Role of matrix protein in cytopathogenesis of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) plays an important structural role in viral assembly, and it also has a regulatory role in viral transcription. We demonstrate here that the M protein has an additional function. It causes visible cytopathic effects (CPE), as evidenced by the typical rounding of polygonal cells after VSV infection. We have analyzed a temperature-sensitive mutant of the M protein of VSV (tsG33) which is defective in viral assembly and which fails to cause morphological changes of the cells after infection at the nonpermissive temperature (40 degrees C). Interestingly, this defect in viral assembly as well as the CPE were reversible. Microinjection of antisense oligonucleotides which specifically inhibit M protein translation also inhibited the occurrence of CPE. Most importantly, when cells were transfected with a cDNA encoding the temperature-sensitive M protein of tsG33, no CPE was observed at the nonpermissive temperature. However, when these cells were shifted to the permissive temperature (32 degrees C), they rounded up and detached from the dish. These results demonstrate that M protein in the absence of the other viral proteins causes rounding of the cells, probably through a disorganization of the cytoskeleton. The absence of CPE at the nonpermissive temperature is correlated with an abnormal dotted staining pattern of M in these cells, suggesting that the mutant M protein may self-aggregate or associate with membranes rather than interact with cytoskeletal elements.

Vesicular stomatitis virus in Drosophila melanogaster cells: regulation of viral transcription and replication.

Vesicular stomatitis virus RNA synthesis was investigated during the establishment of persistent infection in Drosophila melanogaster cells. The transcription rate declined as early as 5 h after infection and was strongly inhibited after 7 h, leading to a decrease in viral mRNA levels and in viral protein synthesis rates. Full-length plus-strand antigenomes and minus-strand genomes were detected after a 3-h lag time and accumulated until 15 h after infection. Short encapsidated plus-strand molecules were also generated corresponding to the 5' end of viral defective antigenomes. Assembly and release of virions were not restricted, but their infectivity was extremely reduced. In persistently infected cells, an equilibrium was reached where the level of intracellular genomes maintained was constant and maximal even after the rate of all viral syntheses had decreased. These results are discussed with regard to the establishment of persistent infection.

Mapping of monoclonal antibody epitopes of the rabies virus P protein.

Thirty-six monoclonal antibodies (MAbs) specific for the rabies virus P phosphoprotein were obtained from mice immunized with recombinant P (PV strain) produced in E. coli. All MAbs reacted against the corresponding rabies virus protein by ELISA and by Western blot analysis and revealed the presence of cytoplasmic inclusions in rabies virus infected cells. The epitopes of seven MAbs were mapped by testing their reactivity with protein fragments expressed from deletion mutants in transfected cells. Western blotting, immunoprecipitation and immunofluorescence assays were performed. These MAbs recognized epitopes in different domains of the P protein: 60% were directed against a region lying between residues 83-172 suggesting a major antigenic determinant of the rabies virus P protein in this region. Most of the antigenic sites appeared to be composed of linear epitopes. These MAbs will be useful as tools to dissect structural and functional properties of the rabies virus P protein.

Vesicular stomatitis virus in Drosophila melanogaster cells: lack of leader RNA transport into the nuclei and frequent abortion of the replication step.

In cultured Drosophila melanogaster cells, vesicular stomatitis virus (VSV) establishes a persistent, noncytopathic infection. No inhibition of host macromolecular synthesis occurs. We studied the synthesis of VSV plus-strand leader RNA, which may be directly involved in vertebrate host synthesis shut-off. Leader RNA accumulated in Drosophila cell cytoplasm, but in low amounts, it was either free or associated to structures larger than the leader RNA-N protein complexes found in vertebrate cells. Only a few leader RNA copies migrated into the cell nucleus; no increase of this transport was observed at any time during the virus cycle. Viral RNAs complementary to the 3' end of the genome and ranging in size from the leader to several hundred nucleotides were found to accumulate in Drosophila cell cytoplasm. Their synthesis was inhibited in the presence of cycloheximide, which blocks all protein synthesis and VSV replication. Correlation between the absence of VSV cytopathogenicity in Drosophila cells and the lack of leader RNA transport into their nuclei is discussed, as well as the possible relationship between the restriction of viral synthesis and the frequent initiation of an abortive replication step.

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.

Insertion of the human immunodeficiency virus CD4 receptor into the envelope of vesicular stomatitis virus particles.

Enveloped virus particles carrying the human immunodeficiency virus (HIV) CD4 receptor may potentially be employed in a targeted antiviral approach. The mechanisms for efficient insertion and the requirements for the functionality of foreign glycoproteins within viral envelopes, however, have not been elucidated. Conditions for efficient insertion of foreign glycoproteins into the vesicular stomatitis virus (VSV) envelope were first established by inserting the wild-type envelope glycoprotein (G) of VSV expressed by a vaccinia virus recombinant. To determine whether the transmembrane and cytoplasmic portions of the VSV G protein were required for insertion of the HIV receptor, a chimeric CD4/G glycoprotein gene was constructed and a vaccinia virus recombinant which expresses the fused CD4/G gene was isolated. The chimeric CD4/G protein was functional as shown in a syncytium-forming assay in HeLa cells as demonstrated by coexpression with a vaccinia virus recombinant expressing the HIV envelope protein. The CD4/G protein was efficiently inserted into the envelope of VSV, and the virus particles retained their infectivity even after specific immunoprecipitation experiments with monoclonal anti-CD4 antibodies. Expression of the normal CD4 protein also led to insertion of the receptor into the envelope of VSV particles. The efficiency of CD4 insertion was similar to that of CD4/G, with approximately 60 molecules of CD4/G or CD4 per virus particle compared with 1,200 molecules of VSV G protein. Considering that (i) the amount of VSV G protein in the cell extract was fivefold higher than for either CD4 or CD4/G and (ii) VSV G protein is inserted as a trimer (CD4 is a monomer), the insertion of VSV G protein was not significantly preferred over CD4 or CD4/G, if at all. We conclude that the efficiency of CD4 or CD4/G insertion appears dependent on the concentration of the glycoprotein rather than on specific selection of these glycoproteins during viral assembly.

Structure of the RNA inside the vesicular stomatitis virus nucleocapsid.

The structure of the viral RNA (vRNA) inside intact nucleocapsids of vesicular stomatitis virus was studied by chemical probing experiments. Most of the Watson-Crick positions of the nucleotide bases of vRNA in intact virus and in nucleoprotein (N)-RNA template were accessible to the chemical probes and the phosphates were protected. This suggests that the nucleoprotein binds to the sugar-phosphate backbone of the RNA and leaves the Watson-Crick positions free for the transcription and replication activities of the viral RNA-dependent RNA polymerase. The same architecture has been proposed for the influenza virus nucleocapsids. However, about 5% of the nucleotide bases were found to be relatively nonreactive towards the chemical probes and some bases were hyperreactive. The pattern of reactivities was the same for RNA inside virus and for RNA in N-RNA template that was purified over a CsCl gradient and which had more than 94% of the polymerase and phosphoprotein molecules removed. All reactivities were more or less equal on naked vRNA. This suggests that the variations in reactivity towards the chemical probes are caused by the presence of the nucleoprotein.