Dataset Information

Proteomic identification of nuclear processes manipulated by cytomegalovirus early during infection.

ABSTRACT: Human cytomegalovirus (HCMV) is a herpesvirus that is ubiquitously distributed worldwide and causes life-threating disease upon immunosuppression. HCMV expresses numerous proteins that function to establish an intracellular environment that supports viral replication. Like most DNA viruses, HCMV manipulates processes within the nucleus. We have quantified changes in the host cell nuclear proteome at 24 h post infection following infection with a clinical viral isolate. We have combined SILAC with multiple stages of fractionation to define changes. Tryptic peptides were analyzed by RP-HPLC combined with LC-MS/MS on an LTQ Orbitrap Velos mass spectrometer. Data from three biological replicates were processed with MaxQuant. A total of 1281 cellular proteins were quantified and 77 were found to be significantly differentially expressed. In addition, we observed 36 viral proteins associated with the nucleus. Diverse biological processes were significantly altered, including increased aspects of cell cycling, mRNA metabolism, and nucleocytoplasmic transport and decreased immune responses. We validated changes for several proteins including a subset of classical nuclear transport proteins. In addition, we demonstrated that disruption of these import factors is inhibitory to HCMV replication. Overall, we have identified HCMV-induced changes in the nuclear proteome and uncovered several processes that are important for infection. All MS data have been deposited in the ProteomeXchange with identifier PXD001909 (http://proteomecentral.proteomexchange.org/dataset/PXD001909).

SUBMITTER: Carter DM

PROVIDER: S-EPMC4556115 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Proteomic identification of nuclear processes manipulated by cytomegalovirus early during infection.

Carter Dominique M DM Westdorp Kristen K Noon Kathleen R KR Terhune Scott S SS

Proteomics 20150428 12

Human cytomegalovirus (HCMV) is a herpesvirus that is ubiquitously distributed worldwide and causes life-threating disease upon immunosuppression. HCMV expresses numerous proteins that function to establish an intracellular environment that supports viral replication. Like most DNA viruses, HCMV manipulates processes within the nucleus. We have quantified changes in the host cell nuclear proteome at 24 h post infection following infection with a clinical viral isolate. We have combined SILAC wit ...[more]

PMID: 25758553

Similar Datasets

Project description:During permissive in vitro infection, the human cytomegalovirus (HCMV) UL36-38 and US3 immediate-early (IE) regions give rise to multiple distinct species of RNA in a temporally regulated manner. We have compared the temporally regulated expression of the UL36-38 and US3 regions with that of the well-characterized major IE (MIE) region. Northern (RNA) blot hybridizations with antisense RNA probes were used to examine RNA isolated from infected cells at IE, early, and late times after infection and from cells infected in the presence of anisomycin (used to block de novo viral protein synthesis) or in the presence of phosphonoformate (used to block HCMV DNA synthesis). Different US3 region transcripts were expressed in the cytoplasm during the IE and late phases of infection, with kinetics similar to those of the MIE region. In contrast, various cytoplasmic transcripts from the UL36-38 region were expressed during each of the IE, early, and late phases of infection, including some expressed from IE through late times. The levels of steady-state RNA from the US3 and MIE regions were increased dramatically by infection in the presence of anisomycin, predominantly because of an increase in multiply spliced transcripts. Two of the three UL36-38 IE transcripts were largely unaffected by anisomycin and were expressed abundantly throughout infection, but a third, multiply spliced UL36-38 IE transcript was abundant only during infection in the presence of anisomycin. Nuclear, cytoplasmic, and polysome-associated transcripts from the three IE regions were not significantly different qualitatively or quantitatively. These results suggest that posttranscriptional controls at the levels of nuclear retention or polysome exclusion of transcripts are not operative for the IE region genes. Overall, these results indicate common features of expression of US3, MIE, and UL36-38, in addition to distinctive expression of the UL36-38 region during all temporal phases of expression.

Project description:Intrinsic immune mechanisms mediated by constitutively expressed proteins termed "restriction factors" provide frontline antiviral defense. We recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. We show here that at an early time point during the in vitro infection of low-passage-number human embryonic lung fibroblasts, IFI16 binds to HCMV DNA. However, during a later phase following infection, IFI16 is mislocalized to the cytoplasmic virus assembly complex (AC), where it colocalizes with viral structural proteins. Indeed, upon its binding to pUL97, IFI16 undergoes phosphorylation and relocalizes to the cytoplasm of HCMV-infected cells. ESCRT (endosomal sorting complex required for transport) machinery regulates the translocation of IFI16 into the virus AC by sorting and trafficking IFI16 into multivesicular bodies (MVB), as demonstrated by the interaction of IFI16 with two MVB markers: Vps4 and TGN46. Finally, IFI16 becomes incorporated into the newly assembled virions as demonstrated by Western blotting of purified virions and electron microscopy. Together, these results suggest that HCMV has evolved mechanisms to mislocalize and hijack IFI16, trapping it within mature virions. However, the significance of this IFI16 trapping following nuclear mislocalization remains to be established.Intracellular viral DNA sensors and restriction factors are critical components of host defense, which alarm and sensitize immune system against intruding pathogens. We have recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. However, viruses are known to evolve numerous strategies to cope and counteract such restriction factors and neutralize the first line of host defense mechanisms. Our findings describe that during early stages of infection, IFI16 successfully recognizes HCMV DNA. However, in late stages HCMV mislocalizes IFI16 into the cytoplasmic viral assembly complex and finally entraps the protein into mature virions. We clarify here the mechanisms HCMV relies to overcome intracellular viral restriction, which provides new insights about the relevance of DNA sensors during HCMV infection.

Dataset Information

Proteomic identification of nuclear processes manipulated by cytomegalovirus early during infection.

Publications

Proteomic identification of nuclear processes manipulated by cytomegalovirus early during infection.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets