Studies on Rabies Virus RNA Polymerase: 1. cDNA Cloning of the Catalytic Subunit (L Protein) of Avirulent HEP‐Flury Strain and Its Expression in Animal Cells

Abstract

To investigate the RNA polymerase of rabies virus, we cloned a cDNA of the catalytic subunit (called L protein because of its large molecular size) of the HEP-Flury strain, an avirulent strain obtained by high frequencies of serial embryonated hen egg passages. Nucleotide sequencing showed that the cDNA encodes a long polypeptide of 2,127 amino acids (Mr. 242,938). A comparison of the deduced amino acid sequence with that of other strains (PV and SAD B19) indicated that the sequence was highly conserved, except for several amino acid substitutions which were accumulated in some limited regions. A fragment of the cDNA was used for expression in Escherichia coli (E. coli) to prepare the L antigen for raising the antibodies in rabbits. Immunoprecipitation studies with the rabbit antiserum showed that the polypeptides produced in the L cDNA-transfected COS-7 cells displayed almost the same electrophoretic mobility as that of authentic L protein.Immunofluorescence studies indicated that both L and P (another subunit of RNA polymerase) proteins displayed colocalized distribution with the nucleocapsid antigen (N) in the cytoplasmic inclusion bodies, where envelope proteins (G and M) were absent. On the other hand, expression of the L protein alone did not cause inclusion body-like granular distribution, suggesting that the inclusion body-like accumulation depends on certain interaction(s) with other viral gene products, probably with the ribonucleoproteins comprising the inclusion bodies.

Studies on the Escape Mutants of Rabies Virus Which Are Resistant to Neutralization by a Highly Conserved Conformational Epitope‐Specific Monoclonal Antibody #1‐46‐12

“…For preparing and sequencing of the M cDNA, we used the same set of oligonucleotide primers that contained the sequence of M gene-flanking regions (shown in Table 1). Nucleotide sequencing was done by an automatic dye terminator method as described previously (17). Computer-assisted analysis of the nucleotide as well as the amino acid sequence was performed by using an application software program, GENETYX Ver.10.0 (Software Development).…”

Studies on the Rabies Virus RNA Polymerase: 2. Possible Relationships between the Two Forms of the Non‐Catalytic Subunit (P Protein)

“…P proteins in the virion as well as in infected cells are mostly associated with the nucleocapsid or N protein (9,13). In addition, the viral L protein (catalytic subunit of viral RNA polymerase) is shown to be associated with P protein or nucleocapsid (28). Although being present abundantly in the cell, the P protein is small in amount in the virion, and accounts for at most 6% of the virion (16).…”

Prokaryotic Expression, Purification, and Polyclonal Antibody Production of a Truncated Recombinant Rabies Virus L Protein

“…It has been previously accepted that the L protein is the largest protein of RABV, and has multifunctional activities including catalytic activities involved in genomic RNA synthesis, viral transcription and phosphorylation of viral phosphoprotein (3). A reverse genetics approach has been applied to make intracellular reconstitution of the transcriptionally active RABV ribonucleoprotein particles from plasmid encoded proteins that addressed the role of conserved L protein sequences of RABV involved in RNA polymerase activity (26).…”

“…The cDNA of L protein encodes a long polypeptide of 2128 amino acids with a relative molecular weight of 243.09 kDa (silver-haired bat-associated strain (SHBRV)). L protein (named after its large molecular weight) is considered to work with the phosphorylated non-catalytic subunit of viral RNA polymerase, P protein, and as a catalytic subunit of RNA polymerase (3). The polymerase complex of L and P displays all the enzymatic activity of transcription, including co-transcriptional modifications of RNAs, such as capping and polyadenylation, and the initiation and elongation of transcripts (4).…”