The Y2H plasmids expressing LexA-UL44 and LexA-Ubc9 were generated by cloning the UL44 and Ubc9 coding sequences from pRSET44 (a gift of P. F. Ertl, GlaxoSmithKline, UK) and pACT2-Ubc9 (from G. Gao, Chinese Academy of Sciences, Beijing, China) respectively, in pBTMK, derived from pBTM116 [25] . The pACT-UL44 and pACT2-Ubc9 plasmids, encoding GAD-UL44 and GAD-Ubc9 fusions, respectively, have been described in [26, 27] . The plasmid expressing GAD-UL54 was created by cloning the UL54 coding sequence from pRSET-Pol (a gift of P. F. Ertl) in pACT2 (Clontech). Plasmid pRSET44 was used to express 6His-UL44 in Escherichia coli. Plasmid pRSET-Ubc9 was constructed by cloning the Ubc9 coding sequence from pACT2-Ubc9 in pRSET (Invitrogen). Plasmid pCDNA3-PB1, used for in vitro transcription of the PB1 subunit of influenza A virus RNA polymerase, was described previously [28] . Plasmids pD15-GST and pD15-UL44, which express GST and GST-UL44, respectively, have been described in [29] . Plasmid pTE1E2S1 [30] was provided by H. Saitoh (Kusamoto University, Japan). Plasmids GFP-UL44, pDESTnV5-UL44, and pDESTnV5-UL44DNLS have been described previously [11, 31] . Plasmids pDsRed2-Ubc9 [27] and pDsRed2-UL53 [32] were kindly provided by G. Gao (Chinese Academy of Sciences, Beijing, China) and D. Camozzi (University of Bologna, Italy), respectively. Plasmid pCDNA3.1-UL44-FLAG (from M. Marschall, Universitat Erlangen-Numberg, Germany) was used to express C-terminally FLAG-tagged UL44 [33] , while plasmid pDEST-nFLAG [34] was used to express N-terminally FLAG-tagged UL44 [35] , UL44Dloop [36] and UL44L86A/L87A in mammalian cells. Plasmids pCDNA3-Ubc9, pCDNA3-Ubc9C93S, pCDNA3-HA-SUMO-1, pCDNA3-HA-SUMO-2, and pCDNA3-HA-SUMO-3 used in overexpression experiments in mammalian cells were a gift of R. T. Hay (University of Dundee, UK). The deletion mutants LexA-UL44 1-100 , LexA-UL44 1-200 , LexA-UL44 1-300 , LexA-UL44 1-350 , LexA-UL44 1-390 , and LexA-UL44 1-420 were generated by PCR amplification of plasmid pBTMK-UL44 with appropriate primers (Table S1 ). The LexA-UL44 114-433 , LexA-UL44 201-433 , and LexA-UL44 313-433 constructs were generated by deleting part of the UL44 coding sequence from pBTMK-UL44 with restriction enzymes. Plasmids pDESTnFLAG-UL44(1-300) and pDESTnFLAG-UL44(313-433) were generated using the Gateway Technology (see Supplementary Material and Methods in Text S1). All other UL44 mutants and the Ubc9C93S mutant were obtained by using the QuikChange mutagenesis kit (Stratagene) with primers containing appropriate nucleotide change(s). More details on plasmid construction and mutagenesis are given in Supplementary Material and Methods in Text S1. The sequences of all primers used in this work are reported in Table S1 . All DNA sequences were confirmed by sequencing.