The Function And Mechanism Of Novel Host Factor In HIV Infection And Replication

BackgroundHuman immunodeficiency virus(HIV) is the pathogen that causes acquired immune deficiency syndrome (AIDS). AIDS is the world's fourth largest cause of death in humans.During 1981-2009,65 million people were infected with HIV globally, of which 25 million had died; According to year 2009 statistics, there were about 0.7 million HIV-infected people in our contury. The number of cumulatively reported AIDS cases was 319,877, of which 102,323 cases were reported as AIDS patients and 49,845 cases reported as death. (1,2) In view of sexual transmission has become the main way of HIV transmission Given the current health care conditions and population distribution pattern, and the trend that sexual transmission is becoming the main route of transmission, in the next decade, the number of AIDS patients in China is expected to increase dramatically.Since the discovery of HIV in 1983, human beings have been suffering and fighting with HIV and AIDS for 30 years, Much progresses have been made in studying HIV biology and pathogenesis of AIDS, and we have gained substantial understandings about the immune mechanism of human resistance to HIV. However, so far we have not found effective therapies to cure AIDS. To deal with the resistance of the virus, Ho and others firstly employed a combination of two reverse transcriptase inhibitors and a protease inhibitor to treat the patients, a therapy termed highly active antiretroviral therapy (HAART, commonly known as "cocktail therapy "). This therapy can significantly reduce the incidence of drug resistance, so that patients achieve significant clinical improvement and prolonged life span. It is still the gold-standard therapy so far. (3)But during longer-term HAART treatment, particularly in patients with poor compliance, the resistant virus strains and opportunistic infections occur frequently which often lead to treatment failure. It was estimated that there are about 50% patients under HAART treatment had encounted the problem of drug resistance. Resistant strains appeared to be inevitable and everlasting, making the fight against HIV an endless battle.(4,5) Therefore, more and more researchers started to turn to an alternative anti-viral strategy, namely to target the host factors (usually proteins and enzymes in human body) which affect certain step(s) of the HIV life cycle. Because compounds inhibiting the host factors usually will not directly interact with any structural components of HIV virus, in principle, it should be very difficult for viruses to develop resistance against the compounds. (6-8) The first antiviral drug targeting the host factor CCR5 (Maraviroc) was approved by FDA in 2007, and some progresses have been made in developing drugs targeting other host factors such as APOBEC3 (9,10), TRIM5 (11).Therefore, targeting host factors as a means to combat HIV is receiving more and more attention in recent years. (6,12,13) The key is to identify which host factors are essential for viral infection and replication, and which host factors will naturally block or promote viral life cycle. As stated above, it will be rather difficult for HIV viruses to develop resistance against a compound that does not directly bind to any viral components, and the virus must gain new capabilities in order to escape the drug action,. Since the discovery of HIV receptor CD4, many host and virus replication-related proteins have been identified.(14-16) In particular, with the application of genome-wide siRNA screening technology, over 200 novel HIV host factors have been identified. (13,17,18) However, more questions remain unanswered. For example, what are the mechanisms of action for those newly-identified host factors? Could the appearent antiviral effect of siRNA treatment actually caused by nonspecific effects on cell viability or cell status? Which identified host factors are drugable? Further work will be needed to fully address these questions before any of those host factors could be considered as an anti-HIV drug target.AimsMy thesis is directed toward testing if two proteins (FBXL20 and RON) identified through preliminary siRNA screening are HIV host factors, and what are their mechanisms of action in affecting viral infection and replication.Methods and MaterialsRT-PCR (reverse transcription-polymerase chain reaction) and Western blot (immunoprecipitation) was used to detect the expression of RON and FBXL20 protein in untreated or treated primary T cells or T cell lines including a T cell line stably transfected with an EGFP gene driven by an HIV LTR promoter (called JLTRG cell). Co-culture of JLTRG cells with HIV-produing H9/HTLV-IIIB cells will result in infection of JLTRG cells which turns on the LTR promoter and subsequent expression of EGFP protein. Specific SiRNAs against FBXL20 were designed, synthesized and delivered into JLTRG cells to knock down the expression of target proteins, and HIV infection and replication in these siRNA-treated JLTRG cells was monitored by flow cytometry using the mean fluorescence intensity of EGFP and the percentage of EGFP-expessing cells as an index. Furthermore,293T cell lines over-expressing the RON protein or HIV Tat protein were established in order to study the mechanism by which HIV Tat and RON work together in regulating HIV life cycle.Results:1. Established a JLTRG-H9/HTLV-IIIB co-culture infectivity assay system which was successfully used to study the function and mechanisms of HIV host factors.2. RT-PCR and Western blot results showed that FBXL20 and RON are highly expressed in T cell lines. Immunofluorescence microscopy experiments revealed that FBXL20 protein is localized in the cytoplasm, while RON is a plasma membrane localized protein.3. RNA interference results showed that endogenous FBXL20 protein level in JLTRG cells was reduced by approximately 70-80%, suggesting a successful knock-down. Under such knock-down conditions, HIV infection and replication was enhanced by 7 fold at an early time point (i.e.,24 h post infection) and the enhancing effect declined gradually over time. This result indicated that FBXL20 may serve as a supressor for HIV infection and replication.4. After HIV infection, the expression and phosphorylation of RON in JLTRG cells was decreased, and HIV inhibits the activity of RON tyrosine kinases via Tat-dependent mechansim.Conclusion:1.Successfully established the HIV infectivity assay system in vitro which provides a useful tool to study HIV host factors.2. Demonstrated that ubiquitin system factor FBXL20 protein is a suppressor for HIV infection and replication; RON protein also played a negative regulatory role in HIV infection and replication. Its inhibitory effect is related with NF-kB signaling pathway.(?)3. HIV can reduce the expression of RON,and inhibit its phosphorylation via Tat-dependent mechanism, and by doing so, overcome RON's effects on viral suppression.