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
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.