In vitro activities of a recombinant foot-and-mouth disease virus replicase expressed in Escherichia coli.
1. The replicase gene of foot-and-mouth disease virus (FMDV) was expressed in Escherichia coli under the control of a tac promoter. The recombinant enzyme was purified by inclusion body precipitation, elution, and poly(U) Sepharose chromatography. 2. The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity. The specific activity of the purified replicase is 1.3 x 10(5). The recombinant replicase synthesizes RNA using FMDV RNA as template, as well as heterologous RNAs, such as globin RNA and synthetic RNAs, polyadenylated or not. In all polymerization reactions, RNA products twice the size of the template are formed, both in the presence and absence of an oligo(U) primer. The enzyme is also capable of incorporating [alpha 32P]UTP in all RNAs tested except the viral template. This activity does not seem to be related to the primer independent polymerization activity. 3. The products from polymerization reactions were characterized by hybridization. In the absence of primer they consist of the template and a complementary strand covalently attached, while in the presence of primer they consist of two complementary strands synthesized de novo. 4. We propose mechanisms of RNA synthesis by the recombinant FMDV replicase in the absence and presence of primer. These mechanisms are discussed in terms of models for in vitro RNA synthesis of other picornaviruses.
Pacheco
A B
AB
Laboratório de Fisiologia Celular, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brasil.
Brindeiro
R M
RM
Soares
M A
MA
de-Almeida
D F
DF
Tanuri
A
A
D005537:Aphthovirus / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics* / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics* / Q000502:physiology; D015967:Gene Expression Regulation, Viral / Q000235:genetics* / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013698:Templates, Genetic; D014779:Virus Replication
D004279:DNA, Viral; D012367:RNA, Viral; D011994:Recombinant Proteins; C023140:DNA replicase; D004259:DNA-Directed DNA Polymerase