Exp Mol Med.  2004 Apr;36(2):145-156.

Evidence that the fully assembled capsid of Leishmania RNA virus 1-4 possesses catalytically active endoribonuclease activity

• Ro YT
• Kim EJ
• Lee HI
• Saiz M
• Jr RC
• Patterson

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.

Keyword

Endoribonuclease; Leishmania virus; mutational analysis; RNA cleavage; viral assembly

MeSH Terms

Amino Acid Substitution
Animals
Anti-Bacterial Agents/pharmacology
Baculoviridae
Capsid/*enzymology
Cell Line
Cysteine/genetics
Endoribonucleases/antagonists & inhibitors/chemistry/genetics/isolation & purification/*metabolism
Enzyme Activation/drug effects
Heat
Insects
Leishmania guyanensis/*virology
RNA/chemistry
RNA Viruses/*enzymology/genetics
Recombinant Proteins/antagonists & inhibitors/genetics/isolation & purification/metabolism
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.
Substrate Specificity/genetics
Transduction, Genetic