Effects And Mechanism Of Latency-associated Gene UL138 On HCMV Latency And Reactivation

Human cytomegalovirus(HCMV)is one of the most frequent causes of congenital infections.It usually causes serious clinical diseases,such as miscarriage,stillbirth,fetal anomaly,intrauterine growth retardation and so on.These pathologies associated with a key characteristic of virus:the ability to establish lifelong latent infection of the human host and reactivate.Previous study showed that UL138 gene was an important HCMV latency-associated gene.On one hand,UL138 encoded protein may help HCMV to limit the presentation of virus proteins to immune cells,thereby restricting T-cell recognition of latently infected cells.On the other hand,UL138 encoded protein could sensitize cells to tumor necrosis factor alpha(TNF-a)signaling to contribute to reactivation.Up to now,the concrete effects of latency-associated gene UL138 on HCMV latency and reactivation are unclear.Some studies showed undifferentiated state of myeloid lineage played a crucial role in HCMV latent infection.Our study would explore the effects of latency-associated gene UL138 on HCMV latency and reactivation and the effects on differentiated state of host’s cells by up-regulating and knocking-down the expression of UL138 gene.This study might provide tantalising glimpses to mechanisms of HCMV latency and reactivation.Objectives(1)To study the effects of latency-associated gene UL138 on HCMV latency and reactivation.(2)To study the effects of latency-associated gene UL138 on differentiated state of host’s cells.Methods and Results(1)Establishment of HCMV latency model THP-1 cells were infected with HCMV Towne as latent group.HEF cells were infected with HCMV Towne as lytic group.① Latency-associated gene UL81-82ast was detected to make sure that host cells were successfully infected.The results showed that HCMV DNA copies kept increasing in both latent group and lytit group during infection.② HCMV DNA copies,the expression of lytic gene UL122 and UL123 were detected to make sure HCMV latency model was successfully established.The results showed that HCMV DNA copies kept increasing in lytic group;HCMV DNA copies were decreasing in latent group.(2)The effects of latency-associated gene UL138 on HCMV latency and reactivation① Two kinds of lentiviral vectors to over-expression and knock-down UL138 gene were construced and transfected into THP-1 cells.② After up-regulate and knock-down UL138 gene,HCMV DNA copies,the expression of lytic genes UL122 and UL123 were detected to evaluate the change of HCMV latent status.The results showed that HCMV DNA copies and the expression of UL122 and UL123 genes kept increasing in up-regulation group;HCMV DNA copies and the expression of UL122 and UL123 genes kept decreasing in knocking-down group.(3)The effects of latency-associated gene UL138 on differentiated state of host’s cells After up-regulate and knock-down UL138 gene,three indexes were detected to evaluate the differential level of host’s cells as follows:① cellular morphologyThe results showed that host cells stretched full,protruded clear in up-regμlation group;VII the cellular morphology was not obviously changed in knocking-down group.② differentiation associated antigensThe results showed that the expression of differentiation associated antigen CD 14 was statistically increased in up-regulation group;the expression of differentiation associated antigen CD45 was statistically increased in knocking-down group.③ differentiation associated cytokines(TNF-α,IL-6,IL-1β,and TGFβ1)The results showed that differentiation associated cytokine TGFβ1 was increased in up-regulation group;the expression of differentiation associated cytokines was not statistically changed in knocking-down group.Conclusions(1)HCMV latecy model was successfully established with HCMV Towne and THP-1 cells.(2 UL138 gene would contribute HCMV to establish or maintain latent infection.(3)UL138 gene would contribute differentiation of host’s cells.