Generation and characterization of P gene-deficient rabies virus

109

113

We propose a reference model of the kinetics of a viral RNA-dependent RNA polymerase (vRdRp) activities and its regulation during infection of eucaryotic cells. After measles virus infects a cell, mRNAs from all genes immediately start to accumulate linearly over the first 5 to 6 h and then exponentially until ϳ24 h. The change from a linear to an exponential accumulation correlates with de novo synthesis of vRdRp from the incoming template. Expression of the virus nucleoprotein (N) prior to infection shifts the balance in favor of replication. Conversely, inhibition of protein synthesis by cycloheximide favors the latter. The in vivo elongation speed of the viral polymerase is ϳ3 nucleotides/s. A similar profile with fivefold-slower kinetics can be obtained using a recombinant virus expressing a structurally altered polymerase. Finally, virions contain only encapsidated genomic, antigenomic, and 5-end abortive replication fragment RNAs.Viruses are obligate intracellular parasites. To understand the complexity of virus replication and to develop potent antiviral drugs, there is a growing need for a systematic analysis of the underlying molecular mechanisms. Although the kinetics of protein expression has been established for many viruses and a model of the dynamics of virus transcription and replication for bacteriophage Q has been proposed (see reference 10 for a review), knowledge in this area is very limited for all eucaryotic viruses. We report the first kinetics model of the transcription and replication of a negative-sense, singlestranded RNA virus.Measles virus (MV) belongs to the order Mononegavirales (33). The replication strategy involves a viral RNA-dependent RNA polymerase (vRdRp), which uses as a template a nucleocapsid (NC) made of a single strand of RNA in tight complex with the nucleoprotein (N). The negative-strand genome contains six transcription units encoding the N, phospho (P), matrix (M), fusion (F), hemagglutinin (H), and large (L) or polymerase protein, in that order (Fig. 1). The transcription units are flanked by short leader (Le) and trailer (Tr) sequences containing the genomic promoter (on the minus strand) and the antigenomic promoter (on the plus strand), respectively. The P gene also encodes the nonstructural V and C proteins by RNA editing and alternative overlapping open reading frame, respectively.Every N protein binds to 6 nucleotides so that the N polymer entirely covers the 15,894-nucleotide genome (5) and makes it resistant to nucleases (33). NC comprises 2,649 N, ϳ300 P, and ϳ20 to 50 L proteins (33) and is enclosed within a membrane envelope containing H and F glycoproteins. After fusion with the plasma membrane at neutral pH, intact NCs are released into the cytoplasm (24), where they serve as a template for both primary transcription from and replication of the genomic RNA (Fig. 1). Transcription is initiated from a single promoter at the 3Ј end of the genome (for a review, see reference 23) by vRdRp made of L and P proteins. Immediately prior to an intergenic junction...

Section: Discussionmentioning

confidence: 99%

Dynamics of Viral RNA Synthesis during Measles Virus Infection

Plumet

Duprex

Gerlier

2005

109

113

“…The highly neurovirulent RV strain, challenge virus standard-11 (CVS-11), and the attenuated strain high egg passage (HEP)-Flury were propagated in NA cells as previously described (57). Preparation of RV virions was performed as described before (35).…”

Section: Methodsmentioning

confidence: 99%

“…Ra2 cells, which had been plated in 24-well culture plates (3 ϫ 10 5 cells/well), were incubated with or without 2 focus-forming units (FFU) per cell of CVS-11 virus suspended in EMEM containing 0.5% FCS, 0.2% glucose, and the above-named antibiotics (hereafter called test medium) for 2 h at 37°C, washed, and overlaid with culture medium. Fluorescent staining of cultured cells was performed as described in previous papers (34,36,57). Briefly, the cells were fixed with 3% formaldehyde in phosphate-buffered saline (PBS) for 10 min and then permeabilized with 0.2% Triton X-100 in PBS for 5 min.…”

Section: Methodsmentioning

confidence: 99%

Rabies Virus-Induced Activation of Mitogen-Activated Protein Kinase and NF-κB Signaling Pathways Regulates Expression of CXC and CC Chemokine Ligands in Microglia

Nakamichi

Saiki

Sawada

et al. 2005

Self Cite

Following virus infection of the central nervous system, microglia, the ontogenetic and functional equivalents of macrophages in somatic tissues, act as sources of chemokines, thereby recruiting peripheral leukocytes into the brain parenchyma. In the present study, we have systemically examined the growth characteristics of rabies virus (RV) in microglia and the activation of cellular signaling pathways leading to chemokine expression upon RV infection. In RV-inoculated microglia, the synthesis of the viral genome and the production of virus progenies were significantly impaired, while the expression of viral proteins was observed. Transcriptional analyses of the expression profiles of chemokine genes revealed that RV infection, but not exposure to inactivated virions, strongly induces the expression of CXC chemokine ligand 10 (CXCL10) and CC chemokine ligand 5 (CCL5) in microglia. RV infection triggered the activation of signaling pathways mediated by mitogen-activated protein kinases, including p38, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase, and nuclear factor B (NF-B). RV-induced expression of CXCL10 and CCL5 was achieved by the activation of p38 and NF-B pathways. In contrast, the activation of ERK1/2 was found to down-regulate CCL5 expression in RV-infected microglia, despite the fact that it was involved in partial induction of CXCL10 expression. Furthermore, NF-B signaling upon RV infection was augmented via a p38-mediated mechanism. Taken together, these results indicate that the strong induction of CXCL10 and CCL5 expression in microglia is precisely regulated by the activation of multiple signaling pathways through the recognition of RV infection.

“…The pathogenic CVS-11 (hereafter called CVS) and nonpathogenic HEP strains of RV were propagated in NA cells as previously described (80). Preparation of RV virions was performed essentially as described before (39).…”

Section: Methodsmentioning

confidence: 99%

Rabies Virus Stimulates Nitric Oxide Production and CXC Chemokine Ligand 10 Expression in Macrophages through Activation of Extracellular Signal-Regulated Kinases 1 and 2

Nakamichi¹,

Inoue

Takasaki³

et al. 2004

Self Cite

Macrophages represent an essential part of innate immunity, and the viral infection of macrophages results in the release of multiple proinflammatory mediators, such as nitric oxide (NO), cytokines, and chemokines. This study was undertaken to define the molecular mechanism of macrophage activation in response to rabies virus (RV) infection. In RAW264 murine macrophage cells, a well-characterized macrophage model, RV replication was strictly restricted, whereas cell proliferation was significantly enhanced upon RV inoculation. Transcriptional analyses for the expression of inducible forms of NO synthase (iNOS), cytokines, and chemokines revealed that RV virions potentiate the gene expression of iNOS and CXC chemokine ligand 10 (CXCL10), a major chemoattractant of T helper cell type 1. However, RV stimulation had little or no effect on the expression profiles of proinflammatory cytokines and other types of chemokines. In macrophages stimulated with UV-inactivated RV virions, as well as infectious viruses, the phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2, members of the mitogen-activated protein kinase family, was significantly induced. Specific inhibitors of MAPK/ERK kinase reduced the RV-induced production of NO and CXCL10. Furthermore, the RV-induced activation of the ERK1/2 pathway was severely impaired by the neutralization of the endosomal and lysosomal pH environment with lysosomotropic agents, indicating that endocytosis is a key step leading to the activation of ERK1/2 signaling. Taken together, these results suggest that the ERK1/2-mediated signaling pathway plays a cardinal role in the selective activation of macrophages in response to RV virions, thereby regulating cellular functions during virus infection.Rabies virus (RV) is a negative-strand RNA virus belonging to the Rhabdoviridae family, genus Lyssavirus. Most RV strains are highly neurotropic, which usually causes a fatal infection in warm-blooded animals, and viral replication primarily occurs in neurons as a cellular target (43). It has been reported that RV replicates in muscle cells prior to the invasion of the peripheral nervous system and central nervous system (CNS) in vivo (62). In vitro, it is known that a highly neurovirulent RV strain, challenge virus standard (CVS), and an attenuated strain, high egg passage (HEP)-Flury (hereafter called HEP), infect a variety of cell types, including nonneuronal cells (26,82,84,85).It is well known that infection of experimental animals with nonpathogenic RV strains triggers a strong antiviral immune response (90). Recent studies have demonstrated that the strong antiviral immune response elicited by attenuated RV infection is closely related to the induction of apoptosis in infected cells due to the expression of viral glycoprotein (61,76). Several groups have attempted to develop vaccine vehicles using RV-based vectors. It has been reported that the RV vectors expressing foreign antigens induce strong humoral and cellular responses against other kinds of viral pathogens, s...