Here we describe functions of human Trim5α that do not depend on recognition of retroviral capsids. We show that human Trim5α, similar to its mouse homolog Trim30, targets TAB2 for degradation, resulting in abrogation of TAB2-dependent NF-kappaB activation. However, unlike mouse Trim30, human and rhesus Trim5α are able to activate NF-kappaB-driven reporter gene expression in a dose-dependent manner. Using splice variants, chimeras and mutants we show that human Trim5α uses distinct domains for the distinct abilities of affecting TAB2 levels, regulating NF-kappaB, and recognizing retroviral capsids. We speculate that the induction of NF-kappaB is the more critical of these activities since it is common between the two primate Trim5 genes tested.
Viruses either escape from or harness innate immunity pathways in order to continue their life cycle. For example, NS1 protein of influenza A virus prevents NF-kappaB-dependent interferon signaling by binding viral dsRNA and preventing PKR activation (Wang et al., 2000
). Based on work reported here, Trim5α, by regulating TAB2 levels or activating NF-kappaB, could be involved in controlling a response to viral infection at various stages. Implications of a TRIM protein in signaling events that affect viruses are not without precedent. For example Trim25, which also has RBCC and PRY-SPRY domains, is able to induce IFN signaling against RNA viruses by activation of RIG-I through K63 polyubiquitination (Gack et al., 2007
). In this example, the antiviral properties of Trim25 appear to be directly linked to its role in RIG-I signaling. Interestingly, human T-cell lymphotropic virus type 1 (HTLV-1) appears to hijack TAB2-dependent signaling through induction of TAB2 levels using its Tax oncoprotein, resulting in transcriptional activation and transformation (Boxus et al., 2008
; Suzuki et al., 2007
; Yu et al., 2008
It is unclear why a rapidly evolving antiviral factor such as Trim5α would be co-opted to play a key role in regulating signaling, or vice versa. One explanation may be that the ability of Trim5α to function as an antiviral factor is linked to its role in signaling, and that these two roles are inseparable even though we show here that genetically they are separable. The recent finding that restriction by Trim5α in the cytoplasmic bodies relies on the presence of p62/sequestosome-1, an interferon-inducible gene involved in signaling pathways such as TRAF6 and NF-kappaB (O'Connor et al., 2010), supports a role of cell signaling in the Trim5α restriction pathway. Furthermore, it is possible that Trim5α signaling and restriction functions are actually linked in some way since infection of cells with a retrovirus that is recognized by Trim5α leads to the degradation of Trim5α (Rold and Aiken, 2008
). Thus, the signaling mechanisms induced by Trim5α could be a cellular response to a viral infection where the “recognition” event is the loss of Trim5α in the cell, which then leads to the absence of those constitutive Trim5α signals.
The ability of Trim5 to target TAB2 and to upregulate NF-kappaB appear to be independent of the PRY-SPRY domain ( and
), however we cannot rule out that the possibility that the PRY-SPRY domain might be involved in moderating these activities. The fact that the PRY-SPRY domain is used by Trim5α for recognizing and binding to the viral capsid, and that this domain contains the region under most positive selection, indicates that Trim5α is able to maintain its other functions while still being able to adaptively evolve in its PRY-SPRY domain. The RBCC domains of Trim5α appear to be involved in the TAB2 and NF-kappaB functions we describe here. Since these functions most likely involve the formation of complexes with other cellular proteins through protein-protein interactions, we would expect evolution to select against amino acid changes that could impede with these functions. Therefore, the functions of Trim5α in TAB2 and NF-kappaB signaling could maintain the evolutionary constraint seen in the RBCC domains of Trim5α. Interestingly, certain amino acids in parts of the RBCC domains are also under positive selection, although not as strongly and as dense as in the PRY-SPRY (reviewed in Johnson and Sawyer, 2009
). Whether these amino acids specifically contribute to the abilities of Trim5α described here remain to be investigated. The inability of rhesus Trim5α to affect TAB2 levels, even though this ability is conserved in humans and mice, could indicate a loss in the ability of rhesus Trim5α to target TAB2, possibly due to amino acid differences in the RBCC domains between human and rhesus Trim5α. It is possible that polymorphisms in rhesus Trim5α will affect this ability, but we have not yet tested other alleles. Likewise, it is possible that human polymorphisms in Trim5α will also affect these activities.
Using chimeras and mutants we were able to separate the ability of human Trim5α to affect TAB2 levels, from its ability to activate NF-kappaB. The interplay of these two abilities () suggests that human Trim5α either upregulates NF-kappaB downstream of where it targets TAB2 in the signaling cascade, that Trim5α is able to upregulate NF-kappaB through a pathway that is independent of TAK1 activation. NF-kappaB is regulated through two pathways known as the canonical and non-canonical pathways (reviewed in Skaug et al., 2009
). TAK1 is involved in the canonical pathway where, upon TNF or IL-1beta stimulation, TAK1 activates IKK, which then targets IkB proteins for degradation, thus allowing the p50/65 NF-kappaB dimmer to enter the nucleus. In the non-canonical pathway, CD40L stimulation results in the activation of the NF-kappaB inducing kinase (NIK) and the IKKalpha subunit. The latter, in turn, targets p100 for degradation, thus allowing the p52/Rel-B dimmer to enter the nucleus. The reporter construct we used in our experiments for NF-kappaB activation is sensitive to both of these pathways. It is also possible that the seeming degradation of TAB2 by human Trim5α is an epi-phenomenon that is simply a readout of a binding activity to TAB2 that is shared with rhesus Trim5α and which results in induction of NF-kappaB in both cases.
Primates, rabbits, cats and dogs have one copy of Trim5
whereas rodents and cows have multiple copies of Trim5
in their locus (Schaller et al., 2007
; McEwan et al., 2009
; Tareen et al., 2009
; Sawyer et al., 2007
; Si et al., 2006
). This diverse nature of the Trim5
locus among mammals should make for interesting strategies in balancing the abilities of viral restriction with regulating TAB2 and NF-kappaB pathways. In the case of rodents and cows, paralogous Trim5
gene expansions may be selected for temporal and spatial sharing of duties; for example, certain Trim5 paralogs may be expressed during certain stages of development and take on functional responsibility. Alternatively, different Trim5 paralogs may complement one another by splitting different functions. Interestingly, two of the mouse Trim5 paralogs, namely Trim12 and Trim30-1, do not encode the PRY-SPRY domain (Tareen et al., 2009
). However, our findings that the PRY-SPRY is not required suggests that mouse Trim12 and Trim30-1 may also be capable of negatively regulating NF-kappaB activation by targeting TAB2 and TAB3 for degradation, similar to Trim30. The fact that the functions for Trim5α we describe here are present in mice and humans suggests that either evolution has maintained this function since the divergence of these two species, or that, although less likely, convergent evolution explains these functions in mouse and human Trim5α.
One problem that rapidly evolving antiviral factors may face is how they are able to adaptively evolve while maintaining sequence conservation for conserved functions. Trim5α appears to have solved this problem by using distinct domains to recognize retroviral capsids versus regulating TAB2 and NF-kappaB signaling: the PRY-SPRY recognizes the capsid and is free to rapidly evolve, while the remaining domains are under evolutionary constraint in order to maintain the functions described here. These functions of Trim5α may have bestowed upon it multiple roles in innate immunity, thus possibly explaining its maintenance over millions of years throughout mammalian evolution even in the absence of retroviral recognition.