Viral calciomics: interplays between Ca2+ and virus

Abstract

Ca(2+) is one of the most universal and versatile signaling molecules and is involved in almost every aspect of cellular processes. Viruses are adept at utilizing the universal Ca(2+) signal to create a tailored cellular environment that meets their own demands. This review summarizes most of the known mechanisms by which viruses perturb Ca(2+) homeostasis and utilize Ca(2+) and cellular Ca(2+)-binding proteins to their benefit in their replication cycles. Ca(2+) plays important roles in virion structure formation, virus entry, viral gene expression, posttranslational processing of viral proteins and virion maturation and release. As part of the review, we introduce an algorithm to identify linear "EF-hand" Ca(2+)-binding motifs which resulted in the prediction of a total of 93 previously unrecognized Ca(2+)-binding motifs in virus proteins. Many of these proteins are nonstructural proteins, a class of proteins among which Ca(2+) interactions had not been formerly appreciated. The presence of linear Ca(2+)-binding motifs in viral proteins enlarges the spectrum of Ca(2+)-virus interplay and expands the total scenario of viral calciomics.

Fig. 1

The choreography of Ca²⁺ signaling and examples of virus-induced perturbations on Ca²⁺ homeostasis. Upon extracellular stimulation, the free cytosolic Ca²⁺ ([Ca²⁺]_CYT) rapidly increases by the entry of extracellular Ca²⁺ across the plasma membrane via Ca²⁺ channels, such as voltage-operated channels (VOC), receptor-operated channels (ROC), transient receptor potential ion-channel (TRP) and store-operated channels (SOC), or by the release of Ca²⁺ from internal stores (e.g., endoplasmic reticulum (ER), Golgi complex, and lysosomes) through inositol-1,4,5-triphosphate receptors (IP₃R) and ryanodine receptors (RyR) due to activation of membrane receptors (G protein coupled receptor [GPCR] and receptor tyrosine kinase [RTK]) and the subsequent synthesis of IP₃. At the resting state, [Ca²⁺]_CYT is maintained at submicromolar range by extruding Ca²⁺ outside of the plasma membrane via plasma membrane Ca²⁺-ATPase (PMCA) and Na²⁺/Ca²⁺ exchanger (NCX), or by pumping Ca²⁺ back into internal stores through sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) or secretory pathway Ca²⁺-ATPase (SPCA). In mitochondria, Ca²⁺ can easily pass through outer mitochondrial membrane pores and cross the inner mitochondrial membrane through the membrane-embedded Ca²⁺ uniporter. Ca²⁺ exits mitochondria through the opening of a non-selective high-conductance channel permeability transition pore (PTP) in the inner mitochondrial membrane and the Na⁺/Ca²⁺ exchanger (NCX). The Ca²⁺ signals are delivered by affecting the activity of Ca²⁺ buffers (e.g., calreticulin), Ca²⁺ effectors (e.g., CaM and S100A10) and Ca²⁺-regulated enzymes. The signals can also have “long-term” effects by modulating the activity of transcriptional factors including nuclear factor of activated T cells (NFAT) or Ca²⁺-dependent transcriptional cotransactivators (e.g., p300). Viral proteins are capable of perturbing the intracellular Ca²⁺ homeostasis by (1) modulating Ca²⁺ pumps and/or channels on the plasma membrane (e.g., Tat and gp120 of HIV-1, HBx of HBV; blue boxes); (2) triggering Ca²⁺ release from internal stores via IP₃R (e.g., NSP4 of rotavirus and Nef of HIV; purple boxes) or altering membrane permeability and pump activity of internal stores (e.g., 2B of coxsakievirus, p7 of HCV, and core protein of HCV; purple boxes,); (3) disrupting mitochondrial membrane permeabilization or potential (e.g., Vpr of HIV-1, p13^II of HTLV-1, core protein of HCV and HBx of HBV; lemon boxes); (4) activating Ca²⁺-responsive transcriptional factors or coactivators, such as p300 (e.g., p12^I of HTLV-1 and Vpr of HIV-1; yellow box) in the nucleus. Moreover, a variety of viral proteins interact with important cellular CaBPs, such as CaM, S100A10 and calreticulin, to remodel the Ca²⁺ signaling network. The gradient color bar on the left indicates free Ca²⁺ concentration in subcellular compartments. Calcium ions are shown as cyan dots.

Fig. 2

Virus infection selectively perturbs pro-apoptotic and pro-survival ER-mitochondria Ca²⁺ signaling. Upon engagement of receptors, the production of IP₃ leads to the activation of IP₃R and release of Ca²⁺ from internal store. The decrease of ER Ca²⁺ concentration is subsequently sensed by the EF-hand-containing ER Ca²⁺ sensor STIM1, which in turn activates the CRAC channels through its direct interaction with the pore-forming subunit Orai1, followed by a second phase of intracellular Ca²⁺ elevation (store-operated Ca²⁺ entry; high frequency cytosolic Ca²⁺ oscillation with low amplitude) and further activation of downstream effectors, including calcineurin, NFAT and NFAT-dependent gene expression (e.g., Bcl-2 and IL-2) , . Overexpression of anti-apoptotic Bcl-2 family members (e.g., Bcl-2) inhibits Ca²⁺ release from ER and thus exerts anti-apoptotic effects to promote survival. NFAT can be activated by viral proteins (e.g., HIV-1 Nef , HTLV-1 p12^I, , HCV core protein , , and HHV-8 K1 , HBV HBx , ; red ovals) to enhance virus replication or to establish persistent infection. It is of great interest to test whether viral proteins (red oval with question mark) can directly target SOC components (STIM1 and Orai1) to affect NFAT activation and ER Ca²⁺ store refilling. A decrease in [Ca²⁺]_ER and [Ca²⁺]_Golgi (for example, induced by viroporin, 2B of Coxsakievirus; red cylinder highlighted in yellow) may inhibit protein trafficking pathways and cause intracellular accumulation of ER or Golgi-derived secretory vesicles, at which viral RNA replication takes place , . Ca²⁺ released from ER can be readily taken up by mitochondria at locations in close proximity to the ER extruding pores (so-called ER-mitochondria Ca²⁺ flux). Abnormal exodus of ER Ca²⁺ could result in Ca²⁺ overloading in mitochondria. Mitochondrial Ca²⁺ uptake is mediated by the mitochondrial voltage-dependent anion channel (VDAC) across outer mitochondrial membrane (OMM) and the Ca²⁺ uniporter of inner mitochondrial membrane (IMM). Ca²⁺ exits mitochondria through the opening of a non-selective high-conductance channel permeability transition pore (PTP) in IMM and the Na⁺/Ca²⁺ exchanger (NCX) , . During virus infection, viral proteins may readily target mitochondria and exert either pro-apoptotic or anti-apoptotic action by altering mitochondrial Ca²⁺ levels. A modest increase of ER-mitochondria Ca²⁺ flux may boost ATP production by activating Ca²⁺-dependent Kreb's cycle dehydrogenases, thereby meeting higher energy demand due to active viral replication (e.g., HCV and CMV [19]). However, Ca²⁺ overloading in mitochondria exerts pro-apoptotic effects. It activates the opening of PTP, causes release of cytochrome c (purple stars) and activation of caspase 9, and eventually commits cells to apoptosis . Virus infection may generate either extrinsic or intrinsic apoptotic stimulus. For example, HIV-1 Tat-induced overexpression of tumor necrosis factor (TNF) and HCV core and NS5A-induced elevation of reactive oxygen species (ROS) may exacerbate mitochondrial Ca²⁺ overloading and cause apoptosis in infected cells. Intracellularly, abnormal increase of mitochondria Ca²⁺ uptake (e.g., HCV core protein , ; red oval) and disruption of IMM permeability (e.g., HIV-1 Vpr , HTLV-1 p13^II and HBV HBx ; red ovals or cylinder) may also lead to apoptotic cell death. Viral proteins induce apoptosis to facilitate virion release and maximize virus dissemination at a later stage of viral life cycle. In contrast, actions that downregulate ER-mitochondria Ca²⁺ flux (e.g., pore-formation on ER by Coxsakievirus 2B [21], [24]) or recruit pro-apoptotic protein (e.g., Bax) within mitochondria (e.g., CMV vMIA [26], [27]), may protect host cells from apoptosis and promote cellular survival signaling . In general, an anti-apoptotic strategy is employed by virus to aid virus replication and effective immune evasion in early or middle stages of infection. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 3

Examples of viral Ca²⁺-binding proteins. (A) 3D representation of the icosahedral asymmetric unit of the cocksfoot mottle virus capsid and the location of the incorporated Ca²⁺ ions (PDB code: 1ng0) . The assembling unit is formed by three subunits, A (blue), B (green) and C (red) that are chemically identical but slightly different in conformational arrangement. Ca²⁺, situated between the interfaces of neighboring subunits (A–B, A–C or B–C), is coordinated by oxygen atoms from the side chains of D136 and D139 in one subunit and oxygen atoms from the main chain of L196, the side chain of N252, and the C-terminal carboxyl group of L253 in the other neighboring subunit (enlarged area). The solid pentagon, triangle and oval represent 5-, 3-, and 2-fold axes of the icosahedron. (B) Ca²⁺ ion located on the 5-fold axis of the capsid of human rhinovirus 3 (HRV3) (PDB code: 1rhi) . The icosahedral capsid of HRV3 is composed of 60 copies of each of the four capsid proteins VP1 (blue), VP2 (green), VP3 (red) and VP4 (black). VP1, VP2 and VP3 are exposed to the external surface of the viral particle, whereas VP4 lies in the internal surface. A Ca²⁺ ion is found situated on the 5-fold axis of the capsid and coordinated by 5 oxygen atoms from the main chain carbonyl group of the 5-fold symmetry-related S1141 on VP1 (enlarged area). With two additional oxygen atoms from water molecules above or below the metal ion as coordinating ligands, the Ca²⁺-binding pocket forms a pentagonal bipyramidal geometry. (C) Overlay of 3D structure of the virion protein VP2 of the single-stranded DNA-containing parvoviruses, feline panleukopenia virus (FPV; PDB code: 1C8F; green) and canine parvorius (CPV; PDB code: 1C8D; magenta). The VP2 proteins of FPV and CPV bind three and two Ca²⁺ ions, respectively, at pH 7.5 (Fig. 2C). Both FPV and CPV contain pH-dependent dual Ca²⁺-binding sites (sites 2 and 3; spheres with mixed green and magenta colors), in which the metal ions are separated by 4.6 Å. The most striking structural difference between VP2 of these two viruses is within a flexible surface loop located within residues 359–375. This loop forms a third Ca²⁺-binding site in FPV (site 1; green sphere), but not in CPV since D375 is replaced by N375 in CPV. This unique Ca²⁺-binding site in FPV VP2 is coordinated by oxygen atoms from the side chain of residues D373 and D375, as well as the main chain carbonyl groups of residues R361 and G362. The binding of Ca²⁺ to this particular site is speculated to cause conformational changes in VP2 and may influence the host range of these two related, but different, parvoviruses. (D) The 3D structure of neuraminidase of influenza B virus (PDB code: 1nsb) . The cartoon only represents half of the tetrameric form of this enzyme. Three Ca²⁺-binding sites are found in two identical subunits, A (blue) and B (red). Each subunit contains one octahedral Ca²⁺-binding site (upper panel) that involves residues D292, T296, D232, G343, G345 and one additional water molecule. Another site (lower panel), coordinated by the 4-fold symmetry-related E167, holds the oligomer together. (E), The core Ca²⁺-binding pocket in the oligomerization domain (aa. 95–137) of NSP4 from rotavirus (PDB code: 2o1j) . The domain self-assembles into a paralleled tetrameric coiled-coil. Chains A, B, C and D are shown in blue, green, orange and red, respectively. The Ca²⁺ ion is coordinated by six oxygen atoms from the side chains of Q123 on chains A to D, as well as the side chains of E120 on chains B and D. Calcium ions are shown as cyan spheres. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 4

The helix-loop-helix EF-hand Ca²⁺-binding motif. (A) Cartoon illustration of the canonical EF-hand Ca²⁺-binding motif. The EF-hand motif contains a 29-residue helix-loop-helix topology, much like the spread thumb and forefinger of the human hand. Ca²⁺ is coordinated by ligands within the 12-residue loop, including seven oxygen atoms from the sidechain carboxyl or hydroxyl groups (loop sequence positions 1, 3, 5, 12), a main chain carbonyl group (position 7), and a bridged water (via position 9). Residue at position 12 serves as a bidentate ligand. The Ca²⁺-binding pocket adopts a pentagonal bipyramidal geometry. n stands for hydrophobic residue. (B) HMM logo for the canonical EF-hand motif (http://pfam.sanger.ac.uk/family?acc=PF00036). The conservation of amino acids at several positions makes it possible to predict EF-hand motifs from genomic sequences. (C) 3D structure of a typical canonical EF-hand motif from calmodulin (PDB code: 3cln). Ca²⁺ is chelated by ligands from a 12-residue loop. (D), 3D structure of an EF-hand-like motif from a soluble fragment of lytic transglycosylase B of Escherichia coli (PDB code: 1qut). This motif contains a 15-residue (instead of 12-residue) Ca²⁺-binding loop flanked by two helices.

Fig. 5

Experimental approaches to validate predicted continuous Ca²⁺-binding motifs and to correlate its biological relevance. Continuous Ca²⁺-binding sites can be predicted either from the primary sequence by using the program CaPS or from 3D modeled structure by using GG and MUG algorithm (available at http://chemistry.gsu.edu/faculty/Yang/Calciomics.htm). As first screen, the predicted Ca²⁺-binding sequences are inserted into a scaffold protein CD2 using the grafting approach. This approach allows one to conveniently monitor the metal binding process with fluorescence spectroscopy by taking advantage of the aromatic residue (W32 in CD2) sensitized Tb³⁺ luminescence resonance transfer (Tb³⁺-LRET). Ca²⁺ competition assay can be further performed based on Tb³⁺-LRET to obtain Ca²⁺-binding affinity. If metal binds to the engineered protein, mutations on the proposed ligand residues will be subsequently introduced to double confirm the binding event. If the mutagenesis results in a decrease in binding, the predicted site has a high chance of binding Ca²⁺. Possible Ca²⁺-induced conformational changes will be examined by expressing the protein of interest. In addition, the functional correlation of Ca²⁺ binding can be followed by comparing the phenotypes of WT and mutant viruses.