Chemotaxis Of Human Cytomegalovirus-Encoded UL128Protein And Its Pathogenic Mechanism In HCMV Associated Hearing Loss

ObjectivesHuman Cytomegalovirus (HCMV) is the leading cause of congenital sensorineural hearing loss (SNHL) in infants. The pathogenesis of infectious related hearing loss is poorly understood is mainly due to the lack of applicable virus infected model. Proliferation of cochlear cells in vitro can provide a basic cell model for studying the mechanism of hearing loss. Researches conducted on animal models display that virally encoded immunomodulatory gene products play a significant role in cytomegalovirus associated hearing loss. HCMV encoded UL128protein has the similar amino acid sequences with human β-chemokine. Verification of the chemotaxis of UL128protein and its impact on cytopathic effect as well as proinflammatory cytokine production can input new information into understanding of this highly evolved virus and provide new targets for the treatment of HCMV associated hearing loss.MethodsThis research includes four parts1) Construction of recombinant plasmid expressing HCMV UL128gene and purification of recombinant UL128protein: The full-length of UL128gene was amplified from the genome of HCMV AD169strain and cloned into pUC57vector with6His-Tag fused into N-terminal amino acid sequences. Recombinant plasmid pUC57-UL128and vector pIRES-AcGFP was fused together with green fluorescence protein for positive selection. His·Bind resin column was used to purify the destined protein and silver stain test manifested its high purity.2) Verification of the chemotactic trait of recombinant UL128protein:Chemotaxis assay, cell membrane receptor ligands cross-linking test and flow cytometry detecting intracellular calcium ion concentration were applied for confirming the chemotaxis of UL128protein.3) Differentiated culture of cochlear epithelial cells and establishment of HCMV infected model:Sensory epithelial specimen grasped from the basilar membrane of cochlear was used to collect primary tissue cells by mechanical dissection and trypsin digestion. Epithelial cells were differentiated from auditory progenitors within a certain culture condition. Immunofluorescent assay was conducted to detect the cellular markers of auditory progenitor and confirm its epithelial origin. When this new cell line was inoculated by HCMV, cell morphology was observed under phase-contrast microscope and HCMV IE gene as well as virally specific protein PP65were detected by polymerase chain reaction and immunofluorescent assay respectively.4) Influences of UL128on virulence and its immune modulatory function:Four pairs of short interference RNA designed against UL128were screened through target sequence analysis and interference sites'prediction as well as homologous sequence analysis before synthesize. The most efficient siRNA was selected by using real-time PCR. Differences in cytopathic effect, virulence and proinflammatory cytokine production were observed by inoculating HCMV (UL128+) and HCMV (UL128-) into sensory epithelial cells. Cell viability of PBMC presenting with rUL128was evaluated by MTT colorimetric method. The influence of rUL128on PBMC signal pathway (MAPKs/P13K) was detected by western blot analysis.ResultsThe rUL128production system was successfully constructed by using eukaryotic expression vector (pIRES-AcGFP-UL128) with6×His-Tag fused to its N-terminal, then transferred into Chinese Hamster Ovary (CHO) cell line. Purified rUL128was about26kilodalton (KD) and was condensed into0.1mg/ml for further use. Silver stain experiment confirmed high purification of the destined protein.Sequence analysis of HCMV UL128protein revealed its β-chemokine trait which was highly conserved in different species of cytomegalovirus. Chemotaxis assay demonstrated that rUL128could recruit PBMC in vitro, the function of which was similar to human β-chemokine (MIP-1α). Chemotaxis index of rUL128was1.69±0.54, close to MIP-la (1.58±0.34, p>0.05), both of which had no concentration dependence. Immunohistochemistry results showed rUL128interacted with cytomembrane receptors of PBMC. The positive binding rate of rUL128and MIP-la were13%and12.6%, respectively. UL128(50ng/ml&100ng/ml) activated PBMC by transiently up-regulating intracellular calcium ion (Ca2+).Cochlear epithelial cells (both mature hair cells and progenitors) were isolated from patient undergoing cochlear implant surgery. Mature hair cells had no regeneration-capability, keeping alive for only3days to7days in vitro. Progenitors derived from basilar membrane was robust and informative, with the potential to expanded into sensory epithelial cell line and neuro-progenitor cells. Cytopathic effect as well as exponential amplification of virus titer were observed when inoculating HCMV into the cochlear epithelial cells. Viral immediate early gene and structural late protein PP65were also detected in HCMV infected cells. It was convinced that HCMV could efficiently multiplied in this new cell line in vitro.UL128specific siRNA was transfected into host cells through lipofectamine. Realtime fluorescence quantitative PCR (RT-PCR) was conducted to detect the transcription level of UL128mRNA. siRNA2with the same reagent (1μl Lipofectamine) and the same concentration (100nM) using in the other duplexes achieved the most efficient interference effect. The suppression rate of siRNA2(50nM) was above75%comparing to negative control. Interference effect was peaked at24h or48h post-transfection. HCMV infected cochlear cells co-cultured with PBMC was observed high levels of IL-6and TNF-α released into supernatants. UL128deficient HCMV had impaired ability to induce the production of proinflammatory cytokines. PBMC presented with rUL128(50ng/ml) got much better cell viability than untreated group. Western blot analysis displayed ERK1/2protein phosphorylated in the cytoplasm of PBMC after30min and60min incubating with UL128protein, which means rUL128could activated the MAPK/ERK transduction pathway, which is closely related with host cell's proliferation and differentiation.Conclusion1. Eukaryotic system constructed by transfecting pIRES-AcGFP-UL128vector into CHO cell line can fulfill the expression of target gene with high through-put and stably expressed in vitro. High purification of rUL128was obtained, structurally and bio-functionally close to the natural protein.2. Recombinant UL128protein could recruit PBMC as well as interacting with the cytomembrane receptor and reactivating PBMC in vitro, which functioned similar to human β-chemokine MIP-1α. UL128was predicted to be another viral encoded immune modulator homologous to human chemokine.3. As a homolog to human β-chemokine, UL128affected host immune responses by promoting the cell proliferation of PBMC through the MAPK/ERK signal transduction pathway and up-regulating the production of pro-inflammatory cytokines (IL-6&TNF-α), which may contribute to the excessive inflammatory processes during occurrence of infection related hearing impairment.4. Postnatal basilar membrane of human cochlear preserved progenitors with robust proliferation ability to be expanded into sensory epithelial cells. Mature hair cells could be isolated from human cochlea tissues, but could only sustained for a very short time with no reproductive capability in vitro.5. Sensory epithelial cells derived from cochlear progenitors was permissive for HCMV productive infection and the basic cell model was established by inoculating HCMV AD169into this new cell line. 6. siRNA synthesized in vitro could be efficiently transfected into host cells. Suppression of the transcription of HCMV UL128gene was specific and apparent by using RNA interference technology. UL128deficient HCMV had impaired ability to induce inflammatory cytokines production. UL128gene can be the potential treatment locus for HCMV associated hearing loss.