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Quantitative temporal viromics: an approach to investigate host-pathogen interaction.

Weekes MP, Tomasec P, Huttlin EL, Fielding CA, Nusinow D, Stanton RJ, Wang EC, Aicheler R, Murrell I, Wilkinson GW, Lehner PJ, Gygi SP - Cell (2014)

Bottom Line: QTV predicted natural killer and T cell ligands, as well as 29 viral proteins present at the cell surface, potential therapeutic targets.Temporal profiles of >80% of HCMV canonical genes and 14 noncanonical HCMV open reading frames were defined.QTV is a powerful method that can yield important insights into viral infection and is applicable to any virus with a robust in vitro model.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA; Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK. Electronic address: mpw1001@cam.ac.uk.

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Further Details of Specific HCMV Proteins and Protein/mRNA Profiles, Related to Figure 5(A) All peptides quantified from the major immediate early region spanning UL122 (IE2) – UL123 (IE1). Peptides from exon 4 are unique to UL123. Peptides from exons 1-3 were assigned to IE1 protein by our data processing software, according to the principles of parsimony. Expression of ten exon 5 peptides corresponding to ORFL265C.iORF1 (lower panel) peaked late at 96h, in comparison to a single peptide N-terminal to this region, which is likely to have derived from UL122 itself. The lower panel shows a map of internal ORFs detected by exon 5 ribosomal footprinting (Stern-Ginossar et al., 2012). ∗ in peptide sequence: methionine oxidation.(B) Relationship between four novel ORFs and their canonical HCMV counterparts, with temporal profiles. Each of the novel ORFs were quantified based only on unique peptides that could only have originated from that ORF. Peptides that could either have originated from the canonical protein or the novel ORF were assigned to the canonical protein.(C) k-means clusters of (i) all quantified canonical HCMV proteins with 9 novel ORFs (experiment WCL2, also shown in Figure 5A) and (ii) all quantified canonical HCMV mRNAs and the same 9 novel ORFs (Stern-Ginossar et al., 2012). 5 clusters were selected in each case. The plots shown represent the average temporal profile for each cluster. As there were no intermediate mRNA time points between 5 and 24 hr, or between 24 and 72 hr, there was insufficient information to make an accurate comparison between the central three mRNA clusters and our Tp2, Tp3, or Tp4 class proteins. We therefore used mRNA data to define 3 classes: Tr1, Tr2-4, and Tr5. See Table S6C for details of the class of each protein.(D) Comparison between temporal protein profiles (this study) and mRNA expression profiles (Stern-Ginossar et al., 2012), grouped according to protein class. The 134 viral genes quantified in both studies are shown. For each protein or transcript, expression was normalized to the maximum across the measured time points. There was extremely good correspondence between protein and mRNA temporal profiles for Tp1 and Tp5 protein classes. Correspondence was less good for Tp2-4 protein classes as there were insufficient intermediate RNA time points to determine when maximal mRNA expression occurred.Red diamonds – 12h after infection with irradiated HCMV.
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figs5: Further Details of Specific HCMV Proteins and Protein/mRNA Profiles, Related to Figure 5(A) All peptides quantified from the major immediate early region spanning UL122 (IE2) – UL123 (IE1). Peptides from exon 4 are unique to UL123. Peptides from exons 1-3 were assigned to IE1 protein by our data processing software, according to the principles of parsimony. Expression of ten exon 5 peptides corresponding to ORFL265C.iORF1 (lower panel) peaked late at 96h, in comparison to a single peptide N-terminal to this region, which is likely to have derived from UL122 itself. The lower panel shows a map of internal ORFs detected by exon 5 ribosomal footprinting (Stern-Ginossar et al., 2012). ∗ in peptide sequence: methionine oxidation.(B) Relationship between four novel ORFs and their canonical HCMV counterparts, with temporal profiles. Each of the novel ORFs were quantified based only on unique peptides that could only have originated from that ORF. Peptides that could either have originated from the canonical protein or the novel ORF were assigned to the canonical protein.(C) k-means clusters of (i) all quantified canonical HCMV proteins with 9 novel ORFs (experiment WCL2, also shown in Figure 5A) and (ii) all quantified canonical HCMV mRNAs and the same 9 novel ORFs (Stern-Ginossar et al., 2012). 5 clusters were selected in each case. The plots shown represent the average temporal profile for each cluster. As there were no intermediate mRNA time points between 5 and 24 hr, or between 24 and 72 hr, there was insufficient information to make an accurate comparison between the central three mRNA clusters and our Tp2, Tp3, or Tp4 class proteins. We therefore used mRNA data to define 3 classes: Tr1, Tr2-4, and Tr5. See Table S6C for details of the class of each protein.(D) Comparison between temporal protein profiles (this study) and mRNA expression profiles (Stern-Ginossar et al., 2012), grouped according to protein class. The 134 viral genes quantified in both studies are shown. For each protein or transcript, expression was normalized to the maximum across the measured time points. There was extremely good correspondence between protein and mRNA temporal profiles for Tp1 and Tp5 protein classes. Correspondence was less good for Tp2-4 protein classes as there were insufficient intermediate RNA time points to determine when maximal mRNA expression occurred.Red diamonds – 12h after infection with irradiated HCMV.

Mentions: We were unable to confidently resolve the profile of UL122 (IE2). UL122 and UL123 are expressed by alternative splicing of a single major immediate-early transcript. Exons 1, 2, 3, and 4 encode UL123 and exons 1, 2, 3, and 5 encode UL122; however, additional transcripts have also been detected from the region of exon 5 (Stenberg et al., 1989; Stern-Ginossar et al., 2012). We examined each peptide quantified from every exon (Figure S5A). The profiles of all peptides from exon 4 peaked at 18–24 hr, corresponding to the predicted expression of UL123 protein. The profiles of ten exon 5 peptides corresponding to the late-expressed internal ORF, ORFL265C.iORF1 (Stern-Ginossar et al., 2012) peaked at 96 hr. A single peptide N-terminal to this ORF that is likely to originate from UL122 had a distinct profile with expression from 6 hr that peaked at 48 hr, similar to UL122 mRNA (Stern-Ginossar et al., 2012). This suggests the existence of at least two proteins arising wholly or in part from exon 5 and corresponds to the detection of an abundant late 40 kDa protein, previously detected from the same internal exon 5 ORF of AD169 strain (Stenberg et al., 1989). It is likely that ORFL265C.iORF1 protein is more abundant than UL122, effectively masking the earlier signal from most UL122 exon 5 peptides. Our quantitation of DNA polymerase processivity subunit UL44 may similarly have been complicated due to multiple transcription start sites (Data S1).


Quantitative temporal viromics: an approach to investigate host-pathogen interaction.

Weekes MP, Tomasec P, Huttlin EL, Fielding CA, Nusinow D, Stanton RJ, Wang EC, Aicheler R, Murrell I, Wilkinson GW, Lehner PJ, Gygi SP - Cell (2014)

Further Details of Specific HCMV Proteins and Protein/mRNA Profiles, Related to Figure 5(A) All peptides quantified from the major immediate early region spanning UL122 (IE2) – UL123 (IE1). Peptides from exon 4 are unique to UL123. Peptides from exons 1-3 were assigned to IE1 protein by our data processing software, according to the principles of parsimony. Expression of ten exon 5 peptides corresponding to ORFL265C.iORF1 (lower panel) peaked late at 96h, in comparison to a single peptide N-terminal to this region, which is likely to have derived from UL122 itself. The lower panel shows a map of internal ORFs detected by exon 5 ribosomal footprinting (Stern-Ginossar et al., 2012). ∗ in peptide sequence: methionine oxidation.(B) Relationship between four novel ORFs and their canonical HCMV counterparts, with temporal profiles. Each of the novel ORFs were quantified based only on unique peptides that could only have originated from that ORF. Peptides that could either have originated from the canonical protein or the novel ORF were assigned to the canonical protein.(C) k-means clusters of (i) all quantified canonical HCMV proteins with 9 novel ORFs (experiment WCL2, also shown in Figure 5A) and (ii) all quantified canonical HCMV mRNAs and the same 9 novel ORFs (Stern-Ginossar et al., 2012). 5 clusters were selected in each case. The plots shown represent the average temporal profile for each cluster. As there were no intermediate mRNA time points between 5 and 24 hr, or between 24 and 72 hr, there was insufficient information to make an accurate comparison between the central three mRNA clusters and our Tp2, Tp3, or Tp4 class proteins. We therefore used mRNA data to define 3 classes: Tr1, Tr2-4, and Tr5. See Table S6C for details of the class of each protein.(D) Comparison between temporal protein profiles (this study) and mRNA expression profiles (Stern-Ginossar et al., 2012), grouped according to protein class. The 134 viral genes quantified in both studies are shown. For each protein or transcript, expression was normalized to the maximum across the measured time points. There was extremely good correspondence between protein and mRNA temporal profiles for Tp1 and Tp5 protein classes. Correspondence was less good for Tp2-4 protein classes as there were insufficient intermediate RNA time points to determine when maximal mRNA expression occurred.Red diamonds – 12h after infection with irradiated HCMV.
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Related In: Results  -  Collection

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figs5: Further Details of Specific HCMV Proteins and Protein/mRNA Profiles, Related to Figure 5(A) All peptides quantified from the major immediate early region spanning UL122 (IE2) – UL123 (IE1). Peptides from exon 4 are unique to UL123. Peptides from exons 1-3 were assigned to IE1 protein by our data processing software, according to the principles of parsimony. Expression of ten exon 5 peptides corresponding to ORFL265C.iORF1 (lower panel) peaked late at 96h, in comparison to a single peptide N-terminal to this region, which is likely to have derived from UL122 itself. The lower panel shows a map of internal ORFs detected by exon 5 ribosomal footprinting (Stern-Ginossar et al., 2012). ∗ in peptide sequence: methionine oxidation.(B) Relationship between four novel ORFs and their canonical HCMV counterparts, with temporal profiles. Each of the novel ORFs were quantified based only on unique peptides that could only have originated from that ORF. Peptides that could either have originated from the canonical protein or the novel ORF were assigned to the canonical protein.(C) k-means clusters of (i) all quantified canonical HCMV proteins with 9 novel ORFs (experiment WCL2, also shown in Figure 5A) and (ii) all quantified canonical HCMV mRNAs and the same 9 novel ORFs (Stern-Ginossar et al., 2012). 5 clusters were selected in each case. The plots shown represent the average temporal profile for each cluster. As there were no intermediate mRNA time points between 5 and 24 hr, or between 24 and 72 hr, there was insufficient information to make an accurate comparison between the central three mRNA clusters and our Tp2, Tp3, or Tp4 class proteins. We therefore used mRNA data to define 3 classes: Tr1, Tr2-4, and Tr5. See Table S6C for details of the class of each protein.(D) Comparison between temporal protein profiles (this study) and mRNA expression profiles (Stern-Ginossar et al., 2012), grouped according to protein class. The 134 viral genes quantified in both studies are shown. For each protein or transcript, expression was normalized to the maximum across the measured time points. There was extremely good correspondence between protein and mRNA temporal profiles for Tp1 and Tp5 protein classes. Correspondence was less good for Tp2-4 protein classes as there were insufficient intermediate RNA time points to determine when maximal mRNA expression occurred.Red diamonds – 12h after infection with irradiated HCMV.
Mentions: We were unable to confidently resolve the profile of UL122 (IE2). UL122 and UL123 are expressed by alternative splicing of a single major immediate-early transcript. Exons 1, 2, 3, and 4 encode UL123 and exons 1, 2, 3, and 5 encode UL122; however, additional transcripts have also been detected from the region of exon 5 (Stenberg et al., 1989; Stern-Ginossar et al., 2012). We examined each peptide quantified from every exon (Figure S5A). The profiles of all peptides from exon 4 peaked at 18–24 hr, corresponding to the predicted expression of UL123 protein. The profiles of ten exon 5 peptides corresponding to the late-expressed internal ORF, ORFL265C.iORF1 (Stern-Ginossar et al., 2012) peaked at 96 hr. A single peptide N-terminal to this ORF that is likely to originate from UL122 had a distinct profile with expression from 6 hr that peaked at 48 hr, similar to UL122 mRNA (Stern-Ginossar et al., 2012). This suggests the existence of at least two proteins arising wholly or in part from exon 5 and corresponds to the detection of an abundant late 40 kDa protein, previously detected from the same internal exon 5 ORF of AD169 strain (Stenberg et al., 1989). It is likely that ORFL265C.iORF1 protein is more abundant than UL122, effectively masking the earlier signal from most UL122 exon 5 peptides. Our quantitation of DNA polymerase processivity subunit UL44 may similarly have been complicated due to multiple transcription start sites (Data S1).

Bottom Line: QTV predicted natural killer and T cell ligands, as well as 29 viral proteins present at the cell surface, potential therapeutic targets.Temporal profiles of >80% of HCMV canonical genes and 14 noncanonical HCMV open reading frames were defined.QTV is a powerful method that can yield important insights into viral infection and is applicable to any virus with a robust in vitro model.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA; Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK. Electronic address: mpw1001@cam.ac.uk.

Show MeSH
Related in: MedlinePlus