Jye-Chian Hsiao

Poxvirus Host Range Protein CP77 Contains an F-Box-Like Domain That Is Necessary To Suppress NF-κB Activation by Tumor Necrosis Factor Alpha but Is Independent of Its Host Range Function

ABSTRACT Tumor necrosis factor alpha (TNF-α) activates the nuclear factor κB (NF-κB) signaling pathway that regulates expression of many cellular factors playing important roles in innate immune responses and inflammation in infected hosts. Poxviruses employ many strategies to inhibit NF-κB activation in cells. In this report, we describe a poxvirus host range protein, CP77, which blocked NF-κB activation by TNF-α. Immunofluorescence analyses revealed that nuclear translocation of NF-κB subunit p65 protein in TNF-α-treated HeLa cells was blocked by CP77. CP77 did so without blocking IκBα phosphorylation, suggesting that upstream kinase activation was not affected by CP77. Using GST pull-down, we showed that CP77 bound to the NF-κB subunit p65 through the N-terminal six-ankyrin-repeat region in vitro. CP77 also bound to Cullin-1 and Skp1 of the SCF complex through a C-terminal 13-amino-acid F-box-like sequence. Both regions of CP77 are required to block NF-κB activation. We thus propose a model in which poxvirus CP77 suppresses NF-κB activation by two interactions: the C-terminal F-box of CP77 binding to the SCF complex and the N-terminal six ankyrins binding to the NF-κB subunit p65. In this way, CP77 attenuates innate immune response signaling in cells. Finally, we expressed CP77 or a CP77 F-box deletion protein from a vaccinia virus host range mutant (VV-hr-GFP) and showed that either protein was able to rescue the host range defect, illustrating that the F-box region, which is important for NF-κB modulation and binding to SCF complex, is not required for CP77s host range function. Consistently, knocking down the protein level of NF-κB did not relieve the growth restriction of VV-hr-GFP in HeLa cells.

Poxviral host range protein CP77 contains a F-box-like domain that is necessary to suppress NF-κB activation by TNF-α but is independent of its host range function

ABSTRACT Tumor necrosis factor alpha (TNF-α) activates the nuclear factor κB (NF-κB) signaling pathway that regulates expression of many cellular factors playing important roles in innate immune responses and inflammation in infected hosts. Poxviruses employ many strategies to inhibit NF-κB activation in cells. In this report, we describe a poxvirus host range protein, CP77, which blocked NF-κB activation by TNF-α. Immunofluorescence analyses revealed that nuclear translocation of NF-κB subunit p65 protein in TNF-α-treated HeLa cells was blocked by CP77. CP77 did so without blocking IκBα phosphorylation, suggesting that upstream kinase activation was not affected by CP77. Using GST pull-down, we showed that CP77 bound to the NF-κB subunit p65 through the N-terminal six-ankyrin-repeat region in vitro. CP77 also bound to Cullin-1 and Skp1 of the SCF complex through a C-terminal 13-amino-acid F-box-like sequence. Both regions of CP77 are required to block NF-κB activation. We thus propose a model in which poxvirus CP77 suppresses NF-κB activation by two interactions: the C-terminal F-box of CP77 binding to the SCF complex and the N-terminal six ankyrins binding to the NF-κB subunit p65. In this way, CP77 attenuates innate immune response signaling in cells. Finally, we expressed CP77 or a CP77 F-box deletion protein from a vaccinia virus host range mutant (VV-hr-GFP) and showed that either protein was able to rescue the host range defect, illustrating that the F-box region, which is important for NF-κB modulation and binding to SCF complex, is not required for CP77s host range function. Consistently, knocking down the protein level of NF-κB did not relieve the growth restriction of VV-hr-GFP in HeLa cells.

A turn-like structure "KKPE" segment mediates the specific binding of viral protein A27 to heparin and heparan sulfate on cell surfaces.

Vaccinia viral envelope protein A27 (110 amino acids) specifically interacts with heparin (HP) or heparan sulfate (HS) proteoglycans for cell surface attachment. To examine the binding mechanism, a truncated soluble form of A27 (sA27-aa; residues 21–84 of A27) with Cys71 and Cys72 mutated to Ala was used as the parent molecule. sA27-aa consists of two structurally distinct domains, a flexible Arg/Lys-rich heparin-binding site (HBS) (residues 21–32; 21STKAAKKPEAKR32) and a rigid coiled-coil domain (residues 43–84), both essential for the specific binding. As shown by surface plasmon resonance (SPR), the binding affinity of sA27-aa for HP (KA = 1.25 × 108 m−1) was approximately 3 orders of magnitude stronger than that for nonspecific binding, such as to chondroitin sulfate (KA = 1.65 × 105 m−1). Using site-directed mutagenesis of HBS and solution NMR, we identified a “KKPE” segment with a turn-like conformation that mediates specific HP binding. In addition, a double mutant T22K/A25K in which the KKPE segment remained intact showed an extremely high affinity for HP (KA = 1.9 × 1011 m−1). Importantly, T22K/A25K retained the binding specificity for HP and HS but not chondroitin sulfate, as shown by in vitro SPR and in vivo cell adhesion and competitive binding assays. Molecular modeling of the HBS was performed by dynamics simulations and provides an explanation of the specific binding mechanism in good agreement with the site-directed mutagenesis and SPR results. We conclude that a turn-like structure introduced by the KKPE segment in vaccinia viral envelope protein A27 is responsible for its specific binding to HP and to HS on cell surfaces.

Structural analysis of the extracellular domain of vaccinia virus envelope protein, A27L, by NMR and CD spectroscopy

This study presents the molecular structure of the extracellular domain of vaccinia virus envelope protein, A27L, determined by NMR and CD spectroscopy. A recombinant protein, eA27L-aa, containing this domain in which cysteines 71 and 72 were replaced with alanine, was constructed to prevent self-assembly due to intermolecular disulfide bonds between these two cysteines. The soluble eA27L-aa protein forms an oligomer resembling that of A27L on vaccinia virions. Heteronuclear correlation NMR spectroscopy was carried out on eA27L-aa in the presence or absence of urea to determine backbone resonance assignments. Chemical shift index (CSI) propensity analysis showed that eA27L-aa has two distinct structural domains, a relatively flexible 22-amino acid random coil in the N-terminal region and a fairly rigid α-helix structure in the remainder of the structure. Binding interaction studies using isothermal titration calorimetry suggest that a 12-amino acid lysine/arginine-rich segment in the N-terminal region is responsible for glycosaminoglycan binding. The rigid α-helix portion of eA27L-aa is probably involved in the intrinsic self-assembly, and CSI propensity analysis suggests that region N37-E49, with a residual α-helix tendency, is probably the self-assembly core. Self-assembly was ascribed to three hydrophobic leucine residues (Leu41, Leu45, and Leu48) in this segment. The folding mechanism of eA27L-aa was analyzed by CD spectroscopy, which revealed a two-step transition with a Gibbs free energy of 2.5 kcal/mol in the absence of urea. Based on these NMR and CD studies, a residue-specific molecular model of the extracellular domain of A27L is proposed. These studies on the molecular structure of eA27L-aa will help in understanding how vaccinia virus enters cells.

Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

ABSTRACT Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm.

Vaccinia Viral Protein A27 Is Anchored to the Viral Membrane via a Cooperative Interaction with Viral Membrane Protein A17

Background: Vaccinia viral protein A27 associates with viral membrane protein A17 for anchoring to the viral membrane. Results: A27 specifically interacts with two binding regions within the N-terminal domain of A17. Conclusion: The A27-A17 interaction is mediated through a specific and cooperative binding mechanism. Significance: As demonstrated, the F1 and F2 bindings are critical for A27 anchoring to the viral membrane and virion assembly. The vaccinia viral protein A27 in mature viruses specifically interacts with heparan sulfate for cell surface attachment. In addition, A27 associates with the viral membrane protein A17 to anchor to the viral membrane; however, the specific interaction between A27 and A17 remains largely unclear. To uncover the active binding sites and the underlying binding mechanism, we expressed and purified the N-terminal (18–50 residues) and C-terminal (162–203 residues) fragments of A17, which are denoted A17-N and A17-C. Through surface plasmon resonance, the binding affinity of A27/A17-N (KA = 3.40 × 108 m−1) was determined to be approximately 3 orders of magnitude stronger than that of A27/A17-C (KA = 3.40 × 105 m−1), indicating that A27 prefers to interact with A17-N rather than A17-C. Despite the disordered nature of A17-N, the A27-A17 interaction is mediated by a specific and cooperative binding mechanism that includes two active binding sites, namely 32SFMPK36 (denoted as F1 binding) and 20LDKDLFTEEQ29 (F2). Further analysis showed that F1 has stronger binding affinity and is more resistant to acidic conditions than is F2. Furthermore, A27 mutant proteins that retained partial activity to interact with the F1 and F2 sites of the A17 protein were packaged into mature virus particles at a reduced level, demonstrating that the F1/F2 interaction plays a critical role in vivo. Using these results in combination with site-directed mutagenesis data, we established a computer model to explain the specific A27-A17 binding mechanism.