Proteomic identification of nuclear processes manipulated by cytomegalovirus early during infection

Carter, Dominique M.; Westdorp, Kristen N.; Noon, Kathleen R.; Terhune, Scott S.

doi:10.1002/pmic.201400599

Cited by 10 publications

(9 citation statements)

References 24 publications

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“…CMV expresses a diverse repertoire of proteins involved in regulating cellular processes, with many of these processes occurring within the nucleus. Our previous studies defined CMV-mediated changes in the nuclear proteome and uncovered several viral nuclear proteins of unknown functions (10). This included three peptides that matched to a protein expressed from the predicted UL31 ORF.…”

Section: Resultsmentioning

confidence: 99%

“…During CMV replication, profound changes occur to the host cell nucleus, including increased size, an altered proteome, and reorganized nuclear domains (9,10,53,54). Our recent studies investigating the nuclear proteome identified numerous viral proteins associated with the nucleus at the onset of viral DNA synthesis (10). This included multiple peptides matching to the predicted ORF of UL31, which contains a conserved dUTPase-like motif (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…1A and Table 1). Proteins containing this signal use the classical nuclear import machinery, importin-␣/␤, and we have previously demonstrated that CMV induces expression of a subset of importin-␣ adaptor proteins starting at 24 hpi to support replication (10). Current annotation of the UL31 ORF in published genomes identifies a downstream methionine as its starting codon, which lacks the NLS motif.…”

Section: Discussionmentioning

confidence: 99%

“…In previous studies, we detected multiple peptides matching to the predicted ORF of UL31 (10). UL31 is in all published genomes of CMV, contains a conserved dUTPaselike motif, and is detected in ribosomal profile studies as CMV ORFL87W (3,30).…”

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confidence: 83%

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Cytomegalovirus Late Protein pUL31 Alters Pre-rRNA Expression and Nuclear Organization during Infection

Westdorp

Sand

Moorman

et al. 2017

J Virol

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The replication cycle of human cytomegalovirus (CMV) leads to drastic reorganization of domains in the host cell nucleus. However, the mechanisms involved and how these domains contribute to infection are not well understood. Our recent studies defining the CMV-induced nuclear proteome identified several viral proteins of unknown functions, including a protein encoded by the UL31 gene. We set out to define the role of UL31 in CMV replication. UL31 is predicted to encode a 74-kDa protein, referred to as pUL31, containing a bipartite nuclear localization signal, an intrinsically disordered region overlapping arginine-rich motifs, and a C-terminal dUTPase-like structure. We observed that pUL31 is expressed with true late kinetics and is localized to nucleolin-containing nuclear domains. However, pUL31 is excluded from the viral nuclear replication center. Nucleolin is a marker of nucleoli, which are membrane-less regions involved in regulating ribosome biosynthesis and cellular stress responses. Other CMV proteins associate with nucleoli, and we demonstrate that pUL31 specifically interacts with the viral protein, pUL76. Coexpression of both proteins altered pUL31 localization and nucleolar organization. During infection, pUL31 colocalizes with nucleolin but not the transcriptional activator, UBF. In the absence of pUL31, CMV fails to reorganize nucleolin and UBF and exhibits a replication defect at a low multiplicity of infection. Finally, we observed that pUL31 is necessary and sufficient to reduce pre-rRNA levels, and this was dependent on the dUTPase-like motif in pUL31. Our studies demonstrate that CMV pUL31 functions in regulating nucleolar biology and contributes to the reorganization of nucleoli during infection. Nucleolar biology is important during CMV infection with the nucleolar protein, with nucleolin playing a role in maintaining the architecture of the viral nuclear replication center. However, the extent of CMV-mediated regulation of nucleolar biology is not well established. Proteins within nucleoli regulate ribosome biosynthesis and p53-dependent cellular stress responses that are capable of inducing cell cycle arrest and/or apoptosis, and they are proposed targets for cancer therapies. This study establishes that CMV protein pUL31 is necessary and sufficient to regulate nucleolar biology involving the reorganization of nucleolar proteins. Understanding these processes will help define approaches to stimulate cellular intrinsic stress responses that are capable of inhibiting CMV infection.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 83%

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Cytomegalovirus Late Protein pUL31 Alters Pre-rRNA Expression and Nuclear Organization during Infection

Westdorp

Sand

Moorman

et al. 2017

J Virol

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“…Amino acid fixed modifications included carbamidomethyl (C) and variable modifications included oxidation (M), Acetyl (Protein N terminus) and phosphorylation (STY). Quantifications were performed as previously described (40). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (41) partner repository with the data set identifier http://proteomecentral.proteomexchange.…”

Section: Methodsmentioning

confidence: 99%

Proteomic Screen for Cellular Targets of the Vaccinia Virus F10 Protein Kinase Reveals that Phosphorylation of mDia Regulates Stress Fiber Formation

Greseth

Carter

Terhune

et al. 2017

Molecular & Cellular Proteomics

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Vaccinia virus, a complex dsDNA virus, is unusual in replicating exclusively within the cytoplasm of infected cells. Although this prototypic poxvirus encodes >200 proteins utilized during infection, a significant role for host proteins and cellular architecture is increasingly evident. The viral B1 kinase and H1 phosphatase are known to target cellular proteins as well as viral substrates, but little is known about the cellular substrates of the F10 kinase. F10 is essential for virion morphogenesis, beginning with the poorly understood process of diversion of membranes from the ER for the purpose of virion membrane biogenesis. To better understand the function of F10, we generated a cell line that carries a single, inducible F10 transgene. Using uninduced and induced cells, we performed tablesotope abeling ofmino acids in ell culture (SILAC) coupled with phosphopeptide analysis to identify cellular targets of F10-mediated phosphorylation. We identified 27 proteins that showed statistically significant changes in phosphorylation upon the expression of the F10 kinase: 18 proteins showed an increase in phosphorylation whereas 9 proteins showed a decrease in phosphorylation. These proteins participate in several distinct cellular processes including cytoskeleton dynamics, membrane trafficking and cellular metabolism. One of the proteins with the greatest change in phosphorylation was mDia, a member of the formin family of cytoskeleton regulators; F10 induction led to increased phosphorylation on Ser Induction of F10 induced a statistically significant decrease in the percentage of cells with actin stress fibers; however, this change was abrogated when an mDia SerAla variant was expressed. Moreover, expression of a SerAsp variant leads to a reduction of stress fibers even in cells not expressing F10. In sum, we present the first unbiased screen for cellular targets of F10-mediated phosphorylation, and in so doing describe a heretofore unknown mechanism for regulating stress fiber formation through phosphorylation of mDia. Data are available via ProteomeXchange with identifier PXD005246.

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TRANSPIRE: A Computational Pipeline to Elucidate Intracellular Protein Movements from Spatial Proteomics Data Sets

Kennedy

Hofstadter

Cristea

2020

J. Am. Soc. Mass Spectrom.

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Protein localization is paramount to protein function, and the intracellular movement of proteins underlies the regulation of numerous cellular processes. Given advances in spatial proteomics, the investigation of protein localization at a global scale has become attainable. Also becoming apparent is the need for dedicated analytical frameworks that allow the discovery of global intracellular protein movement events. Here, we describe TRANSPIRE, a computational pipeline that facilitates TRanslocation ANalysis of SPatIal pRotEomics data sets. TRANSPIRE leverages synthetic translocation profiles generated from organelle marker proteins to train a probabilistic Gaussian process classifier that predicts changes in protein distribution. This output is then integrated with information regarding co-translocating proteins and complexes and enriched gene ontology associations to discern the putative regulation and function of movement. We validate TRANSPIRE performance for predicting nuclear-cytoplasmic shuttling events. Analyzing an existing data set of nuclear and cytoplasmic proteomes during Kaposi Sarcoma-associated herpesvirus (KSHV)-induced cellular mRNA decay, we confirm that TRANSPIRE readily discerns expected translocations of RNA binding proteins. We next investigate protein translocations during infection with human cytomegalovirus (HCMV), a β-herpesvirus known to induce global organelle remodeling. We find that HCMV infection induces broad changes in protein localization, with over 800 proteins predicted to translocate during virus replication. Evident are protein movements related to HCMV modulation of host defense, metabolism, cellular trafficking, and Wnt signaling. For example, the low-density lipoprotein receptor (LDLR) translocates to the lysosome early in infection in conjunction with its degradation, which we validate by targeted mass spectrometry. Using microscopy, we also validate the translocation of the multifunctional kinase DAPK3, a movement that may contribute to HCMV activation of Wnt signaling.

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Proteomic identification of nuclear processes manipulated by cytomegalovirus early during infection

Cited by 10 publications

References 24 publications

Cytomegalovirus Late Protein pUL31 Alters Pre-rRNA Expression and Nuclear Organization during Infection

Cytomegalovirus Late Protein pUL31 Alters Pre-rRNA Expression and Nuclear Organization during Infection

Proteomic Screen for Cellular Targets of the Vaccinia Virus F10 Protein Kinase Reveals that Phosphorylation of mDia Regulates Stress Fiber Formation

TRANSPIRE: A Computational Pipeline to Elucidate Intracellular Protein Movements from Spatial Proteomics Data Sets

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