A Novel Expression Cassette of Lyssavirus Shows that the Distantly Related Mokola Virus Can Rescue a Defective Rabies Virus Genome

Abstract

By comparing three expression vectors for the rabies virus (Rv) minigenome, we show that the characteristic of the Rv RNA is important for efficient rescue despite its not being crucial for replication. Moreover, we show that the coexpression of the viral proteins from helper Rv and Mokola virus could rescue the Rv minigenome while Rv-related European bat lyssavirus 1 could not, suggesting that the signals controlling transcription and replication are conserved in the distantly related Rv and Mokola virus.Rabies virus (Rv) and related viruses from the genus Lyssavirus of the family Rhabdoviridae induce fatal encephalitis in mammals (5). The viral genome is a nonsegmented, singlestranded RNA (about 12 kb long) (8, 21) of negative polarity encapsidated by nucleoproteins. The genome codes for five successive genes. The 3Ј and 5Ј extremities have two short sequences, named the leader and trailer regions, that contain the essential signals promoting genome and antigenome transcription, replication, and encapsidation (20).A rescue method has been developed for biologically active ribonucleocapsid assembly by coexpression in the cell cytoplasm of the N, P, and L proteins with a synthetic RNA minigenome (7,15). This coexpression is under the control of a T7 promoter in which the T7 RNA polymerase (T7RNAP) is provided in trans by a recombinant vaccinia virus (9). By using this method, it was shown with vesicular stomatitis virus (VSV; another rhabdovirus) that the exact 3Ј end of the viral RNA genome is crucial for transcription and replication, whereas extra GGG nucleotides added at the 5Ј end for T7RNAP transcription were tolerated to some extent (14). In this study, we created different minigenome expression vectors to determine whether these extra nucleotides could affect rescue efficiency. Moreover, we developed a novel rescue protocol using a helper virus that allowed us to examine the possibilities of rescuing an Rv minigenome with heterologous lyssavirus.Minigenome expression vectors. All of the minigenome constructs used in this study are under the control of a T7RNAP promoter (Fig. 1A); it includes the trailer and leader sequences with a polylinker between the start and stop transcription signals, and it contains a hepatitis delta virus ribozyme (HdvRz) designed to cut the RNA at the leader-HdvRz junction, producing a minigenome with an exact 3Ј viral end (1, 18).Construct pDI.GGG encoded three extra G nucleotides at the minigenome 5Ј end, since GGG is optimal for transcription initiation by the T7RNAP (10, 17). However, the additional GGG was thought to decrease the minigenome's replication efficiency. Thus, we designed the construct pDI.Ham, in which a hammerhead ribozyme (HamRz) (16) was inserted between the GGG and the minigenome; the ribozyme cleaves the RNA to produce an exact 5Ј viral end. Therefore, pDI.Ham combines efficient T7RNAP transcription (initiation on GGG) with optimal minigenome replication (authentic viral 5Ј end). pDI.Mut was similar to pDI.Ham, but both ribozymes were mutated to insert res...

Hsp70 Protein Positively Regulates Rabies Virus Infection

“…The luciferase gene reporter is framed by 3= leader and 5= trailer sequences and is under the control of rabies transcription signals (21). The production of luciferase activity is the result of the encapsidation of the minigenome followed by its transcription by the L-P complex and by its replication in the presence of N protein.…”

“…The plasmids pTit-N, pTit-P, and pTit-L were described previously (7) and were obtained from K. K. Conzelmann (Ludwig-Maximilians University of Munich). The plasmid pDImut (21) was provided by N. Tordo (Institut Pasteur). The plasmid pCMV-Hsp70, encoding the Hsp70 protein, and pCMV-Neo were kindly provided by G. Trugnan (2).…”

“…The mouse anti-Hsp70 MAb (SPA-810) and rabbit polyclonal anti-Hsp70 (SPA-812) antibody were obtained from Stressgen, and the anti-␣-tubulin MAb (N356) was from Amersham. Quercetin (Qct; Q4951) and the rabbit polyclonal anti-actin (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33) antibody were obtained from Sigma. The mouse anti-green fluorescent protein (GFP) MAb (11814460001) was purchased from Roche.…”

“…The minireplicon system assay was slightly modified from Le Mercier et al (21). BSR cells were grown in a 12-well plate (3 ϫ 10 5 cells per well) in DMEM supplemented with 5% FCS and incubated for 24 h at 37°C (in 5% CO 2 ).…”

Functional Human Immunodeficiency Virus Type 1 (HIV-1) Gag-Pol or HIV-1 Gag-Pol and Env Expressed from a Single Rhabdovirus-Based Vaccine Vector Genome

“…Earlier work indicated that the three extra cytidines introduced at the 5Ј end of the RV antigenome, which are needed for efficient transcription from the T7 RNA polymerase promoter, interfere with the efficient recovery of recombinant RVs (12,48). Our new system is based on the findings of Inoue et al that the creation of infectious RV is more efficient when a CMV promoter and two ribozymes flanking the antigenome are used (23,28). The data presented here showed that the improvement in the recovery frequency is probably due to the gener- ation of the precise 5Ј end of the antigenome.…”