A Comparative Quantitative Proteomic Analysis of HCMV-Infected Cells Highlights pUL138 as a Multifunctional Protein

Abstract

Human cytomegalovirus (HCMV) is a widespread virus that can establish life-long latent infection in large populations. The establishment of latent infection prevents HCMV from being cleared by host cells, and HCMV reactivation from latency can cause severe disease and death in people with immature or compromised immune systems. To establish persistent and latent infection in healthy individuals, HCMV encodes a large array of proteins that can modulate different components and pathways of host cells. It has been reported that pUL138 encoded by the UL133-UL138 polycistronic locus promotes latent infection in primary CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. In this study, recombinant HCMV Han_UL138del was constructed by deleting the UL138 locus of Han, a clinical HCMV strain. Then, a comparative quantitative proteomic analysis of Han- and Han_UL138del-infected MRC5 cells was performed to study the effect of pUL138 on host cells in the context of HCMV infection. Our results indicated that, during the early phase of HCMV infection, the innate immune response was differentially activated, while during the late phase of HCMV infection, multiple host proteins were differentially expressed between Han- and Han_UL138del-infected cells, and these proteins are involved in the oxidation-reduction process, ER to Golgi vesicle-mediated transport, and extracellular matrix organization. Among these proteins, STEAP3, BORCS7, FAM172A, RELL1, and WDR48 were further demonstrated to affect HCMV infection. Our study provides a systematic view of the effect of pUL138 on the host cell proteome and highlights the proposition that multiple biological processes or host factors may be involved in the overall role of the UL133-UL138 polycistronic locus in HCMV persistence.

Keywords: HCMV; UL138; latent infection; quantitative proteomic.

Figure 1

Comparative quantitative proteomic analysis of Han- and Han_UL138del-infected MRC5 cells. (A) Construction of Han_UL138del-BAC. The ORF of UL138 in the ULb’ region was replaced by the kanamycin resistance cassette. (B) Replication kinetics curves of Han and Han_UL138del in MRC5 cells infected at an MOI of 0.1. Cells were harvested separately every 12 h for 7 days post infection. Intracellular levels of HCMV genomic DNA were detected with primers for UL44 by quantitative RT-PCR analysis. Three independent biological replicates were performed, and a representative experiment (with two technical replicates) from three biological replicates is shown. Results are shown as mean ± SD. (C) Replication kinetics curves of Han and Han_UL138del in MRC5 cells infected at an MOI of 5. Three independent biological replicates were performed, and a representative experiment (with two technical replicates) from three biological replicates is shown. Results are shown as mean ± SD. (D) Workflow for quantitative proteomic analysis. Cells were infected with Han or Han_UL138del (at an MOI of 5) or mock-treated. At 12 h p.i. and 96 h p.i., cells were collected and lysed to extract proteins. Proteins in each sample were subjected to parallel denaturation and digestion. Digested samples were labeled with iTRAQ reagents and mixed together. The mixed peptides were fractionated and analyzed by LC-MS/MS analysis. (E) The ratio distribution of quantified proteins.

Figure 2

Validation of MS results. (A) Western blot analysis of MRC5 cells infected with Han or Han_UL138del at an MOI of 5. MRC5 cells were harvested at 12 h p.i. or 96 h p.i., and subjected to Western blot analysis. The intensity of each band was quantitated with Quantity One software and normalized to mock-treated cell band intensity. The numbers with brackets marked below the band represent the relative fold changes. (B) Quantitative real-time PCR analysis of selected proteins in cells (MRC5) infected with Han, HanUL138del (at an MOI of 5) or mock-treated. MRC5 cells were harvested at 12 h p.i. or 96 h p.i. and intracellular RNAs were extracted and reverse transcribed to cDNA. The relative mRNA levels of selected proteins were measured by quantitative RT-PCR analysis. Three replicates were performed, and results are shown as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no significance.

Figure 3

Gene Ontology analysis (biological process) of differentially regulated proteins at 12 h p.i. or 96 h p.i.. Differentially regulated proteins at 12 h p.i. (A) or 96 h p.i. (B) were subjected to DAVID v6.8. The regulated proteins were grouped based on their roles in biological processes, and a statistical overrepresentation test was performed to determine which biological process was overrepresented by differentially regulated proteins. Only the overrepresented categories are presented here. Expected hits indicate the presumed number of hits in this category.

Figure 4

Differential regulation of the innate immune response by Han and Han_UL138del at early stage post infection. (A) Differential expression of ISGs in Han- and Han_UL138del-infected MRC5 cells at 12 h p.i. or 96 h p.i.. Y-axis: protein ratios quantified by mass spectrometry (relative to mock-treated cells). (B) Western blot analysis of SAMD9 levels in Han- and Han_UL138del-infected MRC5 cells at 12 h p.i. or 96 h p.i.. SAMD9 was selected as a representative ISG. (C) Quantitative RT-PCR analysis of selected ISGs in Han and Han_UL138del infected MRC5 cells at 12 h p.i. or 96 h p.i.. ISG15, ISG56, MDA5, SAMD9 and STAT1 were selected as representative ISGs. H: h p.i.. Three replicates were performed, and results are shown as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no significance.

Figure 5

Host proteins differentially regulated by pUL138 at the late stage post HCMV infection can affect HCMV replication. (A) The effect of knockdown of BORCS7, FAM172A, RELL1, WDR48 or STEAP3 on HCMV production. MRC5 cells were transfected with siRNA or NC. At 48 h post transfection, the cells were infected with Han or Han_UL138del at an MOI of 0.1 or mock-treated. At 5 d post infection, cell culture supernatants were collected, and viral titers in the supernatants were measured. (B) The effect of knockdown of BORCS7, FAM172A, RELL1, WDR48 or STEAP3 on intracellular HCMV DNA levels. MRC5 cells were transfected with siRNA or NC. At 48 h post transfection, the cells were infected with Han or Han_UL138del at an MOI of 0.1 or mock-treated. At 5 d post infection, the cells were harvested to extract DNA. Intracellular levels of HCMV genomic DNA were detected with primers for UL44 by quantitative RT-PCR analysis.Three replicates were performed, and results are shown as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no significance.