Characterizing HIV-induced interferon response in the thymus
Arnaud Colantonio, UC Los Angeles
Interferon alpha (IFN-α), a type I interferon, is a potent modulator of immune responses. Its primary function is to prevent the spread of infection through the upregulation of interferon response genes, like MxA, in humans. While any white blood cell in the body can produce IFN-α, it has been reported to be secreted at higher levels by plasmacytoid dendritic cells (pDC). Production and secretion of IFN-α by pDC occurs in response to viral (HSV, Influenza, Sendai, HIV) and bacterial infections (CpG ODN). We have previously shown that pDC are essential for triggering upregulation of MxA in thymocytes. However, while IFN-α is an important factor in the control of viral infections, it does not seem to be enough to control HIV-1 infection in the thymus. Indeed, it seems that IFN-α only delays, rather than blocks, HIV-1 replication.
The first goal of this proposal is to characterize differences between the type I interferon response triggered by CCR5 or CXCR4 tropic HIV-1 infection. Preliminary data suggests that CCR5 tropic HIV-1 infection triggers a stronger type I interferon response than CXCR4 tropic HIV-1 infection. We will monitor the expression and localization of MxA during the course of HIV-1 infection in the thymus. We will use in vitro thymocyte cell cultures to determine if the upregulation of MxA is the result of virus specific factors or whether it is the result of the cell types HIV-1 targets for infection. We will infect thy/liv implants from SCID-hu mice to determine the localization of MxA following infection with HIV-1. Based on the cell surface expression of the HIV-1 coreceptors, we expect CCR5 or CXCR4 tropic viruses to target different regions of the thymus. Both the strength of the interferon response, as measured by upregulation of MxA, and the location of the response may play a role in determining the
pathogenicity of HIV-1.
The second goal of this proposal is to determine if MxA can directly interact with HIV-1 Gag and interfere with HIV-1 replication. MxA has been shown to possess intrinsic antiviral activity and has been linked with resistance to influenza infection. It has been shown to interact with viral nucleocapsids and to interfere with the viral life cycle at three distinct phases: inhibiting nucleocapsid transport to the nucleus, preventing transcription of viral gene products, or interfering with viral assembly. We will use MxA expression vectors to overexpress FLAG-tagged MxA in the CEM cell line. Transfected CEM cells will be infected with CXCR4 tropic HIV-1 and the production of virus will be monitored by ELISA for viral p24. If MxA can inhibit the HIV-1 life cycle then we would expect a reduction in the secretion of p24.
The ability of a virus to trigger the type I interferon response is an important factor in determining the pathogenicity of a virus. Determining how this response is generated by HIV-1 will allow us to understand how HIV-1 establishes a productive infection in the human thymus. Furthermore, the study of the interaction of HIV-1 with natural antiviral proteins, such as MxA, will provide additional information for the development of an effective therapeutic HIV vaccine. Vaccine regimens that are capable of stimulating the correct subset of proteins that can inhibit HIV-1 replication will have the best chance of effectively reducing the viral load of previously infected patients.