A secreted high-affinity inhibitor of human TNF from Tanapox virus

Abstract

A class of secreted poxvirus tumor necrosis factor (TNF)-binding proteins has been isolated from Tanapox-infected cell supernatants. The inhibitor bound to a TNF-affinity column and was identified as the product of the 2L gene. Sequence analysis of 2L family members from other yatapoxviruses and swinepox virus yielded no sequence homology to any known cellular gene. The expressed Tanapox virus 2L protein bound to human TNF with high affinity (K(d) = 43 pM) and exhibits an unusually slow off-rate. However, 2L is unable to bind to a wide range of human TNF family members. The 2L protein can inhibit human TNF from binding to TNF receptors I and II as well as block TNF-induced cytolysis. Thus, Tanapox virus 2L represents an inhibitor of human TNF and offers a unique strategy with which to modulate TNF activity.

Figure 1

TNF-binding activity is encoded by poxvirus orthologs of the YLDV 2L gene. (a) A schematic of the purification and sequencing strategy used to isolate the TNF-binding activity from TPV-infected cell supernatant. (b) Alignment of the YLDV 2L gene with the N-terminal sequence obtained from the TPV TNF-binding protein (blue) and amino acids 185–238 of murine MHC class I histocompatibility antigen H-2 Q5-k α-chain (red). The predicted signal sequence cleavage site is shown as well as four potential N-linked glycosylation sites.

Figure 2

Alignment of the identified 2L orthologs from other poxviruses. An alignment of the vTNF-BP family members from TPV (TPV-2L), YLDV-2L, YMTV (YMTV-2L), and the two copies from SPV (SPV003 and SPV148). The blackened boxes denote conserved cysteine residues.

Figure 3

2L is produced and secreted during TPV and YMTV infections. (a) Supernatants from AcTPV-2L-infected SF-21 cells, TPV-infected OMK cells, or YMTV-infected CV1 cells were concentrated 10-fold, and the proteins were separated on a polyacrylamide gel. Proteins then were transferred to a nitrocellulose membrane that subsequently was probed with rabbit polyclonal antibodies raised against the TPV-2Lmyc/His protein. (b) Supernatants from AcTPV-2L-infected SF-21 cells were passed over a human TNF column. Proteins specifically binding to the column were eluted in 0.2 M acetic acid and subsequently separated on a polyacrylamide gel and visualized by Coomassie blue staining.

Figure 4

TPV-2L binds to human TNF with high affinity but not murine TNF or human IL-2, IL-5, or LT-α. TPV-2L was immobilized on one flow cell of a CM-5 BIAcore sensor chip whereas the other flow cell acted as a blank control surface. Over the TPV-2L sensor chip was passed 100 μl of human TNF, IL-2, IL-5, LT-α, or murine TNF (a) or duplicate injections of 0.3, 0.9, 2.8, or 8.3 nM human TNF at a flow rate of 50 μl/min (b). The amount of protein bound to the surface was recorded as response units as a function of time. After completion of the injection phase (arrow), the dissociation phase was monitored during the injection of buffer alone (HBS-EP). The sensorgrams shown were normalized by subtracting the control surface sensorgram and a blank injection sensorgram. The binding kinetics were determined by using BIAEVALUATION 3.0 and a 1:1 mass transport model to determine the rate of association (K_on), the rate of dissociation (K_off), and the rate constant (K_d).

Figure 5

TPV-2L inhibits TNF binding to both TNFRI and TNFRII. The binding of a fixed concentration of Flag-tagged human TNF (hTNF) (solid symbols) to human TNFRI (hTNFR1) (■ and □) or human TNFRII (hTNFR2) (● and ○) was inhibited by increasing amounts of TPV-2L. In contrast, binding of Flag-tagged murine TNF (muTNF) (open symbols) was unaffected by TPV-2L.

Figure 6

TPV-2L inhibits human but not murine or rabbit TNF. L929 cells were treated with actinomycin D along with 11 pM, 110 pM, or 1.1 nM of either human (a) or murine (b) TNF to induce apoptosis. The cells then were treated immediately with PBS (mock), 3.5 nM purified myxoma T7 protein (mT7), or 3.5 nM purified TPV-2L. (c) L929 cells were treated with 0, 0.001, 0.01, or 0.1 μl of supernatants from wild-type vaccinia virus Western Reserve strain (mock) or vaccinia virus expressing rabbit TNF in the presence (rTNF+TPV-2L) or absence (rTNF) of 3.5 nM TPV-2L. All cells then were incubated for 16 h, and cell viability was determined by staining with crystal violet.