Association of CCR5Δ32 Deletion and Human Cytomegalovirus Infection With Colorectal Cancer in Tunisia.

Background and objectives: Human cytomegalovirus (HCMV) and genetic polymorphisms of the chemokine receptor 5 have been suggested as factors associated with the progression of colorectal cancer (CRC). The aim of the study was to evaluate the associations of both CCR5Δ32 genetic deletion and/or HCMV virus infection with CRC in Tunisia. MATERIALS AND METHODS: The association between HCMV and CRC was validated by Nested PCR technology performed for HCMV and HCMV-specific serum IgG and IgM antibodies were investigated by enzyme-linked immunosorbent assay. Experiments were carried out on 40 tumor and 35 peri-tumor tissues, 100 blood from CRC patients and on 140 blood samples from healthy subjects and finaly serum samples of 80 patients with CRC and 100 healthy individuals. A conventional PCR has been optimized for the detection of CCR5Δ32 in100 CRC patients and 100 healthy subjects. RESULTS: Our results show that HCMV is significantly active in 93% of patients compared to 60% in controls (p < 0.0001, OR = 8.85, 95% CI: 3.82 -20.50). Compared to the healthy controls, the titers of IgG and IgM antiCMV antibodies in CRC patients were significantly higher than in healthy subjects (p value < 0,0001 for IgG and IgM). Statistical analysis revealed a lack of association between CCR5Δ32 mutation and colorectal cancer (p = 0.788, OR = 1.265, 95% CI: 0.228-7.011). CONCLUSION: our data confirmed that the HCMV infection was related to the development of CRC and that CRC cells may be infected more favorably by HCMV. Given the importance of the CCR5 in inflammation and therefore CRC progression, further studies still needed to evaluate CCR5 role as a potential candidate gene for CRC susceptibility under other polymorphisms.

Human Cytomegalovirus miR-US5-2 Downregulation of GAB1 Regulates Cellular Proliferation and UL138 Expression through Modulation of Epidermal Growth Factor Receptor Signaling Pathways.

Regulation of epidermal growth factor (EGF) receptor (EGFR) signaling is critical for the replication of human cytomegalovirus (HCMV) as well as latency and reactivation in CD34+ hematopoietic progenitor cells. HCMV microRNAs (miRNAs) provide a means to modulate the signaling activated by EGF through targeting components of the EGFR signaling pathways. Here, we demonstrate that HCMV miR-US5-2 directly downregulates the critical EGFR adaptor protein GAB1 that mediates activation and sustained signaling through the phosphatidylinositol 3-kinase (PI3K) and MEK/extracellular signal-regulated kinase (ERK) pathways and cellular proliferation in response to EGF. Expression of HCMV UL138 is regulated by the transcription factor early growth response gene 1 (EGR1) downstream of EGFR-induced MEK/ERK signaling. We show that by targeting GAB1 and attenuating MEK/ERK signaling, miR-US5-2 indirectly regulates EGR1 and UL138 expression, which implicates the miRNA in critical regulation of HCMV latency.IMPORTANCE Human cytomegalovirus (HCMV) causes significant disease in immunocompromised individuals, including transplant patients. HCMV establishes latency in hematopoietic stem cells in the bone marrow. The mechanisms governing latency and reactivation of viral replication are complex and not fully understood. HCMV-encoded miRNAs are small regulatory RNAs that reduce protein expression. In this study, we found that the HCMV miRNA miR-US5-2 targets the epidermal growth factor receptor (EGFR) adaptor protein GAB1 which directly affects downstream cellular signaling pathways activated by EGF. Consequently, miR-US5-2 blocks the EGF-mediated proliferation of human fibroblasts. Early growth response gene 1 (EGR1) is a transcription factor activated by EGFR signaling that regulates expression of HCMV UL138. We show that miR-US5-2 regulates UL138 expression through GAB1-mediated downregulation of the signaling pathways that lead to EGR1 expression. These data suggest that miR-US5-2, through downregulation of GAB1, could play a critical role during reactivation from latency by reducing proliferation and UL138 expression.

The Role of the Human Cytomegalovirus UL133-UL138 Gene Locus in Latency and Reactivation.

Human cytomegalovirus (HCMV) latency, the means by which the virus persists indefinitely in an infected individual, is a major frontier of current research efforts in the field. Towards developing a comprehensive understanding of HCMV latency and its reactivation from latency, viral determinants of latency and reactivation and their host interactions that govern the latent state and reactivation from latency have been identified. The polycistronic UL133-UL138 locus encodes determinants of both latency and reactivation. In this review, we survey the model systems used to investigate latency and new findings from these systems. Particular focus is given to the roles of the UL133, UL135, UL136 and UL138 proteins in regulating viral latency and how their known host interactions contribute to regulating host signaling pathways towards the establishment of or exit from latency. Understanding the mechanisms underlying viral latency and reactivation is important in developing strategies to block reactivation and prevent CMV disease in immunocompromised individuals, such as transplant patients.

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

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 HanUL138del was constructed by deleting the UL138 locus of Han, a clinical HCMV strain. Then, a comparative quantitative proteomic analysis of Han- and HanUL138del-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 HanUL138del-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.

The Golgi sorting motifs of human cytomegalovirus UL138 are not required for latency maintenance.

Human cytomegalovirus (HCMV) establishes latency within incompletely differentiated cells of the myeloid lineage. The viral protein UL138 participates in establishing and maintaining this latent state. UL138 has multiple functions during latency that include silencing productive phase viral gene transcription and modulating intracellular protein trafficking. Trafficking and subsequent downregulation of the multidrug resistance-associated protein 1 (MRP1) by UL138 is mediated by one of four Golgi sorting motifs within UL138. Here we investigate whether any of the Golgi sorting motifs of UL138 are required for the establishment and/or maintenance of HCMV latency in model cell systems in vitro. We determined that a mutant UL138 protein lacking an acidic cluster dileucine sorting motif unable to downregulate MRP1, as well as another mutant lacking all four Golgi sorting motifs still silenced viral immediate early (IE) gene expression and prevented progeny virion formation during latency. We conclude that the Golgi sorting motifs are not required for latency establishment or maintenance in model cell systems in vitro.

The Membrane-Spanning Peptide and Acidic Cluster Dileucine Sorting Motif of UL138 Are Required To Downregulate MRP1 Drug Transporter Function in Human Cytomegalovirus-Infected Cells.

The human cytomegalovirus (HCMV) UL138 protein downregulates the cell surface expression of the multidrug resistance-associated protein 1 (MRP1) transporter. We examined the genetic requirements within UL138 for MRP1 downregulation. We determined that the acidic cluster dileucine motif is essential for UL138-mediated downregulation of MRP1 steady-state levels and inhibition of MRP1 efflux activity. We also discovered that the naturally occurring UL138 protein isoforms, the full-length long isoform of UL138 and a short isoform missing the N-terminal membrane-spanning domain, have different abilities to inhibit MRP1 function. Cells expressing the long isoform of UL138 show decreased MRP1 steady-state levels and fail to efflux an MRP1 substrate. Cells expressing the short isoform of UL138 also show decreased MRP1 levels, but the magnitude of the decrease is not the same, and they continue to efficiently efflux an MRP1 substrate. Thus, the membrane-spanning domain, while dispensable for a UL138-mediated decrease in MRP1 protein levels, is necessary for a functional inhibition of MRP1 activity. Our work defines the genetic requirements for UL138-mediated MRP1 downregulation and anticipates the possible evolution of viral escape mutants during the use of therapies targeting this function of UL138.IMPORTANCE HCMV UL138 curtails the activity of the MRP1 drug transporter by reducing its steady-state levels, leaving cells susceptible to killing by cytotoxic agents normally exported by MRP1. It has been suggested in the literature that capitalizing on this UL138-induced vulnerability could be a potential antiviral strategy against virally infected cells, particularly those harboring a latent infection during which UL138 is one of the few viral proteins expressed. Therefore, identifying the regions of UL138 required for MRP1 downregulation and predicting genetic variants that may be selected upon UL138-targeted chemotherapy are important ventures. Here we present the first structure-function examination of UL138 activity and determine that its transmembrane domain and acidic cluster dileucine Golgi sorting motif are required for functional MRP1 downregulation.

The cytomegalovirus protein UL138 induces apoptosis of gastric cancer cells by binding to heat shock protein 70.

It has been hypothesized that human cytomegalovirus (HCMV) could act as a tumor promoter and play an "oncomodulatory" role in the neoplastic process of several human malignancies. However, we demonstrate for the first time that UL138, a HCMV latency-associated gene, could act as a tumor inhibitor in gastric cancer (GC). The expression of UL138 is down-regulated in HCMV positive gastric adenocarcinoma tissues, especially in poorly or none differentiated tumors. Overexpression of UL138 in several human GC cell lines inhibits cell viability and induces apoptosis, in association with the reduction of an anti-apoptotic Bcl-2 protein and the induction of cleaved caspase-3 and caspase-9. Moreover, protein array analysis reveals that UL138 interacts with a chaperone protein, heat shock protein 70 (HSP70). This interaction is confirmed by immunoprecipitation and immunostaining in situ in GC cell lines. In addition, this UL138-mediated cancer cell death could efficiently lead to suppression of human tumor growth in a xenograft animal model of GC. In conclusion, these results uncover a previously unknown role of the cytomegalovirus protein UL138 in inducing GC cells apoptosis, which might imply a general mechanism that viral proteins inhibit cancer growth in interactions with both chaperones and apoptosis-related proteins. Our findings might provide a potential target for new therapeutic strategies of GC treatment.

Constructing a recombinant adenovirus carrying human cytomegalovirus UL138 gene and evaluating the effects of UL138 gene on the functions of monocytes / 中华微生物学和免疫学杂志

Objective To construct a recombinant adenovirus carrying UL138 gene, which was re-lated to the latent infection of human cytomegalovirus, and to investigate the effects of UL138 gene on the functions of THP-1 mononuclear cells. Methods The recombinant adenovirus expressing the UL138 gene was packaged. The titer of the recombinant adenovirus was determined by calculating 50% tissue culture in-fective dose ( TCID50 ) . THP-1 mononuclear cells were infected with the recombinant adenovirus at different multiplicity of infection (MOI) and the optimal MOI was determined (100 PFU/cell) by observing the ex-pression of green fluorescent protein ( GFP ) . Changes in the expression of proinflammatory cytokines by THP-1 mononuclear cells that was induced by overexpressed UL138 were analyzed by quantitative PCR. The expression of chemokines and their receptors were measured by quantitative PCR array. Results The re-combinant adenovirus carrying the UL138 gene was successfully constructed with a titer of 1×1011 PFU/ml. The rate of THP-1 mononuclear cells that was infected with the recombinant adenovirus was 60% at the MOI of 1 ∶ 100. Results of the RT-PCR analysis and Western blot assay further confirmed that the recombinant adenovirus could infect THP-1 mononuclear cells successfully and the expression of UL138 protein increased gradually over time. The overexpressed UL138 in THP-1 mononuclear cells significantly inhibited the expres-sion of IL-18, IL-1β, IL-6, IL-8 and TNF-α as indicated by the results of quantitative PCR. Results ob-tained from the quantitative PCR array analysis showed that most of the chemokines and their receptors were downregulated in the transfected THP-1 mononuclear cells except for the chemokines of CCL17, CCL21, CCL2 and XCL2 and the receptors of CCR2, CXCR1, CXCR2, CXCR4 and CX3CR1 which were upregulat-ed. Conclusion We successfully constructed the recombinant adenovirus carrying UL138 gene which could be used to infect THP-1 mononuclear cells. Overexpressed UL138 in THP-1 mononuclear cells significantly affected the functions of THP-1 mononuclear cells.

Analysis of nucleotide sequences and genetic polymorphisms of UL138 gene of low passage human cy-tomegalovirus strains in Guangzhou / 中华微生物学和免疫学杂志

Objective To analyze the nucleotide sequences and genetic polymorphisms of UL138 gene of low passage human cytomegalovirus ( HCMV) strains isolated from infants in Guangzhou province. Methods The low passage strains of HCMV were isolated from urine samples of 10 infants with HCMV in-fection in Guangzhou province and identified by multiplex PCR.The UL138 genes were amplified, cloned and identified with sequencing.The sequences were analyzed together with the homologous sequences of 10 clinical isolates published in GenBank.The sequences of UL138 genes were analyzed by using bioinformatics softwares for investigation of the post-translational modification sites, isoelectric points and second structures of UL138 proteins.Results Three low passage strains of HCMV ( D2, D3 and D52) were isolated from in-fants with congenital HCMV infection.The complete sequences of UL138 genes of the three strains were sub-mitted to GenBank after sequencing identification with the GenBank accession numbers of DQ180375, DQ180387 and DQ180359, respectively.The UL138 gene sequences of the three clinical isolates were high-ly conservative.Among the 841 base pairs of the UL138 gene sequences, mutations were identified in 16 sites with base substitution, no any insertion and deletion mutation was found.The 16 mutations resulted in 7 amino acid changes.No additional or deleted sites were found with regard to the post translational modifi-cation sites of UL138 protein in all clinical isolates except the Toledo strain.The isoelectric point of UL138 protein was 6.51 for all clinical isolates.Conclusion The UL138 genes and the deduced amino acid se-quences of HCMV strains isolated from infants in Guangzhou were highly conservative, regardless of the poly-morphism of UL138 gene.This study paved the way for further investigation on HCMV infection and its path-ogenic mechanism.

Latent infection of human cytomegalovirus is associated with the development of gastric cancer.

The worldwide contagion, human cytomegalovirus (HCMV), may cause a series of disorders in infected individuals. The aim of the present study was to investigate whether HCMV infection is associated with the development of gastric cancer. In this study, the positive expression of unique long (UL)133-UL138 and immediate-early (IE)1 genes, which are associated with viral latency and replication, respectively, were detected using nested polymerase chain reaction. A χ2 test and logistic regression analysis were performed to further investigate the preliminary data. The data indicated that the positive rate of UL133, UL135 and UL136 expression in cancer tissues was higher than that in paired normal tissues (P=0.01, 0.027 and 0.013, respectively). However, no significant differences were identified in the UL133-138 locus and IE1 gene when associated with clinicopathological features. Furthermore, seven infection patterns were identified, with the UL133 + UL138 infection pattern representing the largest proportion in the cancer (60.34%) and normal tissues (42.11%). In conclusion, it is possible that the UL133-UL138 locus is important in the occurrence of gastric cancer. The mechanism by which UL133-UL138 locus expression differs in human gastric cancer requires further investigation.

Progress in biological functions of encoded proteins in human cytomegalovirus UL/b' region / 国际儿科学杂志

Human cytomegalovirus(HCMV) is a ubiquitous herpesvirus.It is always a serious pathogen in immunosuppressed individuals,and also the most common and harmful agent causing congenital infection and birth defects.Biological functions of the encoded proteins in HCMV UL/b' region,which is deleted in the HCMV laboratory AD169 strain,have been explored in depth in recent years,and the results demonstrate that the proteins have important roles in HCMV virulence,transmission,tropisms and immune escape and so on.UL133-UL138 locus can promote to establish and maintain HCMV latent infections,and pUL1 38 is the first HCMV protein demonstrated to promote infection with the hallmarks of latency in CD34 + hematopoietic progenitor cells (HPC).Therefore,the products expressed by the gene of HCMV UL/b' region are crucial in the course of HCMV infection.This article reviews the genetic structure and biological functions of encoded proteins in HCMV UL/b' region,especially pULl38 which is related with HCMV latent infections.It will be greatly significant to clarify the pathogenesis of HCMV infection.