Autophagy Of THP-1Cell With Latent Infection By Human Cytomegalovirus

ObjectivesHuman cytomegalovirus is widespread in human populations.Although HCMV infection is usually asymptomatic in healthy individuals, it is a major cause of morbidity and mortality in immunocompromised individuals.HCMV congenital infection is also the most common cause of virus induced birth defects.Autophagy is an cellular degradation pathway involving the digestion of intracellular components via the lysosomal pathway. The autophagic pathway constitutively maintains cellular homeostasis by recycling cytoplasmic organelles and long-lived proteins.It also acts as a cellular defense mechanism against microorganism infection and parasitic. In recent years, the studies of relationship between HCMV and autophagy is significant development.But currently, all the researches about the relationships between HCMV infection and autophagy are taken on the cell which cause the lytic infection of HCMV,there are no resports about the relationship between HCMV-latent infection and autophagy.To verification the effect of HCMV latent infection on autophagy of infected cell can input new information into understanding the mechanism of HCMV latent and provide us a target for a new therapeutic approach of HCMV.MethodsThis research includes two parts1Construction of the model of HCMV latency in vitro:Use HEF cell which cause lytic infectin of HCMV as a proliferation control and THP-1as a blank control.HCMV Towne infect THP-1and HEF respectively.Observe the cellular morphology under Inverted Microscope. Discard the cultural supernatant and collect the cell at6h、5d、7d after infection. Extract the HCMV DNA in the THP-1through DNA extraction kit. Fluorescence quantitative PCR detect the HCM VDNA copy number in the THP-1. Extract the total RNA of the cell and detect the expression of HCMV IE1,IE2and UL81-82ast mRNA by RT-PCR. Extract the total protein of cell by RIPA and detect the the level of IE protein. Construction of vitro model of HCMV latency.2) Examination of cells infected with HCMV Towne under the electron microscope to observe the existence of autophosome and compared with the blank group.Detect the transcriptional level of autophagy-related gene LC3,Beclin-1and Atg5mRNA by RT-PCR. Detect the expression level of autophagy-related protein LC3,Beclin-1and Atg5protein by western-blot.ResultsThe model of HCMV latency was successfully constructed.Allthe HCMV IE and latency-associated gene UL81-82ast transcript and expression in HEF and THP-1after infection with HCMV.HCMV IE2express at6h,5d on THP-1after infection with HCMV, without expressing at7dpi.IE1Expression is not obviously,while UL81-82ast consistly expressed after HCMV infection.Western-blot suggest:HCMV IE protein expressed immediately after6h post-infection and increase at5d,7d post-infection on HEF after infection with HCMV. HCMV IE protein expressed at6h,5d post-infection and is not obviously at7d post-infection on THP-1after infection with HCMV.HCMV DNA copy number per THP-1is approximately1.5per cell and increasing at1-5d post-infection and gradually decreasing at5d post-infection.The infection of THP-1with HCMV Towne result in the induction of autophagy. Compared with the control cells, transmission electron microscope revealed:At1dpi, vesicles with the characteristic appearances of autophagosomes were present. At5dpi,there were a large amount of autophagosomes or autolysosomes in the cytoplasm,at the same time, we can also see some cisternal double film structure known as phagophore, the amount of autolysosomes wre more than other autophagy-related vesicles. At9dpi, autophagosomes or autolysosomes can still see in the cytoplasm. In contrast,we can hardly see autophagy-related vesicles in the blank group cells.Then,we randomly choose ten blank group cells and ten cells of the infection of THP-1with HCMV Towne after (1,5,9)dpi respectively and observe under the electron microscope. Counts were made of autophagy-related vesicles in individual cell profiles from multiple fields and nonserial sections. In mock-infected cultures, exhibited a total of2-3vesicles classified as autophagosomes or autolysosomes,whereas cultures infected with HCMV after (1,5,9) dpi revealed approximately10-13,80,30vesicles, respectively.Transcription and expression of the autophagy-related gene LC3,Beclin-1,Atg5obviously increased after infection with HCMV. The intensity of autophagy is different after HCMV establish latent infection:during latent infection the intensity of autophagy is lower than lytic infection,it can be proved by the reduced transcription and expression of LC3,Beclin-1,Atg5gene,but the level is still higher than the control cell.Conclusion1) successfully establishmentof human cytomegalovirus latent infection model in THP-1in vitro. 2) The infection of THP-1with HCMVTowne result in the induction of autophagy,but the intensity of autophagy is lower when HCMV has established latent infection compared with the period of lytic infection.