Identification of protein interacting with DDB1 alone or with the HBx/DDB1

We obtained strong evidence that HBx does not act by preventing DDB1 from performing its normal functions, but that it is the HBx/DDB1 complex by itself that is toxic to the cells. This, together with the finding that HBx and DDB1 must interact through their natural binding regions to interfere with normal cell growth and to stimulate HBV replication, suggests that the HBx/DDB1 complex may function by providing a new surface for interaction with other cellular factors. Other members of the laboratory will perform a biochemical approach aimed at identifying higher order protein complexes containing HBx and DDB1.

Since the laboratory has expertise in yeast genetics, we have set up a genetic screen related to the two-hybrid protein interaction assay that I will call yeast "two-plus-one"

hybrid screen (schematized in Figure S13A). The screen is designed such as to allow selection of cDNA clones encoding VP16 activation domain-tagged proteins that require the presence of either the HBx/DDB1 complex or DDB1 alone to support yeast cell growth under selective conditions. The screen was performed in yeast strain carrying as a selectable marker gene an integrated copy of a his3 allele bearing a single RFX-binding site upstream of the TATA element. As bait we used DDB1 fused to the carboxyl-terminus of RFX (a human DNA binding protein). RFX-DDB1 was expressed from a centromeric plasmid marked with the URA3 gene whereas HBx was expressed in its native form from a centromeric plasmid marked with the ADE2 gene.

Human cDNA libraries fused to VP16 were expressed under the control of galactose-inducible Gal1,10 regulatory sequences from plasmids marked with the TRP1 gene.

The RFX-DDB1 fusion was demonstrated to be suitable as bait since it has no transcriptional activity on its own and it was able to interact with a VP16-HBx fusion protein (data not shown). Additional test showed that a commercially available library fused to the Gal4 activation domain could also be used, since the RFX-DDB1 bait also interacted with a Gal4AD-HBx protein. After transformation of the library, colonies that show DDB1-dependent growth under selective conditions are identified as those that cannot loose the plasmid encoding RFX-DDB1. This has been done by replica-plating the colonies on medium containing 5-fluoroorotic acid (FOA), a drug that kills cells synthesizing the URA3 gene product. HBx-dependency was assessed by

the red phenotype due to accumulation of an intermediate in cells lacking a functional ADE2 gene and reflecting in these settings loss of the HBx-encoding plasmid. Library dependant growth was assessed by galactose induction of the VP16.cDNA. The cDNA clones from FOA-sensitive colonies that scored positive after rescue in E.coli and retransformation back into yeast were sequenced.

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Figure S13: Interaction of DDB1 and the likely ortholog of mouse transient receptor 4-associated protein (hTRP4-AP) in yeast

A) Schematic diagram of the two-plus-one-hybrid assay. DDB1 fused carboxyl-terminally to the transcriptionally inactive human RFX-binding protein was used as bait in a screen for interaction with a cDNA library linked to the VP16 activation domain in presence of HBx expressed in its native form.

B) VP16-hTRP4-AP was tested for interaction with RFX-DDB1 in the presence or absence of HBx (left panel) or SV5-V (right panel). The black bars represent the relative activities of an integrated lacZ reporter gene bearing a single RFX-binding site in strains expressing RFX-DDB1 alone (-) or together (+) with VP16-hTRP4-AP.

Three independent transformations were performed with two VP16 activation domain-tagged human cDNA libraries. From several cDNA clones rescued in bacteria only 2 scored positive after retransformation into a yeast strain carrying an RFX-dependant LacZ reporter gene integrated at the HIS3 locus. The 2 clones derived from 2 different libraries correspond to the same mRNA encoding hTRP4-AP, the likely ortholog of mouse transient receptor protein 4 associated protein (NP_056453). The size of approximate 3,4 kb of the cDNA insert suggests the presence of the entire open reading frame. The yeast protein interaction assay showed that hTRP4-AP could bind to DDB1 in the presence of HBx (Figure S13B). However, HBx has almost no effect on the LacZ activity showing that HBx is not required for DDB1-hTRP4-AP interaction. Although HBx showed only a moderate interaction-promoting activity, we investigated if this interaction is compromised upon SV5-V overexpression as reported for the HBx-DDB1 interaction. Indeed, the LacZ activity dropped to background levels upon high levels of SV5-V expression. This reduction could reflect a mutually exclusive binding of SV5-V and hTRP4-AP to DDB1. However, since

HBx and hTRP4-AP appear to bind simultaneously to DDB1, these findings rather suggest that SV5-V induces a conformational change upon binding to DDB1 such that DDB1 can no longer bind to hTRP4-AP. An alternative explanation for these results is that SV5-V and hTRP4-AP bind to overlapping sites on DDB1, whereas hTRP4-AP and HBx do not. hTRP4-AP was named so because it interacts with transient receptor protein 4 in a yeast two-hybrid screen. Nothing is known about the receptor associated protein whereas the receptor itself was shown to be a calcium channel that can be activated by intracellular calcium store depletion. Calcium signaling by HBx has been shown to be important for HBV replication. A role for hTRP4-AP in the physiological functions of DDB1 or in the pathway, through which HBx acts to induce cell death and stimulation of viral replication, if any, remains to be demonstrated. For this purpose, biochemical (co-immunoprecipitation) and genetic (RNAi, overexpression) approaches will be used.