Hsp70 Interacts with the Retroviral Restriction Factor TRIM5α and Assists the Folding of TRIM5α

Abstract

Tripartite motif (TRIM) protein TRIM5␣ has been shown to restrict human immunodeficiency virus, type 1 infection in OldWorld monkey cells at the early post-entry step by poorly understood mechanisms. Currently, the physiological function of TRIM5␣ is not known. In this study, we showed that transiently overexpressed TRIM5␣ causes a morphological change in HEK293T cells. A proteomics analysis of the protein complexes that were pulled down with hemagglutinin-tagged TRIM5␣ suggested that the heat shock protein 70 (Hsp70) may serve as a TRIM5␣-binding partner. The interaction between Hsp70 and TRIM5␣ was confirmed by co-localization and co-immunoprecipitation assays. Co-expression of Hsp70 reversed the TRIM5␣-induced morphological change in HEK293T cells. Another heat shock protein Hsc70 also bound to TRIM5␣, but unlike Hsp70, Hsc70 was not able to reverse the TRIM5␣-induced morphological change, suggesting that Hsp70 specifically reverses the morphological change caused by TRIM5␣. Studies using a series of TRIM5␣ deletion mutants demonstrate that, although the PRYSPRY domain is critical for binding to Hsp70, the entire TRIM5␣ structure is necessary to induce the morphological change of cells. When the ATPase domain of Hsp70 was mutated, the mutated Hsp70 could not counteract the morphological change induced by TRIM5␣, indicating that the catalytic activity of Hsp70 protein is important for this function. Co-expression of Hsp70 elevated the levels of TRIM5␣ in the detergent-soluble fraction with a concomitant decrease in the detergent-insoluble fraction. Together these results suggest that Hsp70 plays critical roles in the cellular management against the TRIM5␣-induced cellular insults.Retroviruses encounter dominant post-entry restrictions in the cells of particular species. Human immunodeficiency virus type 1 (HIV-1) 2 infection is blocked in the cells of Old World monkeys and simian immunodeficiency virus (SIV mac ) infection is blocked in most New World monkey cells (1-3). These retroviral restrictions are largely mediated by a host protein, tripartite motif 5␣ (TRIM5␣) (4). TRIM5␣ is a member of the large family of tripartite motif proteins (TRIMs) (5). Members of this family commonly have a RING domain, one or two B-box domains and coiled-coil (CC) domain, and thus are also called RBCC proteins (5). The RING domain of many TRIM proteins has been shown to have E3 ubiquitin ligase activity, whereas the B box and CC domains may be involved in protein-protein interactions and homo/heterodimerization (5). The alpha isoforms of many cytoplasmic TRIM proteins, including TRIM5␣, contain the PRYSPRY domain whose function is unknown. To date, about 70 TRIM proteins have been identified in the human genome and some of their homologs have been found in primates and other species. TRIM proteins arose with the metazoans and have expanded in number during vertebrate evolution (5). Many TRIM proteins, including TRIM19, TRIM21, TRIM22, TRIM34, and TRIM5␣, can be up-regulated by interferon, supporting their potential role as ef...

Cytoplasmic Body Component TRIM5α Requires Lipid-enriched Microdomains for Efficient HIV-1 Restriction

“…These data rule out the possible artifacts due to sequestering of an overexpressed TRIM5␣ protein in lipid microdomains. In the course of this study, Hwang et al (57) reported that TRIM5␣rh was present in detergent-insoluble fractions when expressed in 293T cells, which further support our current findings.…”

14-3-3 proteins sequester a pool of soluble TRIM32 ubiquitin ligase to repress autoubiquitination and cytoplasmic body formation

“…Mounting evidence indicates that molecular chaperons such as Hsp90 and Hsp70 play important roles in the formation and/or maintenance of various cytoplasmic foci, including TRIM CBs, processing bodies, and stress granules (Hwang et al, 2010;Johnston et al, 2010). Importantly, the 14-3-3 family participates in the formation/maintenance of processing bodies (Okada et al, 2011) and stress granules (Stoecklin et al, 2004).…”

Recent Insights into the Mechanism and Consequences of TRIM5α Retroviral Restriction

“…Another study has observed TRIM5a associating with components of the autophagic degradation pathway. 65 How and why restrictionsensitive virus alters the degradative fate of TRIM5a remains unclear. However, as p62 mediates both proteasomal 64,66 and autophagic degradation, [67][68][69] it may play a role in dictating the degradative fate of TRIM5a.…”