To further explore the interaction between UL44 and Ubc9, we sought to map the domain of UL44 that interacts with Ubc9. To this aim, several N-and C-terminal deletion mutants of UL44 fused to LexA were generated and tested for the ability to interact with GAD-Ubc9 by Y2H assays. Control western blot experiments with an anti-LexA antibody evidenced protein bands of the expected molecular mass for all mutants (data not shown). As shown in Fig. 1D , the truncated protein UL44 1-300 , lacking most of the C-terminal disordered region of UL44, exhibited interaction with Ubc9. Further C-terminal truncation of UL44 revealed that a protein fragment corresponding to the first 200 amino acids of UL44 (UL44 ) was still capable of interacting with Ubc9 ( Fig. 1D ). In contrast, the N-terminal 100 residues of UL44 (UL44 1-100 ) exhibited no interaction with Ubc9 (Fig. 1D ). Although unfolding of the UL44 1-100 mutant protein cannot be excluded, these results suggested that this region of UL44 may not contain sequences important and/or sufficient for Ubc9 binding. Therefore, we analyzed the effects of N-terminal truncations. Deletion of the N-terminal 113 residues of UL44 (UL44 114-433 ) did not impair the ability of UL44 to bind Ubc9. Similarly, the UL44 201-433 mutant, lacking the N-terminal 200 amino acids, interacted with Ubc9. Interestingly, a mutant that only expresses the C-terminal 121 residues of UL44 (UL44 313-433 ) still retained the ability to bind Ubc9 (Fig. 1D) . Control Y2H experiments showed that none of the truncated UL44 proteins was able to activate transcription by itself (data not shown). Mapping studies in mammalian cells expressing UL44 deletion mutants showed that both the UL44 1-300 and UL44 313-433 mutants could immunoprecipitate endogenous Ubc9, similarly to wild-type UL44 (Fig. S1 ). Thus, our results suggest that UL44 contains two domains capable of independently binding to Ubc9, located at the N-and Cterminus of the protein (likely within residues 1-200 and 313-433, respectively).