Analysis of the impact of TRIM5 polymorphism on retroviral replication

This project aims to improve the SIV/rhesus macaques animal model for AIDS. TRIM5α is an antiviral molecule expressed in mammals including rhesus macaques. The antiviral specificity determinant of TRIM5α is within the B30.2 domain, encoded within the last exon of the TRIM5 gene. This work assessed whether B30.2 polymorphism is responsible for differences in disease progression following retroviral challenge. Exon 8 from TRIM5 was amplified by PCR and sequenced to identify the different alleles of the B30.2 domain, termed Macaca mulatta (Mamu) alleles. These were appended to the remainder of the TRIM5 molecule, creating TRIM5α molecules with varying B30.2 domains. These were expressed in a highly permissive cell line, CRFK. A variety of green fluorescent protein (GFP) expressing retroviruses were generated within HEK 293T cells and used to infect CRFK that express different forms of TRIM5α. Retroviral infection was assessed using a FACS machine, which counts GFP positive cells. This is a method of analysing differences in retroviral restriction potentials due to expression of B30.2 polymorphs. Furthermore, this project examined the dominant negative properties of the identified Mamu alleles against a strong restriction of HIV-1, Mamu1. Cells were protected from infection with FIV, HIV-1, HIV-2 and EIAV by some of the Mamu alleles tested, except Mamu7. In contrast, SIV, MLV-B and MLV-N were not restricted by any of the Mamu alleles tested in this study. Interestingly Mamu4 and Mamu5 protected cells from HIV-1, FIV and EIAV infection but not from HIV-2 infection. These alleles therefore appear to encode functional restriction factors with different antiretroviral specificities. Mamu7, a truncated allele showed the least restricting properties of all tested alleles. However, co-expression of Mamu7 with the wildtype TRIM5α allele was found to slightly abrogate restriction in a dominant negative manner following challenge with HIV-1 and HIV-2. In addition, Mamu4 and 5 exerted a mild dominant negative effect following HIV-2 infection, consistent with their inability to restrict HIV-2 when expressed alone.