The multifunctional character of a geminivirus replication protein is reflected by its complex oligomerization properties

Citation
Bm. Orozco et al., The multifunctional character of a geminivirus replication protein is reflected by its complex oligomerization properties, J BIOL CHEM, 275(9), 2000, pp. 6114-6122
Citations number
56
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
275
Issue
9
Year of publication
2000
Pages
6114 - 6122
Database
ISI
SICI code
0021-9258(20000303)275:9<6114:TMCOAG>2.0.ZU;2-#
Abstract
Tomato golden mosaic virus (TGMV), a member of the geminivirus family, enco des one essential replication protein, AL1, and recruits the rest of the DN A replication apparatus from its plant host. TGMV AL1 is an oligomeric prot ein that binds double-stranded DNA and catalyzes cleavage and ligation of s ingle-stranded DNA. The oligomerization domain, which is required for DNA b inding, maps to a region that displays strong sequence and structural homol ogy to other geminivirus Rep proteins. To assess the importance of conserve d residues, we generated a series of site-directed mutations and analyzed t heir impact on AL1 function in vitro and in vivo. Two-hybrid experiments re vealed that mutation of amino acids 157-159 inhibited AL1-AL1 interactions, whereas mutations at nearby residues reduced complex stability. Changes at positions 157-159 also disrupted interaction between the full-length mutan t protein and a glutathione S-transferase-AL1 oligomerization domain fusion in insect cells. The mutations had no detectable effect on oligomerization when both proteins contained full-length AL1 sequences, indicating that AL 1 complexes can be stabilized by amino acids outside of the oligomerization domain. Nearly all of the oligomerization domain mutants were inhibited or severely attenuated in their ability to support AL1-directed viral DNA rep lication. In contrast, the same mutants were enhanced for AL1-mediated tran scriptional repression. The replication-defective AL1 mutants also interfer ed with replication of a TGMV A DNA encoding wild type AL1, Full-length mut ant AL1 was more effective in the interference assays than truncated protei ns containing the oligomerization domain. Together, these results suggested that different AL1 complexes mediate viral replication and transcriptional regulation and that replication interference involves multiple domains of the AL1 protein.