Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus counterpart inhibits apoptosis independent of Bim

Abstract

Subversion of host cell apoptosis is an important survival strategy for viruses to ensure their own proliferation and survival. Certain viruses express proteins homologous in sequence, structure and function to mammalian pro-survival B-cell lymphoma 2 (Bcl-2) proteins, which prevent rapid clearance of infected host cells. In vaccinia virus (VV), the virulence factor F1L was shown to be a potent inhibitor of apoptosis that functions primarily be engaging pro-apoptotic Bim. Variola virus (VAR), the causative agent of smallpox, harbors a homolog of F1L of unknown function. We show that VAR F1L is a potent inhibitor of apoptosis, and unlike all other characterized anti-apoptotic Bcl-2 family members lacks affinity for the Bim Bcl-2 homology 3 (BH3) domain. Instead, VAR F1L engages Bid BH3 as well as Bak and Bax BH3 domains. Unlike its VV homolog, variola F1L only protects against Bax-mediated apoptosis in cellular assays. Crystal structures of variola F1L bound to Bid and Bak BH3 domains reveal that variola F1L forms a domain-swapped Bcl-2 fold, which accommodates Bid and Bak BH3 in the canonical Bcl-2-binding groove, in a manner similar to VV F1L. Despite the observed conservation of structure and sequence, variola F1L inhibits apoptosis using a startlingly different mechanism compared with its VV counterpart. Our results suggest that unlike during VV infection, Bim neutralization may not be required during VAR infection. As molecular determinants for the human-specific tropism of VAR remain essentially unknown, identification of a different mechanism of action and utilization of host factors used by a VAR virulence factor compared with its VV homolog suggest that studying VAR directly may be essential to understand its unique tropism.

Structural Insight into African Swine Fever Virus A179L-Mediated Inhibition of Apoptosis

“…Interestingly, the A179L E76:ssBid R81 ionic interaction is also found in the variola virus F1L:Bid complex in the form of F1L E143:Bid R83 (20), unlike the Bcl-x L , Mcl-1, A1, and Bcl-w complexes with Bid, where this interaction is absent (37,45,47,48). In contrast, the two additional ionic interactions present in the A179L:Bax BH3 complex formed by D80:K64 and K79:E61 are also conserved in the Bcl-2:Bax BH3 complex as D140:R64 and R139:E61 (36), whereas in the poxviral DPV022:Bax BH3 complex (26) and Mcl-1:Bax complex they are absent (49).…”

“…2B) or the unrelated viral Bcl-2 protein variola virus F1L ( Fig. 2C) (20). Bax BH3 binds to A179L in an equivalent manner (Fig.…”

“…Other viruses encode antiapoptotic proteins that display weak or no overt sequence identity with Bcl-2. A number of these distant orthologous Bcl-2 proteins are encoded by viruses that are members of the Poxviridae family, including vaccinia virus and variola virus, which both encode F1L, vaccinia virus N1L, E3L, and GAAP, and myxoma virus-encoded M11L (13)(14)(15)(16)(17)(18)(19)(20)(21), as well as the more recently identified fowlpox virus FPV039, orf virus ORF125, deerpox virus DPV022, and sheeppox virus SPPV14 (22)(23)(24)(25)(26). The use of sequence and/or structural homologs of Bcl-2 for immune modulatory purposes is not limited to the pox viruses and herpesviridae.…”

Inflammasomes and its importance in viral infections

“…Additionally, viral proteins can prevent the recruitment of ASC molecules, thus inhibiting the innate immune response and promoting a better environment for viral replication [137]. Numerous reports have shown that VACV encodes viral homologs of cellular proteins; for example, it encodes anti-apoptotic proteins with homology to the BCL-2 family that regulate the innate immune response by interacting with NLRP1 [138]. In detail, the F1L protein of VACV plays a key role in inhibiting the function of NLRP1, and this function depends on a specific region of F1L (residues 1-47).…”

The N Terminus of the Vaccinia Virus Protein F1L Is an Intrinsically Unstructured Region That Is Not Involved in Apoptosis Regulation

“…Both EVM025 and variola virus F1L were shown to be apoptosis inhibitors with EVM025 inhibiting Bak directly, whereas Bax-mediated apoptosis was inhibited via the sequestration of Bim (70). In contrast, variola virus F1L was only able to inhibit Bak-mediated apoptosis and did not show any ability to engage Bim (71). Both EVM025 and variola virus F1L harbor long N-terminal extensions prior to their Bcl-2 fold; however, neither has been functionally characterized.…”