Journal: Viruses

Article Title: Role of PARP-1 in Human Cytomegalovirus Infection and Functional Partners Encoded by This Virus

doi: 10.3390/v14092049

Figure Lengend Snippet: HCMV UL76 binds to the BRCT domain of PARP-1 through its C terminal. ( A ) NAD + levels declined to varying degrees upon HSV-1 and HCMV infection. Fibroblasts were infected with HSV-1 (F strain) or HCMV (AD169 strain) at an MOI of 1. Cells were collected at 24 hpi by trypsin digestion and lysed in a NAD + extraction buffer. NAD + levels in these cells were normalized to total protein and compared to mock-infected cells. Values are averages of triplicate experiments (±1 SD), * p < 0.05. ( B ) Protein PARylation and the protein levels of PARP-1 and PARG were compared between HSV-1- and HCMV-infected cells. MRC-5 cells were infected with HSV-1 or HCMV at an MOI of 1. Pp65 of HCMV and glycoprotein C of HSV-1 served as infection markers for each virus. The whole cell lysates were analyzed by indicated antibodies. ( C ) HCMV tegument protein UL76 was a PARP-1 binding protein. MRC-5 cells were infected or mock infected with HCMV at an MOI of 1 for 96 h. Cells were harvested and lysed in NETN 420 in the presence of protease inhibitors. Cell lysates were prepared and subjected to immunoprecipitation with PARP-1 antibody or control IgG. The presence of PARP-1 and UL76 in the immune complexes was determined by Western blot analysis. * indicates nonspecific band. ( D ) UL76 directly interacts with PARP-1 in vitro. Purified His-PARP-1 was incubated with equal amount of GST or GST-UL76. Samples were subjected to GST pull-down, and bound complexes were analyzed by SDS-PAGE. ( E ) Schematic depiction of PARP-1 and its deletion mutants. Results from domain mapping experiments indicated that UL76 binds to the BRCT domain of PARP-1. ( F ) BRCT domain of PARP-1 was responsible for the association between UL76 and PARP-1. SFB vector or SFP-UL76 was co-transfected with GFP-tagged PARP-1 WT or each deletion mutant. UL76 did not bind with the PARP-1 BRCT domain deletion mutant. ( G ) Schematic depiction of HCMV UL76. Results from domain mapping experiments indicated that PARP-1 binds to the C terminal of UL76. ( H ) HEK293T cells were transfected with a construct for SFB vector, SFB-tagged UL76, UL76 truncates or UL76 homologs in HSV-1 and KSHV, respectively. Cell lysates were subjected to SFB immunoprecipitation, and the presence of PARP-1 was analyzed by immunoblotting.

Article Snippet: HCMV AD169 strain (ATCC-VR538) was propagated in MRC-5 cells.

Techniques: Infection, Binding Assay, Immunoprecipitation, Western Blot, In Vitro, Purification, Incubation, SDS Page, Plasmid Preparation, Transfection, Mutagenesis, Construct

Journal: Viruses

Article Title: New Insights into Human Cytomegalovirus pUL52 Structure

doi: 10.3390/v13081638

Figure Lengend Snippet: Identification of a putative zinc-finger motif within pUL52 protein. ( a ) Structure of HCMV pUL52 with a putative zinc-finger (ZF) pattern (VRI and VRII: variable regions I and II). ( b ) Sequence alignment of the conserved region I from 18 herpesviruses and residues involved in a metal-binding site. Sequence numbering is consistent with that of HCMV reference strain AD169 residues. Key residues involved in the formation of the zinc-finger motif are shown as white letters on black background. ( c ) The region I was extracted from the pUL52 theoretical structure. Cysteines are represented in purple, histidine H276 in yellow. ( d ) Impact of HCMV-BAC- UL52 mutants on growth in cell culture at day 11 (fibroblasts MRC-5). ( e ) Growth curves of the recombinant virus strain HCMV-BAC- UL52 -C229S in comparison to the parental strain HCMV-BAC AD169 WT. Fluorescent foci were counted daily from day 1 to day 7. Curves are the average of three independent experiments. ** p < 0.01; *** p < 0.001 (Mann–Whitney test). VRI and VRII: variable regions I and II. aa: amino acid.

Article Snippet: Five reference strains—AD169 (ATCC VR-538), Davis (ATCC VR-807), Towne (ATCC VR-977) Merlin and Toledo—and 75 HCMV clinical isolates collected from various hospitals in France for the National Reference Center for Herpesviruses were studied.

Techniques: Sequencing, Binding Assay, Cell Culture, Recombinant, MANN-WHITNEY

Journal: Viruses

Article Title: New Insights into Human Cytomegalovirus pUL52 Structure

doi: 10.3390/v13081638

Figure Lengend Snippet: CXXC-like motif in conserved Region II. ( a ) Structure of HCMV pUL52 with a CXXC motif. ( b ) Sequence alignment of the conserved region II from 18 herpesviruses and residues involved in a CXXC motif. Sequence numbering is consistent with that of HCMV reference strain AD169 residues. Key residues involved in the formation of the CXXC motif are shown as white letters on black background. ( c ) Impact of HCMV-BAC- UL52 mutants on growth in cell culture at day 11 (fibroblasts MRC-5). VRI and VRII: variable regions I and II. aa: amino acid.

Article Snippet: Five reference strains—AD169 (ATCC VR-538), Davis (ATCC VR-807), Towne (ATCC VR-977) Merlin and Toledo—and 75 HCMV clinical isolates collected from various hospitals in France for the National Reference Center for Herpesviruses were studied.

Techniques: Sequencing, Cell Culture

Journal: Viruses

Article Title: New Insights into Human Cytomegalovirus pUL52 Structure

doi: 10.3390/v13081638

Figure Lengend Snippet: HXXCXXXC motif in conserved Region III. ( a ) Structure of HCMV pUL52 with an HXXCXXXC motif. ( b ) Sequence alignment of the conserved region III from 18 herpesviruses and residues involved in an HXXCXXC motif. Sequence numbering is consistent with that of HCMV reference strain AD169 residues. Key residues involved in the formation of the HXXCXXC motif are shown as white letters on black background. The residue in red matches the position of the mutation V580I found in one of the 61 naive strains. ( c ) Region 3 was extracted from the pUL52 theoretical structure. Cysteines and histidine are represented in purple and yellow, respectively. ( d ) Impact of HCMV-BAC-UL52 mutants on growth in cell culture at day 11 (fibroblasts MRC-5). ( e ) Growth curves of the recombinant virus strain HCMV-BAC- UL52 -C570S in comparison to the parental strain HCMV-BAC AD169 WT. Fluorescent foci were counted daily from day 1 to day 7. Curves are the average of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 (Mann–Whitney test). VRI and VRII: variable regions I and II. aa: amino acid.

Article Snippet: Five reference strains—AD169 (ATCC VR-538), Davis (ATCC VR-807), Towne (ATCC VR-977) Merlin and Toledo—and 75 HCMV clinical isolates collected from various hospitals in France for the National Reference Center for Herpesviruses were studied.

Techniques: Sequencing, Mutagenesis, Cell Culture, Recombinant, MANN-WHITNEY

Journal: Frontiers in Pediatrics

Article Title: Analysis of Thermal Sensitivity of Human Cytomegalovirus Assayed in the Conventional Conditions of a Human Milk Bank

doi: 10.3389/fped.2021.640638

Figure Lengend Snippet: Effects of treatment at 60°C for 30 min on HCMV strain AD169 infectivity at two different titers in a pasteurizer-like model. (A) Heat treatment and infection protocol for HCMV. (B,C) In the graphs, HCMV infectivity of heat-treated milk samples spiked with HCMV AD169 at two titers (1 × 10 4 TCID50 and 3 × 10 5 TCID50) is reported as number of infected cells/ml at 5 days post infection (B) and as FFU/ml at 10 days post infection (C) . (D,E) . Representative figures of HFF-1 cells treated with milk samples spiked with HCMV (3 × 10 5 TCID50) and treated at 4°C for 30 min (unpasteurised, left) or at 60°C for 30 min (pasteurized, right) at 5 days post infection (D) and 10 days post infection (E) . Infected cells and foci are brown by immunostaining. UP, unpasteurised.

Article Snippet: Human cytomegalovirus strain AD169 was purchased from the ATCC (VR-538).

Techniques: Infection, Immunostaining