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cmi12080-sup-0001-fs1.pdf368K

Fig. S1. Tagging the M50 and M53 genes of MCMV at their native loci.

A. Overview of the galK-kn-based BAC recombineering in E. coli. This combined cloning and selection procedure is divided into two steps (positive and negative selection), and allows seamless site-specific modifications of BAC cloned genomes. The process is based on the activity of the λ phage encoded proteins Redα (exo), Redβ (bet) and Red γ (gam), which mediate recombination between homologous DNA sequences. To generate MCMV BACs coding for the tagged viral proteins pM50HA and pFLAGM53, galK-kn targeting cassettes with flanking homologies (H1 and H2) specific either to the M50- or the M53-ORF were synthesized by PCR and introduced into the wild type MCMV BAC pSM3fr. Recombinants harbouring the galK-kn cassette at the targeted locus were selected for the second cloning step. Here, the galK-kn cassettes were replaced by linear DNA fragments containing the M50HA or FLAGM53 coding sequences plus the flanking homologies H1 and H2. The resulting MCMV BACs pSM3fr-M50HA, pSM3fr-FLAGM53 and pSM3fr-M50HA-FLAGM53 were verified by restriction pattern analysis and sequencing of the modified loci.

B. Sequence comparison of the M50 ORF within the wild type MCMV BAC before (pSM3fr) and after (pSM3fr-M50HA) genetic tagging of the M50 with a HA tag at the C-terminus.

C. Sequence comparison of the M53 ORF within the wild type MCMV BAC before (pSM3fr) and after (pSM3fr-FLAGM53) genetic tagging of the M53 at the N-terminus with a FLAG tag.

cmi12080-sup-0002-ts1.pdf399K

Table S1. Primers used in this study for the genetic tagging of M50 and M53.

cmi12080-sup-0003-Data S1.xlsx19K

Data S1. Viral proteins unspecifically interacting with the NEC.

The mass spectrometric data from eight independent experiments (samples plus controls) were analysed using the Scaffold 3 Proteome Software. The data interpretation was exported into excel files. The file contains following information for all listed hits: percentage of the protein sequence identified during the mass spectrometric analysis (Sequence Coverage), percentage of total spectra in the samples represented by the spectra that contributed to the protein identification (Percentage of Spectra), calculated probability of correct protein identification (Protein Probabilities), normalized peptide spectrum counts for the identified proteins (Spectrum Count), number of unique peptides on which the identification is based (Unique Peptide Counts), number of unique spectra associated with this protein (Unique Spectrum Count).

cmi12080-sup-0004-Data S2.xls76K

Data S2. Cellular proteins specifically interacting with the NEC. For more information see legends for Data S1.

cmi12080-sup-0005-Data S3.xlsx18K

Data S3. Viral proteins specifically interacting with the NEC. For more information see legends for Data S1.

cmi12080-sup-0006-Data S4.xlsx142K

Data S4. Cellular proteins unspecifically interacting with the NEC. For more information see legends for Data S1.

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