ABSTRACT
In this study, Leishmania RNA virus 1-4 (LRV1-4) particles purified from host Leishmania guyanensis promastigotes were examined for capsid endoribonuclease. Temperature optimum for the endoribonulease activity was found to be at 37degrees C to 42degrees C and the activity was specifically inhibited by the aminoglycoside antibiotics, neomycin, kanamycin, and hygromycin and by 100 mM levels of NaCl or KCl. To determine the catalytic domain of the capsid endoribonuclease activity, three point-mutation at cysteine residues at C47S (P1), C128/ 133S (P2), and C194R (P3) were prepared and each gene was constructed into baculoviruses and expressed in Sf9 insect cells. LRV1-4 capsid N- terminus (N2 and N3) and C-terminus (C1 and C2) deletion mutants (Cadd et al., 1994) were also examined by in vitro RNA cleavage assay. The results showed that the capsid mutants; C1, C2, N3, P1, and P2 were capable of forming proper virus-like particles (VLPs) and they all possessed the specific endoribonuclease activity. However, two assembly-defective capsid mutants, N2 (N- terminus 24-amino acids deletion) and P3 mutants, did not retain the specific endoribonuclease activity. Taken together, the results suggest that at least 24 amino acids from the N-terminal region and C194 residue in LRV1-4 capsid protein are functionally important for LRV1-4 viral assembly and the capsid endoribonuclease activity may be dependent upon the properly assembled LRV1-4 virus particles.
Subject(s)
Animals , Amino Acid Substitution , Anti-Bacterial Agents/pharmacology , Baculoviridae , Capsid/enzymology , Cell Line , Cysteine/genetics , Endoribonucleases/antagonists & inhibitors , Enzyme Activation/drug effects , Hot Temperature , Insecta , Leishmania guyanensis/virology , RNA/chemistry , RNA Viruses/enzymology , Recombinant Proteins/antagonists & inhibitors , Substrate Specificity/genetics , Transduction, GeneticABSTRACT
Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi, the causative agent of Chagas' disease, was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. LADH lipoamide reductase and diaphorase activities decreased as a function of incubation time and composition of the MPO/H2O2/halide system, a transient increase preceding the loss of diaphorase activity. Iodide, bromide, thiocyanide and chloride were effective components of MPO/H2O2 or MPO/NADH systems. Catalase prevented LADH inactivation by the MPO/NADH/halide systems in agreement with H2O2 production by NADH-supplemented LADH. Thiol compounds (L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine) and Captopril prevented LADH inactivation by the MPO/H2O2/NaCl system and by NaOCl, thus supporting HOCl as agent of the MPO/H2O2/NaCl system. MPO/H2O2/NaNO2 and MPO/NADH/NaNO2 inactivated LADH, the reaction being prevented by MPO inhibitors and thiol compounds. T. cruzi LADH was affected by MPO-dependent systems like myocardial LADH, allowance being made for the variation of the diaphorase activity and the greater sensitivity of the T. cruzi enzyme to MPO/H2O2/halide systems.