Title: TRIM5alpha(hu)
Description: Host cell restriction factors influence retroviral tropism in mammals. A dominant cellular restriction factor limits N-tropic murine leukemia virus (N-MLV) infection in several primate species, including human. Restriction of N-MLV infection occurs at an early post-entry step and prevents the accumulation of full-length viral cDNA. The viral determinant of susceptibility to restriction has been mapped to residue 110 of the viral capsid protein. Recently, a genetic screen identified tripartite motif 5α (TRIM5α) as a major intracellular restriction factor in mammals that prevents retroviral infection. TRIM5α is a member of the tripartite motif family of proteins, characterized by the presence of a RING, B-box 2 and coiled-coil domains. The TRIM5α isoform also includes a C-terminal B30.2 domain. The expression of human TRIM5α (TRIM5α hu ) accounts for N-MLV restriction in human cells. Permissive cells transduced with a vector expressing TRIM5α hu potently block N-MLV infection at an early post-entry step. TRIM5α hu restriction prevents the accumulation of full-length viral cDNA and is dependent upon residue 110 of the MLV capsid. Knockdown of TRIM5α hu expression in human cells, using siRNA, relieves N-MLV restriction and allows productive infection of the cells. The TRIM5α from some other primate species, such as rhesus macaque (TRIM5α rh ), exhibit a modest N-MLV restriction potential. To identify species-specific potency determinants within TRIM5α hu , numerous chimera between TRIM5α rh and TRIM5α hu were generated and assayed for acquisition of potent N-MLV restriction. This study identified two regions within the TRIM5α hu B30.2 domain required for potent N-MLV restriction. The potency determinant within TRIM5α hu v1 maps to a small stretch of amino acids, residues 335-340, that share limited sequence conservation with TRIM5α rh.

The TRIM5α hu v3 determinant was identified as two acidic residues at positions 405 and 406. To study the interaction between TRIM5α and the retroviral capsid, we develop an in vitro binding assay using assembled HIV-1 capsid-nucleocapsid complexes. We demonstrate that the TRIM5α from Old World monkeys, which restrict HIV-1 infection, efficiently interacts with the HIV-1 capsid-nucleocapsid complexes. Conversely, the TRIM5α from unrestricted New World monkeys failed to interact with the HIV-1 capsid-nucleocapsid complexes. To identify the mechanism by which TRIM5α mediates retroviral restriction, we developed a "fate of capsid" assay that allows us to monitor the steady-state levels of particulate retroviral capsids within the cytoplasm of infected cells. Using this assay we demonstrate that TRIM5α promotes the premature conversion of particulate retroviral capsids into soluble capsid proteins. TRIM5α rh and TRIM5α hu promote the rapid disassembly of particulate HIV-1 and N-MLV capsids, respectively. TRIM5α hu -mediated disassembly of particulate N-MLV capsids is dependent upon residue 110 of the MLV capsid and requires all four domains of TRIM5α hu.