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. 2014 Dec;11(6):697-711.
doi: 10.1586/14789450.2014.971116. Epub 2014 Oct 18.

The life cycle and pathogenesis of human cytomegalovirus infection: lessons from proteomics

Pierre M Jean Beltran  1 , Ileana M Cristea
Affiliations

The life cycle and pathogenesis of human cytomegalovirus infection: lessons from proteomics

Pierre M Jean Beltran et al. Expert Rev Proteomics. 2014 Dec.

Abstract

Viruses have coevolved with their hosts, acquiring strategies to subvert host cellular pathways for effective viral replication and spread. Human cytomegalovirus (HCMV), a widely-spread β-herpesvirus, is a major cause of birth defects and opportunistic infections in HIV-1/AIDS patients. HCMV displays an intricate system-wide modulation of the human cell proteome. An impressive array of virus-host protein interactions occurs throughout the infection. To investigate the virus life cycle, proteomics has recently become a significant component of virology studies. Here, we review the mass spectrometry-based proteomics approaches used in HCMV studies, as well as their contribution to understanding the HCMV life cycle and the virus-induced changes to host cells. The importance of the biological insights gained from these studies clearly demonstrate the impact that proteomics has had and can continue to have on understanding HCMV biology and identifying new therapeutic targets.

Keywords: CMV; mass spectrometry; post-translational modifications; protein–protein interactions; proteomics; virus–host interactions.

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Figures

Figure 1
Figure 1. Overview of the HCMV life cycle
(A) Infectious particles enter the cell through interaction with cellular receptors. Capsid and tegument proteins are delivered to the cytosol. (B) The capsid travels to the nucleus, where the genome is delivered and circularized. Tegument proteins regulate host cell responses and initiate the temporal cascade of the expression of viral I immediate early (IE) genes, followed by delayed early (DE) genes, which initiate viral genome replication, and late (L) genes. (C) Late gene expression initiates capsid assembly in the nucleus, followed by nuclear egress to the cytosol. Capsids associate with tegument proteins in the cytosol and are trafficked to the viral assembly complex (AC) that contains components of the endoplasmic reticulum (ER), Golgi apparatus and endosomal machinery. The capsids further acquire tegument and viral envelope by budding into intracellular vesicles at the AC. (D) Enveloped infectious particles are released along with non-infectious dense bodies.
Figure 2
Figure 2. Proteomic approaches used for studying HCMV infection
The first step involves the selection of a relevant cell type, the virus strain, and the time(s) of infection for the study, as all these factors will influence the proteome content and the virus-host interactions. These decisions are commonly based on a priori knowledge and/or experimental evidence. To date, mass spectrometry-based proteomics methods have been used for protein identification and quantification in studies of virion composition, whole cell or subcellular proteomes, and virus-host protein interactions during HCMV infection. While posttranslational modifications have been analyzed in subcellular proteomes and protein affinity purification studies, their characterization has the potential to be expanded to virion and whole cell studies.
Figure 3
Figure 3. Quantitative proteomic methods used for studying HCMV-infected cells
Both label-free and labeling methods have been used in protein quantification during HCMV infection. Label-free quantification can be readily integrated in studies of subcellular fractions, whole cell lysates (WCL), or affinity purified (AP) proteins. The usual workflow involves the digestion of proteins and their analysis by LC-MS/MS using data-dependent acquisition mode. Quantitative values for each protein are obtained by extracting the protein spectral counts or the integrated peptide intensities. Labelling quantification approaches that are commonly used are metabolic labeling and isobaric tag labeling. Stable isotope labeling by amino acids in cell culture (SILAC) incorporates “light” or “heavy” amino acids, allowing the comparison of uninfected and infected cells. Quantification is done at the MS level by comparing the ion intensities of the heavy and light peptides. For labeling with isobaric tag, the samples that are to be compared are processed in parallel, similar to label-free quantification up to the proteolysis steps. The resulting peptides are then labeled using isobaric tags and then mixed. The quantification is done at the MS/MS level, as the spectra contain both peptide fragments for amino acid sequence information and a set of reporter ions from the isobaric tags that illustrate the relative abundances of the peptides originating from the different samples. These compared samples can include cells collected at different times after infection, cells infected with various HCMV strains, or multiple biological replicates.
Figure 4
Figure 4. Examples of critical virus-host interactions during the HCMV replication cycle
(A) Host and virus control of immediate early (IE) gene expression. pUL38 and pUL29/28 interact with the HDAC1-NuRD complex to help induce expression of IE genes from the MIEP. pUL97 phosphorylates HDAC1 and inhibits its deacetylase activity, promoting viral gene expression by potentially de-stabilizing HDAC1 association with the NuRD complex. The DNA sensor IFI16 binds the HCMV genome to induce antiviral cytokine production. pUL83 interacts with IFI16 to restrict its oligomerization and cytokine production. Additionally, IFI16 is repurposed to activate the MIEP. (B) Protein complexes involved in HCMV genome replication. pUL84 is part of the HCMV DNA replication machinery, interacting with oriLyt and host and viral proteins. To function within the replication complex, pUL84 has to be phosphorylated (P) by CKII. pUL84 also interacts with the ubiquitin-conjugating enzyme E2 and becomes mono ubiquitinated (Ub). The DNA processivity factor, pUL44, is thought to be a part of multiple protein complexes and is important for efficient DNA replication. (C) Viral modulation of cellular stress response through TSC1/2. pUL38 interacts with TSC1/2 to block its activity and maintain an active mTOR pathway following infection. (D) Control of cell cycle progression and induction of the DNA damage response. pUL21a interacts with APC members, leading to their degradation, reduced APC activity, and dysregulation of the cell cycle. pUL27 interacts with Tip60, targeting this protein for degradation and contributing to cell-cycle arrest. pUL35 interacts with subunits of the Cul4 ubiquitin ligase complex, causing cell cycle arrest and induction of DNA damage response.
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