Insertion of enhanced green fluorescent protein in a hinge region of vesicular stomatitis virus L polymerase protein creates a temperature-sensitive virus that displays no virion-associated polymerase activity in vitro.
The RNA-dependent RNA polymerase of viruses belonging to the order Mononegavirales is part of a large multifunctional L protein that also catalyzes viral mRNA capping and cap methylation. The L protein of this diverse group of agents displays six blocks of conserved sequences. The precise relationship between these conserved regions and individual functions is largely unknown, except for "domain" VI that clearly encodes a viral mRNA cap methylase. The L protein of morbilliviruses (family Paramyxoviridae) was reported to tolerate insertion of the enhanced green fluorescent protein (EGFP) in a region just upstream of domain VI. Recombinant viruses with this insertion grow well in cell culture but are highly attenuated in animal hosts. We show here that the L protein of vesicular stomatitis virus (VSV), the prototype of the Rhabdoviridae family, also tolerates insertion of EGFP at a similar site. The modified protein (L(EGFP)) and the resultant recombinant virus both demonstrated a sharp temperature-sensitive phenotype for polymerase activity, with reduced activity at 37 degrees C and no activity at 37.5 degrees C. Neither translation nor methylation of mutant virus transcripts was affected at 37 degrees C. Curiously, mutant virus grown at permissive temperature contained about threefold-less L protein than the wild-type virus did and displayed no virion-associated polymerase activity in vitro. These findings support the notion that a flexible "hinge" region separates the cap methylase domain of L proteins from upstream functions and open up a number of avenues for studies of L-protein function in the more-tractable VSV model system.
Department of Biology and Center for Microbial Sciences, San Diego State University, San Diego, California 92182, USA.
John B
JB
Ruedas
Jacques
J
Perrault
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D049452:Green Fluorescent Proteins / Q000235:genetics*; D016254:Mutagenesis, Insertional; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D013696:Temperature; D018116:Vesiculovirus / Q000235:genetics* / Q000254:growth & development*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D014764:Viral Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase