Proteasome-Dependent Disruption of the E3 Ubiquitin Ligase Anaphase-Promoting Complex by HCMV Protein pUL21a

This work is licensed under a Creative Commons Attribution 4.0 International License.The anaphase-promoting complex (APC) is an E3 ubiquitin ligase which controls ubiquitination and degradation of multiple cell cycle regulatory proteins. During infection, human cytomegalovirus (HCMV), a widespread pathogen, not only phosphorylates the APC coactivator Cdh1 via the multifunctional viral kinase pUL97, it also promotes degradation of APC subunits via an unknown mechanism. Using a proteomics approach, we found that a recently identified HCMV protein, pUL21a, interacted with the APC. Importantly, we determined that expression of pUL21a was necessary and sufficient for proteasome-dependent degradation of APC subunits APC4 and APC5. This resulted in APC disruption and required pUL21a binding to the APC. We have identified the proline-arginine amino acid pair at residues 109–110 in pUL21a to be critical for its ability to bind and regulate the APC. A point mutant virus in which proline-arginine were mutated to alanines (PR-AA) grew at wild-type levels. However, a double mutant virus in which the viral ability to regulate the APC was abrogated by both PR-AA point mutation and UL97 deletion was markedly more attenuated compared to the UL97 deletion virus alone. This suggests that these mutations are synthetically lethal, and that HCMV exploits two viral factors to ensure successful disruption of the APC to overcome its restriction on virus infection. This study reveals the HCMV protein pUL21a as a novel APC regulator and uncovers a unique viral mechanism to subvert APC activity

Human Cytomegalovirus UL29/28 Protein Interacts with Components of the NuRD Complex Which Promote Accumulation of Immediate-Early RNA

Histone deacetylation plays a pivotal role in regulating human cytomegalovirus gene expression. In this report, we have identified candidate HDAC1-interacting proteins in the context of infection by using a method for rapid immunoisolation of an epitope-tagged protein coupled with mass spectrometry. Putative interactors included multiple human cytomegalovirus-coded proteins. In particular, the interaction of pUL38 and pUL29/28 with HDAC1 was confirmed by reciprocal immunoprecipitations. HDAC1 is present in numerous protein complexes, including the HDAC1-containing nucleosome remodeling and deacetylase protein complex, NuRD. pUL38 and pUL29/28 associated with the MTA2 component of NuRD, and shRNA-mediated knockdown of the RBBP4 and CHD4 constituents of NuRD inhibited HCMV immediate-early RNA and viral DNA accumulation; together this argues that multiple components of the NuRD complex are needed for efficient HCMV replication. Consistent with a positive acting role for the NuRD elements during viral replication, the growth of pUL29/28- or pUL38-deficient viruses could not be rescued by treating infected cells with the deacetylase inhibitor, trichostatin A. Transient expression of pUL29/28 enhanced activity of the HCMV major immediate-early promoter in a reporter assay, regardless of pUL38 expression. Importantly, induction of the major immediate-early reporter activity by pUL29/28 required functional NuRD components, consistent with the inhibition of immediate-early RNA accumulation within infected cells after knockdown of RBBP4 and CHD4. We propose that pUL29/28 modifies the NuRD complex to stimulate the accumulation of immediate-early RNAs

An immuno-enrichment free, validated quantification of tau protein in human CSF by LC-MS/MS

Tau protein is a key target of interest in developing therapeutics for neurodegenerative diseases. Here, we sought to develop a method that quantifies extracellular tau protein concentrations in human cerebrospinal fluid (CSF) without antibody-based enrichment strategies. We demonstrate that the fit-for-purpose validated method in Alzheimer\u27s Disease CSF is limited to quasi quantitative measures of tau surrogate peptides. We also provide evidence that CSF total Tau measures by LC-MS are feasible in the presence of monoclonal therapeutic antibodies in human CSF. Our Tau LC-MS/MS method is a translational bioanalytical tool for assaying target engagement and pharmacodynamics for anti-tau antibody drug development campaigns

Abrogation of both pUL21a APC regulatory activity and pUL97 results in a more severe attenuation in HCMV growth than pUL97 deletion alone.

<p>(A) Abrogation of pUL21a-APC binding alone is not sufficient to alter HCMV replication. MRC-5 cells in serum-containing (cycling condition) or serum-free (G0 condition) media were infected with AD<i>gfp</i>, AD<i>sub</i>UL21a, AD<i>pm</i>UL21a_PH-AA, or AD<i>pm</i>UL21a_PR-AA at an MOI of 0.01. Production of cell-free virus at indicated times was determined by plaque assay. (B) Abrogation of both UL97 and the pUL21a-APC binding site markedly reduced the efficiency of HCMV reconstitution as compared to abrogation of UL97 alone. To reconstitute AD<i>pm</i>UL21a_PR-AA/<i>sub</i>UL97 and pAD<i>pm</i>UL21a_PH-AA/<i>sub</i>UL97 viruses, MRC-5 fibroblasts were transfected with their corresponding BAC clones. For each recombinant virus, three independent clones were tested. Shown are representative images of virus spread indicated by virus-driven GFP expression at indicated days post transfection of two of the three clones. Images were taken under a Leica fluorescent microscope. (C) Abrogation of both UL97 and the pUL21a-APC binding site markedly reduced HCMV replication as compared to abrogation of UL97 alone. MRC-5 cells were infected with indicated recombinant viruses at an input genome number equivalent to that of 0.03 infectious units of wild type virus/cell. Production of cell-free virion DNA at indicated times was determined by qPCR analysis and normalized to input levels of AD<i>pm</i>UL21a_PH-AA, which was set to 1. (D) Multi-step growth analysis of double mutant viruses that carried the UL117 deletion and point mutation in the UL21a-APC binding site. Cells were infected with indicated recombinant viruses and analyzed as described in panel C. The input value of AD<i>gfp</i> was set to 1.</p

pUL21a interacts with the APC.

<p>(A) Identification of pUL21a interacting partners. MRC-5 cells were infected with AD<i>gfp</i> or AD<i>gfp</i>UL21a at an MOI of 5, collected at 48 hpi, and were immunoprecipitated with GFP antibody. Eluted proteins were run on an SDS-containing polyacrylamide gel and silver stained. The bands indicated with an arrow were identified by mass spectrometry as APC3 (100 kDa), APC7, and APC8 (both at 65 kDa). (B) GFP-tagged pUL21a interacts with the APC in HCMV infection. MRC-5 cells were infected as described in panel A, and lysates were subjected to co-immunoprecipitation with GFP or APC3 mouse monoclonal antibodies. Cell lysates and eluted proteins were analyzed by immunoblotting with indicated antibodies. GFP blots were cropped to save space but were from the same lane and exposed film. Non-specific cross-reacting bands are indicated by asterisk (see text). Partial proteolysis was often seen with the GFP-tagged UL21a protein, particularly in cell lysate samples. (C) Native pUL21a interacts with the APC in HCMV infection. MRC-5 cells were infected with AD<i>gfp</i> or AD<i>sub</i>UL21a (as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002789#s4" target="_blank">Materials and Methods</a>) in the presence (+) or absence (−) of 10 µM MG132. Cell lysates were prepared at 24 hpi and immunoprecipitated with APC3 antibody. Cell lysates and eluted proteins were analyzed by immunoblotting. (D) Interaction of pUL21a with the APC does not require other viral proteins. 293T cells were transfected with plasmids expressing <i>gfp</i>UL21a_wt or <i>gfp</i>UL21a_stop. Cells were collected 72 hours later and cell lysates were immunoprecipitated as in panel B. Cell lysates and eluted proteins were analyzed by immunoblotting. PCNA and pUL44 were used as cellular and viral negative controls, respectively.</p

pUL21a binding to the APC promotes degradation of APC subunits and APC dissociation.

<p>(A) pUL21a is required for HCMV to reduce APC4 and APC5 accumulation during infection. MRC-5 cells were infected in the presence or absence of PAA with AD<i>gfp</i> or AD<i>sub</i>UL21a. Cell lysates were analyzed by immunoblotting at 24 hpi. pUL21a, IE1-72, and actin were used as infection and loading controls, respectively. (B) HCMV infection does not alter APC4 or APC5 transcript levels. MRC-5 cells were infected with AD<i>gfp</i> or AD<i>sub</i>UL21a, total RNA was collected at indicated times, and APC4 and APC5 transcripts were measured by reverse transcription-coupled quantitative PCR (RT-qPCR) and normalized to that of GAPDH. (C) APC binding activity of pUL21a is required for proteasome-dependent degradation of APC4 and APC5 in HCMV infection. MRC-5 cells were infected with AD<i>gfp</i>, AD<i>sub</i>UL21a, AD<i>pm</i>UL21a_PH-AA, or AD<i>pm</i>UL21a_PR-AA. MG132 was added at 6 hpi, and cell lysates were analyzed at 20 hpi by immunoblotting. (D) APC binding ability of pUL21a is required for APC dissociation in HCMV infection. MRC-5 cells were infected with AD<i>pm</i>UL21a_PH-AA or AD<i>pm</i>UL21a_PR-AA, and treated with MG132 as described in panel C. Cells were collected at 20 hpi, cell lysates were immunoprecipitated with APC3 antibody, and both lysates and eluted proteins were analyzed by immunoblotting.</p

pUL21a reduces APC4 and APC5 protein levels and inhibits APC activity in an inducible cell line.

<p>(A) pUL21a expression is sufficient to reduce APC4 and 5 protein accumulation. Hela cells constitutively expressing GFP-tagged TetR were transduced with pLKO-derived lentivirus that expressed pUL21a_PH-AA, pUL21a_stop, or pUL21a_PR-AA under a Tet operator (TetO)-regulated CMV promoter. Transduced cells were enriched by antibiotic selection and pUL21a expression was induced by tetracycline treatment for 72 hours. Cell lysates were collected and analyzed by immunoblotting. (B) pUL21a expression is sufficient to induce an M-phase cell cycle arrest. Cells carrying pUL21a_PH-AA (+/− tetracycline) were treated with nocodazole (100 ng/µl) for 16 hours to synchronize cells in G2/M phase. The cells were then released from nocodazole, collected at indicated time points, and analyzed for DNA content by flow cytometry. (C) pUL21a expression is sufficient to regulate APC activity. Cells from panel B were also analyzed for APC subunit and substrate levels by immunoblotting.</p

The carboxyl-terminus of pUL21a contains the APC binding site.

<p>(A) Amino acid alignment of UL21a proteins from human, chimpanzee, and rhesus CMVs. Boxes above aligned proteins divide the protein into N-terminal, Middle, and C-terminal regions. Conserved residues at the C-terminal region targeted for alanine substitution are boxed. (B) Diagram of UL21a truncation mutants analyzed in this study. (C) The C-terminus of pUL21a binds to the APC. GFP-tagged UL21a truncation mutant proteins were expressed in 293T cells by transfection, cells were collected at 72 hours, and lysates were immunoprecipitated with GFP antibody. Cell lysates and eluted proteins were analyzed by immunoblotting. (D) Identification of residues critical for APC binding. C-terminal conserved residues indicated in panel A were mutated by alanine substitution, and GFP-tagged UL21a mutant proteins were tested for APC binding as described in panel C. (E) The APC binding site of pUL21a was validated during HCMV infection. MRC-5 cells were infected with recombinant HCMV virus carrying the GFP-tagged UL21a_PH-AA or UL21a_PR-AA point mutant. Cells were collected at 48 hpi and lysates were immunoprecipitated with GFP or APC3 antibody. Cell lysates and eluted proteins were analyzed by immunoblotting. Non-specific cross-reacting bands are indicated by asterisk.</p