The absence of glycoprotein gL, but not gC or gK, severely impairs pseudorabies virus neuroinvasiveness.

Penetration and propagation of herpesviruses in the nervous system require the action of several glycoproteins. To assay for a function of glycoproteins gC, gK, and gL in the neuroinvasiveness of pseudorabies virus (PrV), deletion mutants lacking one of these glycoproteins and corresponding rescuants were inoculated in the nasal cavity of adult mice. We demonstrate that the lack of gL almost prevented the virus from penetrating and propagating in trigeminal, sympathetic, and parasympathetic tracks innervating the nasal cavity, while the lack of gC and gK only slowed the invasion of the nervous system. The conclusion of this and previous studies is that only gB, gD, gH, and gL are indispensable for penetration into neurons, while gB, gH, and gL (and, in some categories of neurons, also gE and gI) are necessary for transneuronal transfer in the mouse model. The deletion of other glycoprotein genes has little effect on PrV neuroinvasiveness although it may affect the dissemination of the virus.

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.

Neuronal propagation of HSV1 from the oral mucosa to the eye.

PURPOSE: To identify possible neuronal pathways leading to herpetic ocular disease after primary oral infection in mice. METHODS: The SC16 strain of herpes simplex virus (HSV)-1 (10(6) plaque-forming units) was injected into the mucocutaneous border of the left upper lip. Animals were killed 2 to 10 days postinoculation (DPI). Spread of the virus in neural structures was studied by immunochemistry. RESULTS: HSV1 first replicated at the site of inoculation and then at the superior cervical ganglion (at 2 DPI). The trigeminal ganglion and the facial nerve fibers were infected by 4 DPI. Infection of the ciliary body and iris occurred at 6 DPI, together with several brain stem nuclei belonging to the autonomic or sensory pathways. Between 8 and 10 DPI, the neural infection gradually cleared up, except for the ipsilateral sympathetic ganglion, and ipsilateral keratitis appeared in some animals. CONCLUSIONS: The pattern of viral dissemination in this mouse model suggests that infection of iris and ciliary body results from transfer of virus in the superior cervical ganglion from sympathetic neurons innervating the lip to neighboring neurons innervating the anterior uvea. Later, zosteriform spread of virus from the trigeminal system may have contributed to the clinical and histologic findings.

Neuronal pathways for the propagation of herpes simplex virus type 1 from one retina to the other in a murine model.

Herpetic retinitis in humans is characterized by a high frequency of bilateral localization. In order to determine the possible mechanisms leading to bilateral retinitis, we studied the pathways by which herpes simplex virus type 1 (HSV-1) is propagated from one retina to the other after intravitreal injection in mice. HSV-1 strain SC16 (90 p.f.u.) was injected into the vitreous body of the left eye of BALB/c mice. Animals were sacrificed 1, 2, 3, 4 and 5 days post-inoculation (p.i.). Histological sections were studied by immunochemical staining. Primary retinitis in the inoculated eye (beginning 1 day p.i.) was followed by contralateral retinitis (in the uninoculated eye) starting at 3 days p.i. Infected neurons of central visual pathway nuclei (lateral geniculate nuclei, suprachiasmatic nuclei and pretectal areas) were detected at 4 days p.i. Iris and ciliary body infection was minimal early on, but became extensive thereafter and was accompanied by the infection of connected sympathetic and parasympathetic pathways. The pattern of virus propagation over time suggests that the onset of contralateral retinitis was mediated by local (non-synaptic) transfer in the optic chiasm from infected to uninfected axons of the optic nerves. Later, retinopetal transneuronal propagation of the virus from visual pathways may have contributed to increase the severity of contralateral retinitis.

The UL25 protein of pseudorabies virus associates with capsids and localizes to the nucleus and to microtubules.

The UL25 gene of pseudorabies virus (PrV) can encode a protein of about 57 kDa which is well conserved among herpesviruses. The UL25 protein of herpes simplex virus type 1 is a capsid constituent involved in virus penetration and capsid maturation. To identify and characterize the UL25 gene product of PrV, polyclonal mouse anti-UL25 antibodies were raised to a bacterially expressed fusion protein. In immunoblotting and immunoprecipitation assays of PrV-infected cell lysates, these anti-UL25 antisera specifically recognized a protein of the expected size with late expression kinetics. This 57-kDa product was also present in purified virions and was found to be associated with all types of capsids. Synthesis of a protein migrating at the same size point was directed from the eukaryotic expression plasmid pCG-UL25. To determine the subcellular localization of UL25, immunofluorescence studies with anti-UL25 antisera were performed on Nonidet P-40-extracted COS-7 cells infected with PrV or transfected with pCG-UL25. In PrV-infected cells, newly synthesized UL25 is directed mainly to distinct nuclear compartments, whereas UL25 expressed in the absence of other viral proteins is distributed more uniformly in the nucleus and colocalizes also with microtubules. To study the fate of UL25 at very early stages of infection, immunofluorescence experiments were performed on invading PrV particles in the presence or absence of drugs that specifically depolymerize components of the cytoskeleton. We found that the incoming nucleocapsids colocalize with microtubules during their transport to the nucleus and that UL25 remains associated with nucleocapsids during this transport.

Glycoproteins gM and gN of pseudorabies virus are dispensable for viral penetration and propagation in the nervous systems of adult mice.

Glycoproteins gM and gN are conserved throughout the herpesviruses but are dispensable for viral replication in cell cultures. To assay for a function of these proteins in infection of an animal, deletion mutants of pseudorabies virus lacking gM or gN and corresponding revertants were analyzed for the ability to penetrate and propagate in the nervous systems of adult mice after intranasal inoculation. We demonstrate that neither of the two glycoproteins is required for infection of the nervous systems of mice by pseudorabies virus.

Soluble ectodomain of rabies virus glycoprotein expressed in eukaryotic cells folds in a monomeric conformation that is antigenically distinct from the native state of the complete, membrane-anchored glycoprotein.

Rabies virus glycoprotein (G) is a trimeric type I transmembrane glycoprotein that mediates both virus receptor recognition and low pH-induced membrane fusion. G can assume three different states: the 'native' state (N) detected at the virus surface, which is responsible for receptor binding, the activated hydrophobic state (A), which interacts with the target membrane as a first step in the fusion process, and the fusion-inactive conformation (I). These three states, which are structurally different, are in a pH-dependent equilibrium. This equilibrium is shifted toward the I state at low pH. This paper includes an investigation of the structure of the ectodomain of the PV strain of rabies virus when it is synthesized as a soluble form (G1-439) lacking the transmembrane and intracytoplasmic domains (residues 440-505). It is shown that, whatever the extracellular pH, G1-439 is secreted as a monomer that has the antigenic characteristics of the I state. This I-like state is not acquired in the acidic compartments of the Golgi but directly in the endoplasmic reticulum. Finally, membrane anchorage by the G transmembrane domain (G1-461) is sufficient for the G ectodomain to be folded into the native N form. These results emphasize the role of the G transmembrane domain in the correct folding of the ectodomain.

Rabies virus-induced membrane fusion.

Rabies virus is a member of the rhabdovirus family. It enters cells by a process of receptor mediated endocytosis. Following this step, the viral envelope fuses with the endosomal membrane to allow release of the viral nucleocapsid into the cytoplasm. Fusion is induced by the low pH of the endosomal compartment and is mediated by the single viral glycoprotein G, a homotrimeric integral membrane protein. Rabies virus fusion properties are related to different conformational states of G. By different biochemical and biophysical approaches, it has been demonstrated that G can assume at least three different states: the native (N) state detected at the viral surface above pH 7, the activated (A) hydrophobic state which interacts with the target membrane as a first step of the fusion process, and the fusion inactive (I) conformation. Differently from other fusogenic viruses for which low pH-induced conformational changes are irreversible, there is a pH dependent equilibrium between these states, the equilibrium being shifted toward the I-state at low pH. The objective of this review is to detail recent findings on rhabdovirus-induced membrane fusion and to underline the differences that exist between this viral family and influenza virus which is the best known fusogenic virus. These differences have to be taken into consideration if one wants to have a global understanding of virus-induced membrane fusion.

The left border of the genomic inversion of pseudorabies virus contains genes homologous to the UL46 and UL47 genes of herpes simplex virus type 1, but no UL45 gene.

The genome of pseudorabies virus (PrV) is collinear with the herpes simplex virus type 1 (HSV1) genome, except for an inversion in the unique long region, the right extremity of which resides within the BamHI fragment 9 and the left within the BamHI fragment 1. We previously sequenced the right border of the inversion which is situated next to the UL44-gC gene and found that it encodes the UL24, UL25, UL26 and UL26.5 gene counterparts of HSV1. We have now sequenced 5317 base pairs of the BamHI fragment 1, upstream of the UL27-gB gene. We found two open reading frames homologous to UL46 and UL47 of HSV1 yet UL45 was absent and replaced by a set of strictly repeated sequences. PrV UL46 and UL47 are transcribed into two 3' co-terminal messenger RNAs with early and late kinetics, respectively. Comparison of the PrV UL46 and UL47 protein sequences with their counterparts from alphaherpesviruses indicated a strong similarity. The genome is rearranged in this region with respect to HSV1 and the inversion must have taken place, on the left side, within the UL46-UL27 intergenic region. Thus, the inversion should include genes UL27 to UL44.

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.

Neuronal cell surface molecules mediate specific binding to rabies virus glycoprotein expressed by a recombinant baculovirus on the surfaces of lepidopteran cells.

The existence of specific rabies virus (RV) glycoprotein (G) binding sites on the surfaces of neuroblastoma cells is demonstrated. Spodoptera frugiperda (Sf21) cells expressing G of the RV strain CVS (Gcvs-Sf21 cells) bind specifically to neuroblastoma cells of different species but not to any other cell type (fibroblast, myoblast, epithelial, or glioma). Attachment to mouse neuroblastoma NG108-15 cells is abolished by previous treatment of Gcvs-Sf2 cells with anti-G antibody. Substitutions for lysine at position 330 and for arginine at position 333 in RV G greatly reduce interaction between Gcvs-Sf21 cells and NG108-15 cells. These data are consistent with in vivo results: an avirulent RV mutant bearing the same double mutation is not able to infect sensory neurons or motoneurons (P. Coulon, J.-P. Ternaux, A. Flamand, and C. Tuffereau, J. Virol. 72:273-278, 1998) after intramuscular inoculation into a mouse. Furthermore, infection of NG108-15 cells by RV but not by vesicular stomatitis virus leads to a reduction of the number of binding sites at the neuronal-cell surface. Our data strongly suggest that these specific attachment sites on neuroblastoma cells represent a neuronal receptor(s) used by RV to infect certain types of neurons in vivo.

An avirulent mutant of rabies virus is unable to infect motoneurons in vivo and in vitro.

An antigenic double mutant of rabies virus (challenge virus standard [CVS] strain) was selected by successive use of two neutralizing antiglycoprotein monoclonal antibodies, both specific for antigenic site III. This mutant differed from the original virus strain by two amino acid substitutions in the ectodomain of the glycoprotein. The lysine in position 330 and the arginine in position 333 were replaced by asparagine and methionine, respectively. This double mutant was not pathogenic for adult mice. When injected intramuscularly into the forelimbs of adult mice, this virus could not penetrate the nervous system, either by the motor or by the sensory route, while respective single mutants infected motoneurons in the spinal cord and sensory neurons in the dorsal root ganglia. In vitro experiments showed that the double mutant was able to infect BHK cells, neuroblastoma cells, and freshly prepared embryonic motoneurons, albeit with a lower efficiency than the CVS strain. Upon further incubation at 37 degrees C, the motoneurons became resistant to infection by the mutant while remaining permissive to CVS infection. These results suggest that rabies virus uses different types of receptors: a molecule which is ubiquitously expressed at the surface of continuous cell lines and which is recognized by both CVS and the double mutant and a neuron-specific molecule which is not recognized by the double mutant.

Phage-displayed and soluble mouse scFv fragments neutralize rabies virus.

A phage-display technology was used to produce a single-chain Fv antibody fragment (scFv) from the 30AA5 hybridoma secreting anti-glycoprotein monoclonal antibody (MAb) that neutralizes rabies virus. ScFv was constructed and then cloned for expression as a protein fusion with the g3p minor coat protein of filamentous phage. The display of antibody fragment on the phage surface allows its selection by affinity using an enzyme-linked immunosorbent assay (ELISA); the selected scFv fragment was produced in a soluble form secreted by E. coli. The DNA fragment was sequenced to define the germline gene family and the amino-acid subgroups of the heavy (VH) and light (VL) chain variable regions. The specificity characteristics and neutralization capacity of phage-displayed and soluble scFv fragments were found to be identical to those of the parental 30AA5 MAb directed against antigenic site II of rabies glycoprotein. Phage-display technology allows the production of new antibody molecule forms able to neutralize the rabies virus specifically. The next step could be to engineer and produce multivalent and multispecific neutralizing antibody fragments. A cocktail of multispecific neutralizing antibodies could contain monovalent, bivalent or tetravalent scFv fragments, for passive immunoglobulin therapy.

Identification of amino acids controlling the low-pH-induced conformational change of rabies virus glycoprotein.

The glycoprotein (G) of rabies virus assumes at least three different conformations: the native state detected at the viral surface above pH 7, the activated state involved in the first step of the fusion process, and the fusion-inactive conformation (I). A new category of monoclonal antibodies (MAbs) which recognized specifically the I conformation at the viral surface has recently been described. These MAbs (17A4 and 29EC2) became neutralizing when the virus was preincubated at acidic pH to induce the conformational change toward the I state of G. Mutants escaping neutralization were then selected. In this study, we have investigated the fusion and the low-pH-induced fusion inactivation properties of these mutants. All of these mutants have fusion properties similar to those of the CVS parental strain, but five mutants (E282K, M44I, M44V, V392G, and M396T) were considerably slowed in their conformational change leading to the I state. These mutants allow us to define regions that control this conformational change. These results also reinforce the idea that structural transition toward the I state is irrelevant to the fusion process. Other mutations in amino acids 10, 13, and 15 are probably located in the epitopes of selecting MAbs. Furthermore, in electron microscopy, we observed a hexagonal lattice of glycoproteins at the viral surface of mutants M44I and V392G as well as strong cooperativity in the conformational change toward the I state. This finding demonstrates the existence of lateral interactions between the spikes of a rhabdovirus.

Glycoprotein gH of pseudorabies virus is essential for penetration and propagation in cell culture and in the nervous system of mice.

Glycoprotein H (gH) of pseudorabies virus (PrV) is a structural component of the virion and forms a complex with another glycoprotein, gL. For a detailed analysis of the function of PrV gH, we isolated a gH-deficient mutant on trans-complementing gH-expressing cells after insertion of a beta-galactosidase expression cassette into a partially deleted gH gene. The absence of gH did not affect primary or secondary attachment of PrV but the mutant was not infectious. The defect in infectivity could partially be overcome by experimentally induced membrane fusion using PEG, which suggests that gH was necessary for fusion between virion and cellular membranes. After intranasal inoculation into mice, the LD50 of complemented gH- PrV was more than four orders of magnitude higher than that of wild-type PrV. Infection of the respiratory epithelium was much less efficient with complemented gH- PrV as compared with rescued PrV, reflecting the lack of direct cell-to-cell spread. Complemented gH- PrV was able to penetrate into a few trigeminal and sympathetic first order neurons accessible from the nasal cavity, whereas transneuronal transfer in the second order neurons was not observed. In summary, gH is essential for entry and cell-to-cell spread in cell culture, and for propagation in the nervous system of mice. This substantiates the hypothesis that transneuronal spread in vivo and direct cell-to-cell spread in cell culture are governed by similar mechanisms.

The BamHI fragment 9 of pseudorabies virus contains genes homologous to the UL24, UL25, UL26, and UL 26.5 genes of herpes simplex virus type 1.

The genomes of pseudorabies virus (PrV) and of herpes simplex virus type 1 (HSV1) are colinear, excepting an inversion in the unique long region, of which one extremity resides within the BamHI fragment 9. This fragment (4088 bp) encodes the counterparts of HSV1 UL24, UL25, UL26 and UL26.5 that are transcribed into four 3'-coterminal mRNAs. Multiple alignments of UL24, UL25 and UL26 protein homologs from alpha-, beta- and gamma-herpesviruses were performed. The PrV UL24 protein is shorter than its counterparts, missing the non-conserved COOH-terminal region. The region which is common to all viruses contains a basic NH2-terminus and a hydrophobic COOH-end, suggesting that UL24 may function as a matrix protein. The UL25 proteins are well conserved, particularly among the alpha-herpesviruses. All the domains involved in the proteolytic activity of theUL26 protein are highly conserved, as well as the two cleavage sites. Thus, its function and processing may be similar in PrV as in other herpesviruses. Due to the fact that in PrV the UL26 and UL44 genes are adjacent and their ends are conserved, the right border of the inversion must lie within their intergenic region.

Deletion of glycoprotein gE reduces the propagation of pseudorabies virus in the nervous system of mice after intranasal inoculation.

A pseudorabies virus (PrV) mutant, deficient in the nonessential glycoprotein E (gE) and expressing the LacZ gene (gE- beta gal+ PrV), and its rescued virus were inoculated intranasally in mice. The median lethal dose of gE- beta gal+ PrV was similar to that of the parental Kaplan strain, but mice survived longer and did not develop symptoms of pseudorabies. In the nasal mucosa, gE- beta gal+ PrV replicated less efficiently than rescued virus. gE- beta gal+ PrV could infect first-order trigeminal and sympathetic neurons innervating the nasal mucosa. However, transneuronal transfer to second-order cells groups did not occur in trigeminal pathways and was severely reduced in sympathetic pathways. The mutant was also unable to propagate in the parasympathetic system. In contrast, gE-rescued virus was transferred transneuronally in trigeminal, sympathetic, and parasympathetic pathways, like wild-type PrV. These findings provide further evidence that deletion of gE specifically affects transneuronal transfer of PrV more than penetration and multiplication of the virus in first-order neurons.

Immunodominant epitopes defined by a yeast-expressed library of random fragments of the rabies virus glycoprotein map outside major antigenic sites.

Nineteen yeast colonies secreting rabies virus glycoprotein (G) peptides immunoreactive with polyclonal anti-rabies virus sera were selected from a random expression library. The peptides, around 80 amino acids long, spanned amino acids 54-494 of the G protein. These peptides, together with two constructions including, respectively, immunodominant sites II and III, were analysed for their immunoreactivity with 40 anti-G protein monoclonal antibodies (MAbs) composed of 12 MAbs that reacted with SDS-treated protein in Western blot under reducing conditions (WB+) and 28 representative MAbs that did not react after denaturation (WB-). This last category represents 98% of anti-rabies virus G MAbs. None of the WB- MAbs bound peptides. Of the 12 WB+ MAbs, one bound two peptides situated before the transmembrane domain of the protein and six bound peptides overlapping a region situated between amino acids 223 and 276. These six MAbs define a new antigenic region that would be considered 'immunodominant' if the peptide strategy had been used to study the antigenicity of the protein; however, this region is only recognized by about 1% of our MAbs. Three of these WB+ MAbs had significant neutralizing activity; two were used for the selection of antigenic mutants (MAR mutants). Some mutants had a substitution within the region delimited by the peptides, confirming the pertinence of both the peptide and escape mutant approaches. However, a few mutants had a substitution outside the peptide-delimited region, suggesting that remote mutation(s) could affect epitope accessibility in the native protein.

Biological function of the low-pH, fusion-inactive conformation of rabies virus glycoprotein (G): G is transported in a fusion-inactive state-like conformation.

The glycoprotein (G) of rabies virus can assume at least three different conformations: the native (N) state detected at the viral surface above pH 7; the activated (A) hydrophobic state, which is probably involved in the first steps of the fusion process; and the fusion-inactive (I) conformation. There is a pH-dependent equilibrium between these states, the equilibrium being shifted towards the I state at low pH. It has been supposed that the transition from the N to the I state mediates membrane fusion. By using a lipid-mixing assay, we studied the kinetics of fusion and fusion inactivation for two rabies virus strains, PV and CVS. In addition, by using electron microscopy and a trypsin sensitivity assay, we analyzed the kinetics of the conformational change towards the I state for both strains. Although the PV strain fuses faster, inactivation and the conformational change of PV G occur more slowly than for the CVS strain. This suggests that the structural transition towards the I state is irrelevant to the fusion process. Immunofluorescence and immunoprecipitation experiments performed with infected cells and two different monoclonal antibodies, one specific for the N form of G and one which recognizes both the N and the I states, suggest that G is transported in an I state-like conformation through the Golgi apparatus and acquires its N structure only near or at the cell surface. We propose that the role of the I state is to avoid unspecific fusion during transport of G in the acidic Golgi vesicles.

Monoclonal antibodies which recognize the acidic configuration of the rabies glycoprotein at the surface of the virion can be neutralizing.

Around 15% of our anti-glycoprotein monoclonal antibodies (MAbs) failed to neutralize the infectivity of the rabies virus during a 1-hr incubation at room temperature. In previous studies, we have demonstrated that it is possible to induce a massive conformational change of the glycoprotein population by incubating the virus at acidic pH. The conformational change is reversible and consequently viral infectivity is not affected by transient exposure at acidic pH. The proportion of glycoproteins in acidic or neutral configuration depends on the pH which means that even at neutral pH some glycoproteins transiently adopt the acidic configuration and vice versa. Here we report that some of our nonneutralizing MAbs recognize the acidic form of the glycoprotein at the virion surface. After incubation of the virus at pH 6.4, most glycoproteins are in the acidic configuration. Further 1-hr incubation with these MAbs at the same pH resulted in more immunoglobulins being attached to the virus and consequently neutralization was induced. It was also possible to induce neutralization with the same MAbs by incubation at neutral pH for a longer period or at a higher temperature. Mutants resistant to neutralization by these MAbs could be selected. Mutations confering resistance to neutralization were not localized in previously described antigenic sites and did not modify these sites at distance. They had no effect on the pathogenic power of the virus. Either they are situated in the epitope or they modify the epitope, so that it is no longer recognized by the antibody on the acidic configuration of the protein. Alternatively, these mutations may stabilize the protein in its neutral configuration. In addition, these experiments confirm our previous finding that neutralization requires the fixation of a large number of immunoglobulins on the virus, irrespective of the region of the protein recognized by the antibody.