Retroviral Restriction Factor Research Articles

Arsenic counteracts human immunodeficiency virus type 1 restriction by various TRIM5 orthologues in a cell type-dependent manner.

Arsenic trioxide (As(2)O(3)) increased human immunodeficiency virus type 1 (HIV-1) infectivity when particular Homo sapiens and Cercopithecus aethiops cell lines were used as targets. Knockdown of human TRIM5alpha by RNA interference eliminated the As(2)O(3) effect, demonstrating that the drug acts by modulating the activity of this retroviral restriction factor. In contrast, HIV-1 infectivity in target cell lines from other primate species (Cercopithecus tantalus, Macaca mulatta, and Aotus trivirgatus) was not increased by As(2)O(3), despite the potent TRIM5-dependent HIV-1 restriction activity that these cells exhibit. To determine if As(2)O(3) responsiveness is characteristic of particular TRIM5 orthologues and not others, TRIM5 cDNAs from these five primate species were transduced into cat fibroblasts, which lack endogenous HIV-1 restriction activity and, therefore, responsiveness to As(2)O(3). In this context, the HIV-1 restriction activity conferred by all TRIM5 orthologues was largely eliminated by As(2)O(3). The effect of As(2)O(3) on HIV-1 restriction is thus shared by different TRIM5 orthologues but dependent on factors specific to the cell line in which TRIM5 is expressed.

757. Engineered Expression of Novel TRIM5 Isoforms Inhibits HIV-1 Infection in CD34 Hematopoietic Stem Cell Derived Macrophages

Top of pageAbstract Species specific innate resistance against viral infections offers novel avenues for antiviral therapeutic and prophylactic approaches. The retroviral and lentiviral restriction factors Ref1 and Lv1 are variants of the tripartite motif protein, TRIM5a, a component of cytoplasmic bodies. TRIM5a severely restricts productive retroviral infections at the level of post-entry and pre-integration by destabilizing the incoming viral capsid via ubiquitination. Using this approach, resistance to HIV-1 infection could be conferred by TRIM5arh expression in otherwise susceptible cells. Here we show that stable expression of simian TRIM5arh via a lentiviral vector in a permissive cell culture line, Magi-CXC4, conferred resistance to HIV-1. To translate these findings into a stem cell gene therapy setting, the TRIM5arh transgene was stable introduced into CD34+ hematopoietic progenitor cells to derive transgenic macrophages. Upon viral challenge, TRIM5arh expressing macrophages were highly resistant to HIV-1 infection compared to control cells. Human macrophages expressing TRIM5arh were also found to be phenotypically and functionally normal expressing the characteristic surface markers CD14, CD4, CCR5, CXCR4, MHCII, and B7.1. Based on these promising results, we sought to further improve the TRIM5a inhibiting action by designing more novel constructs by modifying the native human isoform of TRIM5a. These modified isoforms have also been shown to be effective in conferring viral resistance. These results demonstrate that the species specific restriction factor TRIM5arh is effective in conferring HIV-1 resistance in a stem cell setting thus paving the way for its application in AIDS gene therapy.

Restriction of human immunodeficiency virus type 1 by TRIM-CypA occurs with rapid kinetics and independently of cytoplasmic bodies, ubiquitin, and proteasome activity.

TRIM-CypA is an owl monkey-specific variant of the retrovirus restriction factor TRIM5alpha. Here, we exploit its modular domain organization and cyclosporine sensitivity to probe the kinetics and mechanism of TRIM5-mediated restriction. Time of addition/withdrawal experiments reveal that inhibition of incoming human immunodeficiency virus type 1 capsids by TRIM-CypA occurs within minutes of their delivery to the target cell cytoplasm. However, while TRIM-CypA restriction is partly dependent on a RING domain, restriction occurs independently of the ubiquitin/proteasome system. Moreover, tagged TRIM-CypA proteins can be fully active as restriction factors without forming cytoplasmic bodies.

Retroviral Restriction Factor TRIM5α Is a Trimer

The retrovirus restriction factor TRIM5alpha targets the viral capsid soon after entry. Here we show that the TRIM5alpha protein oligomerizes into trimers. The TRIM5alpha coiled-coil and B30.2(SPRY) domains make important contributions to the formation and/or stability of the trimers. A functionally defective TRIM5alpha mutant with the RING and B-box 2 domains deleted can form heterotrimers with wild-type TRIM5alpha, accounting for the observed dominant-negative activity of the mutant protein. Trimerization potentially allows TRIM5alpha to interact with threefold pseudosymmetrical structures on retroviral capsids.

A retrovirus restriction factor TRIM5α is transcriptionally regulated by interferons

TRIM5α is a member of tripartite motif protein family and recently identified as a restriction factor for retroviral infection in a species-specific manner. Human TRIM5α gene is located on chromosomal position 11p15 in a cluster with other TRIM genes including TRIM6, 21, 22, and 34. We show here that interferon (IFN) upregulates TRIM5α mRNA expression in HeLa and HepG2 cells by performing Northern blot analysis and quantitative real-time PCR. TRIM5α promoter activity was IFN inducible as confirmed by luciferase assay using a reporter plasmid that contained the 5′-flanking region of TRIM5α. Mutational analysis has revealed that IFNs activate TRIM5α promoter activity through a putative interferon-stimulated response element (ISRE). Intriguingly, another IFN-responsive protein signal transducer and activator of transcription factor 1 (STAT1) binds to the ISRE sequence as shown by electrophoretic mobility shift assay using HeLa cell extracts. We have raised a specific polyclonal antibody against TRIM5α and confirmed that TRIM5α protein expression is inducible by IFN-β in HeLa cells. These results lead us to define that the transcription and protein synthesis of TRIM5α could be modulated by IFN, suggesting that TRIM5α may play a role in an IFN-induced antiviral state against retrovirus infection.

TRIM5αrh expression restricts HIV-1 infection in lentiviral vector-transduced CD34+-cell-derived macrophages

Species-specific innate resistance against viral infections offers novel avenues for antiviral therapeutic and prophylactic approaches. The retroviral and lentiviral restriction factors Ref1 and Lv1 are variants of the tripartite motif protein TRIM5α, a component of cytoplasmic bodies. TRIM5α severely restricts productive retroviral infections at the postentry and preintegration steps by destabilizing the incoming viral capsid via ubiquitination. Using this approach, resistance to HIV-1 infection could be conferred by TRIM5αrh expression in otherwise susceptible cells. Here we show that stable expression of simian TRIM5αrh via a lentiviral vector in a permissive cell culture line, Magi-CXCR4, conferred resistance to HIV-1. To translate these findings into a stem cell gene therapy setting, the TRIM5αrh transgene was stably introduced into CD34+ hematopoietic progenitor cells to derive transgenic macrophages. Upon viral challenge, TRIM5αrh-expressing macrophages were highly resistant to HIV-1 infection compared to control cells. Human macrophages expressing TRIM5αrh were also found to be phenotypically and functionally normal, expressing the characteristic surface markers CD14, CD4, CCR5, CXCR4, MHC II, and B7.1. These results demonstrate that the species-specific restriction factor TRIM5αrh is effective in conferring HIV-1 resistance in a stem cell setting, thus paving the way for its application in AIDS gene therapy.

A Single Amino Acid Change in the SPRY Domain of Human Trim5α Leads to HIV-1 Restriction

Retroviral restriction factors are cellular proteins that interfere with retrovirus replication at a postpenetration, preintegration stage in the viral life cycle. The first restriction activity described was the mouse Fv1 gene. Three different alleles of Fv1, capable of restricting different murine leukaemia viruses (MLV), have been characterized at the molecular level. Two further activities, Ref1, which acts on MLV, and Lv1, which acts on lentiviruses, have been identified in other mammalian species. Recently, it has become clear that Ref1 and Lv1 are encoded by the same gene, Trim5alpha, which inhibits retrovirus replication in a species-specific manner. A series of chimeras between the human and rhesus monkey Trim5 genes were created to map and identify these specificity determinants. The Trim5alpha SPRY domain was found to be responsible for targeting HIV-1 restriction. By contrast, N-MLV restriction appears dependent on both the coiled-coil domain and the SPRY domain. A single amino acid substitution (R332P) in the human Trim5alpha can confer the ability to restrict HIV-1, suggesting that small changes during evolution may have profound effects on our susceptibility to cross-species infection.

Cyclophilin interactions with incoming human immunodeficiency virus type 1 capsids with opposing effects on infectivity in human cells.

Cyclophilin A (CypA) is a peptidyl-prolyl isomerase that binds to the capsid protein (CA) of human immunodeficiency virus type 1 (HIV-1) and by doing so facilitates HIV-1 replication. Although CypA is incorporated into HIV-1 virions by virtue of CypA-Gag interactions that occur during virion assembly, in this study we show that the CypA-CA interaction that occurs following the entry of the viral capsid into target cells is the major determinant of CypA's effects on HIV-1 replication. Specifically, by using normal and CypA-deficient Jurkat cells, we demonstrate that the presence of CypA in the target and not the virus-producing cell enhances HIV-1 infectivity. Moreover, disruption of the CypA-CA interaction with cyclosporine A (CsA) inhibits HIV-1 infectivity only if the target cell expresses CypA. The effect of CsA on HIV-1 infection of human cells varies according to which particular cell line is used as a target, and CA mutations that confer CsA resistance and dependence exert their effects only if target cells, and not if virus-producing cells, are treated with CsA. The differential effects of CsA on HIV-1 infection in different human cells appear not to be caused by polymorphisms in the recently described retrovirus restriction factor TRIM5alpha. We speculate that CypA and/or CypA-related proteins affect the fate of incoming HIV-1 capsid either directly or by modulating interactions with unidentified host cell factors.

The human and African green monkey TRIM5alpha genes encode Ref1 and Lv1 retroviral restriction factor activities.

The rhesus macaque tripartite motif containing protein TRIM5alpha specifically restricts HIV-1 infection at an early post-entry step before reverse transcription [Stremlau, M., Owens, C. M., Perron, M. J., Kiessling, M., Autissier, P. & Sodroski, J. (2004) Nature 427, 848-853]. Here, we show that the human and African green monkey (AGM) TRIM5alpha genes encode Ref1 and Lv1 antiretroviral activities, respectively. Expression of TRIM5alpha in permissive cat cells renders them resistant to restriction-sensitive murine leukemia virus but not closely related insensitive virus. Disruption of TRIM5alpha expression in human and AGM cells with small interfering RNA rescues infectivity of restricted virus without affecting unrestricted virus. We also demonstrate that the activity of the murine restriction factor Fv1 depends on TRIM5alpha expression when Fv1 is expressed in human cells. Furthermore, a drug that modifies the behavior of the related promyelocytic leukemia protein PML specifically rescues infection by viruses restricted by human TRIM5alpha. Alignment of the TRIM5alpha proteins from rhesus macaque and AGM indicates an 18-aa insertion. We speculate that this insertion may contribute to the broader specificity of the AGM TRIM5alpha restriction as compared with the human and rhesus macaque proteins.