NP and L proteins of lymphocytic choriomeningitis virus (LCMV) are sufficient for efficient transcription and replication of LCMV genomic RNA analogs.
The genome of lymphocytic choriomeningitis virus (LCMV) consists of two negative-sense single-stranded RNA segments, designated L and S. Both segments contain two viral genes in an ambisense coding strategy, with the genes being separated by an intergenic region (IGR). We have developed a reverse genetic system that allows the investigation of cis-acting signals and trans-acting factors involved in transcription and replication of LCMV. To this end, we constructed an LCMV S minigenome consisting of a negative-sense copy of the chloramphenicol acetyltransferase (CAT) reporter gene flanked upstream by the S 5' untranslated region (UTR) and IGR and downstream by the S 3' UTR. CAT expression was detected in LCMV-infected cells transfected with the minigenome RNA. Intracellular coexpression of the LCMV minigenome and LCMV L and NP proteins supplied from cotransfected plasmids driven by the T7 RNA polymerase provided by the recombinant vaccinia virus vTF7-3 resulted in high levels of CAT activity and synthesis of subgenomic CAT mRNA and antiminigenome RNA species. Thus, L and NP represent the minimal viral trans-acting factors required for efficient RNA synthesis mediated by LCMV polymerase.
Lee
K J
KJ
Division of Virology, Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037, USA.
Novella
I S
IS
Teng
M N
MN
Oldstone
M B
MB
de La Torre
J C
JC
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D016679:Genome, Viral; D006378:Helper Viruses / Q000235:genetics*; D015151:Immunoblotting; D008217:Lymphocytic choriomeningitis virus / Q000235:genetics* / Q000378:metabolism; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D010957:Plasmids / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D012367:RNA, Viral; D014758:Viral Core Proteins; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases