Institut für Infektionsmedizin, Abteilung Virologie, Freie Universität Berlin, Hindenburgdamm 27, D-12203 Berlin, Germany1
Author for correspondence: Regine Heilbronn. Fax +49 30 8445 4485. e-mail heilbronn{at}ukbf.fu-berlin.de
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Abstract |
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Main text |
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To identify additional cellular Rep targets relevant for the diverse effects of Rep78 and Rep68 we employed the yeast two-hybrid system as outlined in Weger et al. (1999) . The majority of Rep68 interaction partners corresponded to the transcriptional coactivator PC4 (Weger et al., 1999
). To identify Rep-interacting cellular proteins that do not correspond to PC4, positive clones obtained from a yeast two-hybrid screen (5x106 yeast transformants) of a HeLa cDNA library (1x106 independent clones) with the central part of the large AAV-2 Rep proteins (pGBT9M172/530; Fig. 1A
) as bait were subjected to colony hybridization with a PC4 hybridization probe. Three PC4-negative clones were identified, which were positive for interaction with pGBT9Rep68 and negative with unrelated bait proteins after retransformation. The coding region of one of these clones corresponded to the C-terminal 192 amino acids of a protein recently identified both as a novel p53 binding protein, named p53BP3 (Zhou et al., 1999
), and as a DNA topoisomerase I binding protein, named Topors (Haluska et al., 1999
), in yeast two-hybrid screens. Sequence analysis had predicted an ORF of 815 amino acids for p53BP3 (Zhou et al., 1999
) and an ORF with 230 additional amino acids at the N terminus for Topors (Haluska et al., 1999
). We cloned the entire cDNA sequence by RTPCR from HeLa mRNA and obtained nucleotide sequence data corresponding to that published for Topors (Haluska et al., 1999
). Thus we will refer to this protein as Topors. To demonstrate direct binding of Rep to the C terminus of Topors, a GST fusion protein encoding the 192 C-terminal amino acids of Topors was expressed in E. coli. The GSTTopors fusion protein and GST alone as a control were purified on glutathioneSepharose beads and incubated with in vitro-transcribed/translated and 35S-labelled Rep78 or Rep52, respectively, as described in Weger et al. (1999)
. Rep78 (Fig. 1B
, left panel), but not Rep52 (Fig. 1B
, right panel), was specifically retained by the GSTTopors(8541045) fusion protein. These findings demonstrate binding of the large AAV-2 Rep proteins to the C terminus of Topors both in vivo and in vitro.
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To address the question of which regions of Rep and Topors are involved in the RepTopors interaction, a series of pGBT9Rep constructs containing various parts of the Rep ORF fused to the Gal4 DNA binding domain and a series of pGAD424Topors constructs containing different parts of the Topors ORF fused to the Gal4 transactivation domain were generated. Interaction studies were then performed in yeast SFY526 cells with a quantitative liquid culture -galactosidase assay using CPRG as a substrate (Clontech).
The Rep sequences required for interaction were analysed after cotransformation with the pGAD424Topors(8541045) construct comprising the 192 C-terminal Topors amino acids. Rep M172/530 (Fig. 2A) demonstrates that Rep amino acids 1 to 171 were not absolutely necessary for the RepTopors interaction. Further deletion of 53 amino acids in Rep40, however, led to a complete loss of interaction (Fig. 2A
). The reduced reporter gene activity observed for M1/369 and M1/243 (Fig. 2A
) implies an involvement of amino acids in the central part of the Rep coding region. However, a reduced expression level or improper folding of the corresponding fusion proteins cannot be excluded. A point mutation in the Rep nucleotide binding site changing lysine 340 to histidine, which abolishes the ATPase and helicase activities of the large Rep proteins, did not abolish binding to the C terminus of Topors (Fig. 2A
, Rep68K340H).
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Since the RING finger motif of Topors is closely related to that of the ICP0 family of viral transactivators (Haluska et al., 1999 ), we examined the impact of Topors overexpression on AAV gene expression in the absence of helper virus. Together with a cloned wild-type AAV genome (pTAV2-0; Heilbronn et al., 1990
), increasing amounts of a human cytomegalovirus promoter-driven Flag-tagged Topors construct (pCATCHTopors) were cotransfected into HeLa cells. AAV gene expression was monitored as steady state mRNA level and Rep and Cap protein expression levels 24 h post-transfection. The low levels of Rep78 and Rep52 protein expressed from pTAV2-0 in the absence of helper virus were increased in parallel to increasing amounts of cotransfected pCATCHTopors (Fig. 3A
, lanes 25). Faint bands corresponding in size to Rep68 and Rep40 could be detected after prolonged exposures (not shown). AAV-2 capsid proteins VP1, VP2 and VP3 could only be detected after cotransfection of high amounts of pCATCHTopors (Fig. 3B
, lane 5). The reactive bands observed in Fig. 3
(B, lanes 1 to 4) are due to cross-reacting cellular proteins, since they were also observed in the absence of pTAV2-0 (not shown). A concomitant increase in the steady state level of p5, p19 and p40 transcripts was also found at high concentrations of pCATCHTopors (Fig. 3C
, lanes 4 and 5), suggesting that the stimulatory effect of Topors overexpression on AAV protein levels takes place at a level preceding translation, such as transcription or RNA stability. Expression of Flag-tagged Topors protein was assayed with an anti-Flag antibody and could consistently only be detected at the highest concentration of cotransfected pCATCHTopors plasmid (Fig. 3E
), which may be due to a limiting sensitivity of the anti-Flag antibody. Neither AAV DNA replication nor the formation of infectious AAV particles was detected upon overexpression of Topors in the absence of helper virus (not shown).
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Received 3 October 2001;
accepted 23 November 2001.