Studies On HIV-1 Entry Into Retinal Pigment Epithelial Cells And Subsequent Cell-to-cell Transmission

BACKGROUND:AIDS (Acquired immunodeficiency syndrome) is caused by HIV-1, which is a retrovirus.HIV infects human immune system, which leads tothe disruption of the immune system. Defects of the immune system will result invarious opportunistic infections and tumors. With the improvement of the antiretroviral therapy for the treatment of AIDS and all kinds of investment on the treatment, AIDS now is a chronic and controable infectious disease and the life of the infected patienthas been greatly extended. Because AIDS patients now are living longer and the number of people infected with HIV is increasing, the number of HIV infected aged patients is also rising. In addition, about 1/5 of the patients now can access to medical treatment. Therefore, various complications affect the patient’s quality of life.Ocular complication is one of the most common symptoms of people living with HIV-1 and the oclular symptoms is often the first expression in HIV-1 patient. About 45%～75% of HIV-1 patients are affected by ocular complication. Ocular complication is also an indicator of the symptoms of HIV-1 testing in clinical. The majority of people living with HIV-1 will suffer from retinal micro vascular leakage, nerve fiber layer thining, swollen endothelial cells and pathological characteristics of structure of vascular network. Retinopathy is a serious threat to the AIDS patients, which will lead to color vision defects, decreased ocular contrast sensitivity and even blindness. Because the HIV-1 antigen p24 could be detected in all layers of retinal tissuein HIV patients with or without ocular complication, the entry of HIV-1 into the retina is considered to be the leading cause of HIV retinal complications.HIV/AIDS cannot be cured and the main reason is that the HIV-1 sets up latency in the various tissues and organs, in which and the antiretroviral therapy cannot access to the viruses.The treatment failure of "functionally cured" Mississippi girl and experimental results from macaque showed that even if antiretroviral treatment is effective at the early stage of infection, once the treatment is stopped, the virus could recover.These results suggest that the virus can set up its latency in the resovior at the early stage of HIV infection. To clear the latent viruses is the key to cure HIV. Apart from CD4+T lymphocytes, nervous system, retina, testis are also the ideal place for HIV-1 to set up its latency.Human retinal pigment epithelial cells (human retinal pigment epithelium, HRPE) lie between optic nerve retinal and choroid blood capillary. Due to its tight junctions, HRPE constitute the outside-blood retina barrier. Similar with the blood-brain barrier, HRPE is vital to maintain the retinal microenvironment and protect the retinal nerve cells from the damage caused by inflammatory cells and cytotoxic chemicals in the blood. So the outside-blood retina barrier is important to regulate the retinal microenvironment.In our previous study, when HRPE was exposed to HIV-1 for 48 hours, theHIV gp120 induced an inflammatory state in HRPE cells, which could result in impairment of HRPE monolayer integrity. Further study showed that when HRPE was treated with HIV even for 2 hours, the core HIV antigen p24 could be detected in the HRPE. Therefore, HIV-1 could affect the barrier function of HRPE and, it also could directly infect the HRPE. In this study, we selected the retinal pigment epithelium as the research object. We investigated the pathway that exploited by HIV to enter into the retinal pigment epithelial cells and the molecular events that occurred after the vrial entry into the cells. We intented to elucidate the pathogenic mechanism of retinal disorder and the mechanism of viral latency, so as tofind potential therapeutic targets.OBJECTIVE:We cultured the D407 cells in vitro. By flow cytometry, western blotting, confocal microscope and other methods to study the entry pathway of HIV-1 into D407 cells. Using various chemical inhibitors, we explored the mechanism of HIV-1 enter into D407 cell. PCR was explored to examine the integreation of HIV in D407 cells. Using of lysosomal marker and inhibitors, we detected the molecular events occurred after HIV-1 entry into D407 cells. A co-culture model was used to examine HIV-1 cell-to-cell transmission. We intented to elucidate the pathogenic mechanism of retinal disorder and the mechanism of viral latency, to find potential therapeutic targets.METHODS:1. The preparation of the clone virus and the detection of its infection.1) We transfected the SF162 (R5 receptor tropism) and NL4-3 (X4 receptor tropism) virus plasmids with full-length of proviral DNA into the 293TX cells.2) We serially diluted the clone virus supernatent after a 48-hour transfection and the mixtures were added into 96-well cell culture plate,100 μl per well. After infection for 48 hs, the TZM-bl cells were detected by luciferase activity.2. Detection the protein expression on the surface of D407 cell.1) Flow cytometry assay:D407 cell were seeded in 6-well cell culture plate and were clultured for 24 h.Then, we digested the cells with 0.025% EDTA and resuspended the cells with PBS and the cells were incubated with 1 μg/ml anti-CD4, anti-CCR5 and anti-CXCR4 antibody for 1 h on 4 ℃ respectively. After resuspended by PBS, the cells were incubated with 1 μg/ml second antibody for 40 mins on 4 ℃. Finally, the cells were detected by flow cytometry.2) RT-PCR assay:According to the kit protocols, we extracted the genome of D407 cell and detected the mRNA level of CD4, CXCR4 and CCR5 by qRT-PCR.3) ELISA assay:The CEMX174 5.25M7 cells and D407 cells were incubated with R5-tropic virus for 6 h and then the cells were digested with trypsin. We then collected the cells and washed the cells for three times with PBS. After resuspented by culture medium, the cells were seeded in 6-well cell culture plate in a density of 1×105 cells/well, which were cultured. Culture supernatants were collected at day 1, 3,5,7 respectively and determined the level of p24.3. HIV-1 can integrate into D407 cell.1) Flow cytometry assay:D407 cell were seeded in 6-well cell culture plate in density of 2 × 105 cells/well and cultured for 12 hs. The D407 cells were incubated with the EGFP labelled HIV-1 for 2 hs at 37 ℃ and then digested with trypsin. Cells were washed three times with PBS. Finally, the percentage of endocytozed HIV was analyzed by Flow cytometer.2) Western blotting:D407 cell were seeded in 6-well cell culture plate in a density of 2×105 cells/well and were clutured for 24 h. After the D407 cells were incubated by various strain of HIV for 1 h at 37 ℃, the cells were digested by trypsin and were washed three times with PBS. Cells were harvested in RIPA buffer containing protein and phosphotase inhibitors as described previously. Equal amounts of proteins were resolved with SDS-PAGE and transferred on PVDF membrane. The membrane was probed with primary antibodies overnight at 4 ℃ and incubated for 1 h with secondary peroxidase-conjugated anti-body at room temperature. Chemiluminescent signals were then developed with Lumiglo reagent and exposed to X-ray film.3) Confocal microscope assay:D407 cell were seeded in 6-well cell culture plate in a density of 2 × 105 cells/well and were cultured for 24 hs. The cells were incubated with EGFP labeled virus for 2 hs at 37 ℃. After washed three times with PBS, the cells were incubated with DiI (Red fluorescent dye for cell membranes). Then the cells were fixed with 4%(v/v) paraformaldehyde for 15 mins and then washed three times with PBS. Finally, cells were incubated with 4, 6-diamidino-2-phenylindole (DAPI) at room temperature for 5 mins. Fluorescent signals were detected using a confocal microscope (C1-si, Nikon, Japan). The acquired images were analyzed by the Nikon software (NIS-Elements AR, Nikon, Japan).4. To investigate that mechanism by which HIV enters into D407 cells.1) Flow cytometry assay:D407 cell were seeded in 6-well cell culture plate in a density of 2 × 105 cells/well and were clutured for 24 h. The D407 cells were incubated with various chemical inhibitors for 1 h.Then the cells were incubated with EGFP labeled virus for 2 hs at 37℃. Finally, cells were digested with trypsin and washed three times by PBS. After the above treatment, the percentage of the endocytozed HIV was analyzed by flow cytometer.2) Western blotting:D407 cell were seeded in 6-well cell culture plate in a density of 2 × 105 cells/well and were cultured for 24 hs. The D407 cells were challenged with various chemicals for 1 h. Then the cells were incubated with HIV-1 for 2 hs at 37 ℃. Finally, cells were digested with trypsin and washed three times with PBS. Cells were harvested in radioimmunoprecipitation buffer containing protein and phosphotase inhibitors at room temperature as described previously.5. To detect the retention of virus in D407 cells.1) Western blotting:D407 cell were seeded in 6-well cell culture plate in a density of 2 × 105 cells/well and cells were cultured for 24 hs. Then, D407 cells were incubated with Pepastatin A (50 μM) and Leupeptin (50 pM) for 1 h at 37 ℃. The cells were incubated with HIV-1 for 1 h at 37 ℃. Cells were then harvested in radioimmunoprecipitation buffer containing protein and phosphotase inhibitors as described previously. Chemiluminescent signals were then developed with Lumiglo reagent and exposed to X-ray film.2) Fluorescent microscope assay:D407 cells were seeded in 6-well cell culture plate in a density of 2 x 105 cells/well and were cultured for 24 hs. The cells were incubated with Pepastatin A (50 μM) and Leupeptin (50μM) for 2 hs at 37 ℃. After washing three times with PBS, the cells were fixed with 4%(v/v) paraformaldehyde for 15 mins and then with 100% ethanol for another 10 mins. After this procedure, cells were incubated with anti-EEAl antibody. Cells were washed three times with PBS. Finally, the fluorescent signals were detected by using a fluorescent microscope.3) RT-PCR assay:D407 cell were seeded in 6-well cell culture plate in a density of 2 x 105 cells/well and were cultured for 24 hs. After incubating with HIV-1, the genome of D407 cell was extracted and detected its integration by both common PCR and Real-time-PCR.6. To detect HIV-1 cell-to-cell transmission.1) Transwell assay:D407 cells were seeded in 6-well cell culture plate in a density of 2 × 105 cells/well and were cultured for 24 h. Then, the R5-tropic virus was added into the D407 for 1 h. Finally, cells were digested with trypsin and washed three times with PBS. Cells were then resuspended with the culture medium. The D407 cells were re-seeded in the upper layer of a transwell. At the same time, the CEMX174 5.25M7 were seeded in the under layer of the transwell. After 48 hs, the virus released from D407 was detected by flow cytometry and ELISA.RESULTS:1. We found that EGFP labelled virus entered into D407 cells. The amount of virus entering into the D407 increased within the first one hourand reached a maximum at 1 h. There’s significant difference between the virus entering into D407 at 37 ℃ and those entering at 4 ℃. We detected little expression of CD4, CCR5, CXCR4 at the cellular suface of D407.2. The results of western blotting and flow cytometry demonstrated that chemicals, including chlorpromazine, ammonium chloride, chloroquine and sucrose, could inhibit the entry of HIV-1 into D407 cells in a dose-dependent manner, while progestone, amiloride, T-20 and maraviroc couldn’t.3. The amount of HIV-1 virus decreased after vrial entry into D407 cells. Virus integrated it genome into the D407 genome, which could be inhibited by AZT and chlorpromazine. The expression of protein EEA1 gradually increased after viral entering into D407 cells.4. In the transwell model, virus in D407 cells could not infect the CEMX174 5.25M7, while HIV-1 in D407 could infect CEMX174 5.25M7 in a contact-dependant cell-to-cell manner.CONCLUSION:1. HIV-1 can endocyte into D407 cells in a CD4 receptor and CXCR4 or CCR5 independent pathway.2. The entry of HIV-1 into retinal pigment epithelial cells is a Clathrin mediated endocytosis process.3. After HIV-1 enterring into retinal pigment epithelial cells, the HIV-1 integrates its genome into D407 and HIV-1 was degraded in lysosome.4. HIV-1 can transmit from D407 cells to CD4 T cells in a cell-to-cell manner.