Biochemical and Biophysical Characterization of a Chimeric TRIM21-TRIM5α Protein

After entering the host cell, retroviruses must evade intracellular restriction factors to establish a successful infection. The life cycle of certain retroviruses is blocked in the cells of some mammalian species prior to the initiation of reverse transcription (2, 3, 8, 42). In primates, these early blocks are mediated by TRIM5α, which binds the incoming viral capsid and promotes its premature uncoating (10, 31, 32, 38, 42, 43). Differences among the TRIM5α proteins of primate species account for the different restrictions against infection by particular retroviruses (15, 26, 35, 36, 40, 41, 44, 48). For example, the TRIM5α proteins of Old World monkeys block human immunodeficiency virus type 1 (HIV-1), whereas the TRIM5α proteins of most New World monkeys inhibit simian immunodeficiency virus (SIVmac). TRIM5α is a tripartite motif (TRIM) family protein (28, 34). TRIM proteins contain RING, B-box 2, and coiled-coil domains. The TRIM5α protein also contains a carboxy-terminal B30.2(SPRY) domain. The contribution of the TRIM5α domains to retroviral restriction has been assessed (12, 18, 19, 30, 36, 44). The RING domain contributes to the potency of restriction but is not absolutely essential for virus inhibition (12). The RING domains of some TRIM proteins have been associated with protein ubiquitylation and targeting to the proteasome (5, 6, 16, 45). Unlike the RING domain, the B-box 2, coiled-coil, and B30.2(SPRY) domains of TRIM5α are essential for antiretroviral activity (29, 30). The coiled coil mediates TRIM5α oligomerization (13, 25), which increases the avidity of TRIM5α for the retroviral capsid (13). Capsid binding is also dependent on the B30.2(SPRY) domain (19, 37, 40). Species-specific differences in retroviral restriction often result from variation in the B30.2(SPRY) domain (18, 20, 30, 36, 44). Although the B-box 2 domain of TRIM5α is not required for capsid recognition, some changes in the B-box 2 domain result in proteins that are deficient in restricting viral infection (4, 43). It has been suggested that the B-box 2 domain contributes to an effector function involved in mediating the premature uncoating of the retroviral capsid (4). A tendency to aggregate and low levels of TRIM5α expression present barriers to its biochemical and structural characterization. Many TRIM proteins are aggregation-prone and, when overexpressed, form pre-aggresomal cytoplasmic bodies (39). TRIM5 is expressed at low levels due to its rapid turnover (5). TRIM5 turnover is dependent on RING and B-box 2 sequences and is largely resistant to proteasome inhibitors (4). The rapid turnover of TRIM5α is not required for its antiretroviral activity (4). Substitution of the RING domain of TRIM5α with that of related TRIM proteins, which exhibit significantly longer half-lives than that of TRIM5α, results in long-lived chimeric proteins with antiretroviral function (5, 18). For example, in the present study, we examine a TRIM5α protein from rhesus monkeys (TRIM5αrh) in which the RING domain has been replaced by that of human TRIM21. This chimeric protein, designated TRIM5Rh-21R, can potently inhibit HIV-1 infection (18). Here we report on our efforts to overexpress and characterize the chimeric TRIM5Rh-21R protein.