The current study has identified a highly virus-specific TRIM protein, TRIM79α, as a key mediator of the innate cellular response to TBEV infection. TRIM79α expression was dependent on type I IFN and was required for effective restriction of TBEV replication by IFN-β. The mechanism of TRIM79α-dependent restriction of TBEV was direct, targeting NS5, the viral polymerase and an essential component of the RC, for degradation. The few TRIM proteins previously demonstrated to have direct antiviral activity including TRIM5α and TRIM22 generally require the RING domain and may use the proteasome to restrict virus replication (Barr et al., 2008
; Diaz-Griffero et al., 2007
; Eldin et al., 2009
; Gao et al., 2009
; Maegawa et al., 2010
; Wu et al., 2006
). However, TRIM79α mediated degradation of NS5 through lysosomes independently of the RING catalytic site. TRIM79α-mediated restriction was specific to flaviviruses belonging to the TBEV serogroup because NS5 derived from the mosquito-borne flaviviruses WNV or JEV was not recognized by TRIM79α and WNV replication was unimpeded by TRIM79α expression. This high degree of specificity demonstrated by TRIM79α reveals a remarkable ability of the innate IFN response to discriminate between closely related flaviviruses.
Ectopic expression of TRIM79α in 293 cells resulted in a 50–90% reduction of both LGTV and TBEV replication, despite the fact that TRIM79α expression resulted in lower expression of IFN-β. The degree of inhibition observed here is highly reminiscent of similar experiments evaluating virus restriction by proteins with dominant roles in IFN- dependent antiviral responses. Notable examples of these proteins include P56 inhibition of human papilloma virus (Terenzi et al., 2008
), IRF-1 as a general antiviral molecule (Schoggins et al., 2011
) and 2′-5′-oligoadenylate synthetase 1b (OAS1b), encoded by the flavivirus resistance gene Flv
. In the latter case, ectopic expression of OAS1b in cells derived from susceptible mice resulted in approximately 50% reduction in WNV titers. However, WNV titers in resistant mice are 103
fold lower than in susceptible mice (Perelygin et al., 2002
). While a limited number of additional gene products may contribute to flavivirus susceptibility, the Flv
studies suggest that in vitro
experiments examining ectopically expressed protein may underestimate the importance of individual ISGs in controlling virus replication in vivo
Lysosomes are cellular organelles critical for macromolecule degradation and are the final destination of material undergoing phagocytosis, endocytosis or autophagy (Schroder et al., 2010
). Thus, a switch from proteasome-dependent degradation of normal TRIM79α to lysosome-dependent degradation of NS5 observed in this study may represent an antiviral mechanism to target large protein complexes for destruction. In support of this, TRIM79α facilitated the degradation of protein complexes containing at least NS5, NS2B and NS3. However, despite the fact that NS5 is expressed on the cytosolic side of ER membranes, flavivirus RCs are shielded by virus-generated membrane proliferations thought to prevent recognition of viral replication intermediates by the host cell (Hoenen et al., 2007
; Overby et al., 2010
). NS5 is also anchored to membranes through its interactions with other viral NS proteins. Thus, it is unclear how TRIM79α would access NS5 in RCs and transport it to lysosomes. We did not find clear evidence that TRIM79α functions in concert with autophagy to drive destruction of the TBEV RC. Therefore, further studies will be required to elucidate the precise mechanism by which TRIM79α mediates TBEV restriction.
Although central to viral RC function, not all NS5 produced during flavivirus replication is found in membrane-bound RCs. NS5 is also present free in the cytoplasm or nucleus of cells infected with some flaviviruses (Davidson, 2009
). Thus, multiple populations of NS5 exist over the course of infection that function indirectly in virus replication by modulating cellular processes such as suppression of IFNα/β-dependent signal transduction or host gene expression (Medin et al., 2005
). These populations can be defined by the viral and cellular proteins bound to NS5, or by post-translational modifications such as phosphorylation and ubiquitination. We observed at least two species of NS5, a non-ubiquitinated form and a Ub-conjugated form that was stabilized by MG132. Thus NS5 degradation occurs by at least two pathways, the TRIM79α-dependent lysosome and the TRIM79α-independent proteasome. The presence of separable populations of NS5 suggests that TRIM79α may also target a population involved in functions other than in the RC. Understanding the molecular determinants required for TRIM79α recognition of TBEV NS5 would help illuminate the complexity of NS5 function in virus replication and pathogenesis.
The structure of NS5 is highly conserved between flaviviruses despite the fact that NS5 proteins share only ~40% identity at the amino acid level. The discovery of TRIM79α by yeast two-hybrid analysis of the NS5 MTase domain suggests that differences within this domain between TBEV and WNV may determine specificity and will be the subject of future studies. We also demonstrated a further level of specificity in TRIM recognition as TRIM30α shares 82% identity with TRIM79α but failed to directly interact with LGTV NS5. Taken together, these observations suggest that the NS5/TRIM79α interaction exists as a consequence of virus-host coevolution. The enzootic transmission cycle of TBEV occurs predominantly between tick vectors and their rodent hosts without causing obvious morbidity in the rodent (Bakhvalova et al., 2009
; Ebel, 2010
). This clearly differentiates the evolutionary pressures of TBEV from those of WNV and JEV that cycle between mosquitoes and either birds or pigs (Mackenzie et al., 2004
; van den Hurk et al., 2009
). Hence, suppression of TBEV replication by the rodent-specific TRIM79α may represent an example of virus-host coevolution whereby type I IFN dampens virus replication, thereby contributing to reservoir host tropism and virus maintenance in nature.
For every antiviral measure utilized by the host, viruses have evolved strategies of evasion. TBEV delays production of type I IFN (Overby et al., 2010
) and antagonizes IFN signaling (Best et al., 2005
; Werme et al., 2008
), strategies that would suppress TRIM79α expression. In addition, TRIM79α protein levels may be a target of virus antagonism. A loss in TRIM79α protein was evident late in infection with LGTV coincident with the detection of viral proteins by western blot (). A similar reduction in TRIM79α was seen in the presence of both NS5 and NS2B/3 (). TRIM79α does not appear to be degraded with NS5 in the lysosome as only proteasome inhibitors could stabilize TRIM79α expression and ectopically expressed NS5 did not impact TRIM79α levels. A protein complex containing TRIM79α and NS5 may simply be degraded more efficiently in the presence of NS2B/3. However, since NS2B/3 is the viral protease, TBEV may also evade restriction through the cleavage and inactivation of TRIM79α. A similar phenomenon was recently reported for TRIM56-mediated restriction of bovine viral diarrhea virus (BVDV), a Pestivirus
and member of the Flaviviridae
family. While the viral target of TRIM56 is unknown, expression of the BVDV small N-terminal protease (Npro
) was associated with reduced TRIM56 protein levels suggesting Npro
might directly antagonize this TRIM (Wang et al., 2011
). Hence, interference of TRIM function may be an unexplored mechanism contributing to flavivirus evasion of innate immunity and virus pathogenesis.
In addition to direct roles in virus restriction, TRIM proteins are required to regulate signaling pathways such as toll-like receptors (TLRs) and RIG-I-like receptors (RLR) leading to virus detection and innate immune responses (Ozato et al., 2008
). Both TRIM79α and TRIM30α have been linked to lysosomal degradation of the signaling components TAB2 and TAB3 (Shi et al., 2008
; Tareen and Emerman, 2011
), thus acting as negative regulators of the TLR/NFκB pathway. This function is consistent with reduced IFN-β expression observed during LGTV replication in TRIM79α-expressing cells. As has been demonstrated for influenza NS1 that binds to TRIM25 to inhibit RIG-I activity (Gack et al., 2009
), the function of NS5-bound TRIM79α may provide a benefit to TBEV replication in vivo
. For example, suppression of TRIM79α cellular function may increase production of inflammatory cytokines to recruit monocytes and macrophages to sites of infection and facilitate TBEV transmission to feeding ticks or dissemination in the vertebrate host. Alternatively, NS5 may potentiate TRIM79α function to suppress IFN expression. Experiments are currently in progress to evaluate the impact of TBEV infection and NS5 expression on the cellular roles of TRIM79α.
Despite the importance of host IFNα/β responses in the control of flavivirus infections, IFN is ineffective as a clinical therapy, likely compromised by virus-encoded antagonists of IFN-dependent JAK-STAT signaling (Robertson et al., 2009
). Thus, understanding the precise antiviral mechanisms of ISGs may enable development of therapeutics effective against viruses like the flaviviruses that have evolved to target IFN-dependent signal transduction. Moreover, although hundreds of antiviral genes are expressed in response to IFN, this work demonstrates that antiviral activity can be tailored to individual pathogens by the activity of virus-specific ISGs. The fact that the TRIM proteins often target necessarily conserved structures such as the viral RNA polymerase suggests that resistance to TRIM mimetics as therapeutics may not be easily acquired through virus mutation. Therefore, further studies to identify additional TRIM molecules that specifically target flaviviruses as well as to understand TRIM mechanisms of restriction are warranted.