TRIM5alpha(hu): An innate cellular defense against N-tropic murine leukemia virus infection in humans

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 5alpha (TRIM5alpha) as a major intracellular restriction factor in mammals that prevents retroviral infection. TRIM5alpha 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 TRIM5alpha isoform also includes a C-terminal B30.2 domain. The expression of human TRIM5alpha (TRIM5alphahu) accounts for N-MLV restriction in human cells. Permissive cells transduced with a vector expressing TRIM5alpha hu potently block N-MLV infection at an early post-entry step. TRIM5alpha hu restriction prevents the accumulation of full-length viral cDNA and is dependent upon residue 110 of the MLV capsid. Knockdown of TRIM5alpha hu expression in human cells, using siRNA, relieves N-MLV restriction and allows productive infection of the cells.; The TRIM5alpha from some other primate species, such as rhesus macaque (TRIM5alpharh), exhibit a modest N-MLV restriction potential. To identify species-specific potency determinants within TRIM5alpha hu, numerous chimera between TRIM5alpharh and TRIM5alpha hu were generated and assayed for acquisition of potent N-MLV restriction. This study identified two regions within the TRIM5alphahu B30.2 domain required for potent N-MLV restriction. The potency determinant within TRIM5alphahu v1 maps to a small stretch of amino acids, residues 335-340, that share limited sequence conservation with TRIM5alpharh . The TRIM5alphahu v3 determinant was identified as two acidic residues at positions 405 and 406.; To study the interaction between TRIM5alpha and the retroviral capsid, we develop an in vitro binding assay using assembled HIV-1 capsid-nucleocapsid complexes. We demonstrate that the TRIM5alpha from Old World monkeys, which restrict HIV-1 infection, efficiently interacts with the HIV-1 capsid-nucleocapsid complexes. Conversely, the TRIM5alpha from unrestricted New World monkeys failed to interact with the HIV-1 capsid-nucleocapsid complexes.; To identify the mechanism by which TRIM5alpha 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 TRIM5alpha promotes the premature conversion of particulate retroviral capsids into soluble capsid proteins. TRIM5alpharh and TRIM5alphahu promote the rapid disassembly of particulate HIV-1 and N-MLV capsids, respectively. TRIM5alphahu-mediated disassembly of particulate N-MLV capsids is dependent upon residue 110 of the MLV capsid and requires all four domains of TRIM5alphahu.