**1. Introduction**

Viruses exploit the environment of host cells to replicate, thereby inducing host cells' dysfunction. Virus–host interaction is the foundation of pathogenesis and closely associated with disease severity and incidence. The prevention and therapy of virus infections are often confounded by the high mutation rates that facilitate the viral evasion of antiviral strategies that target virally encoded proteins. Modulations of the intracellular environment have become an important strategy in antiviral drug discovery and development. In mammalian cells, Ca2+, as an important second messenger, mediates the sensor input and responses output for almost all known cellular progress, such as stress responses, synaptic plasticity, immunodefenses, protein transport, and endosome formation [1,2]. It has been demonstrated that the host cell dysfunction following infection with a virus is accompanied by abnormal intracellular Ca2+ concentration [3]. A virus can hijack the host intracellular Ca2+ system to achieve successful replication via multiple routes; for instance, viral proteins directly bind to Ca2+ or disturb the membrane permeability for Ca2+ by manipulating Ca2+ apparatus.

The host cell plasma membrane is the first barrier against the invasion of viruses. Various Ca2+ channels and pumps are distributed on the cell plasma membrane. Therefore, these membrane proteins become the direct target of virus infection. Interaction between viruses and these membrane proteins is the foremost approach of viruses perturbing the host cell calcium signal system. This interaction may inhibit or stimulate calcium influx and modulate free cytosolic Ca2+ concentrations. After entry into the host cell, viruses stimulate or inhibit the calcium release from internal stores via an effect on calcium-permeable channels, transporters, and exchangers on organellar membranes. Then, the change in cytosolic calcium concentration may trigger further distortion of the host cell system, which benefits virus survival and replication.

This review concentrates on host cell membranes' calcium channels and pumps in viral infection. Blockers for these membrane proteins or preventing viruses from grabbing these host calcium-signaling components may lower the probability of virus stability, replication, and release, as well as infection-related host–cell apoptosis and reactive oxygen species production, neurotoxicity, and enterotoxin, making these membrane proteins potential targets for antiviral drugs.

### **2. Calcium Channels and Pumps in Host Ca2**<sup>+</sup> **Homeostasis**

Cellular Ca2+ is from two major sources: the internal Ca2+ store (mainly endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR)) and the extracellular medium. Calcium channels on cell plasma membrane mediate the entry of Ca2+ from the extracellular medium. These channels are activated by specific stimuli, such as voltage-gated calcium channels (VGCCs), which are stimulated by membrane depolarization, specific receptor-operated channels (ROC), which are stimulated by external agonists, or intracellular messengers and store-operated calcium channel (SOC), which are stimulated by the depletion of internal Ca2+ stores. The IP3 receptor (IP3R) and the ryanodine receptors (RyR) are the main players in mediating the release of Ca2+ from the internal stores. Inositol-1,4,5-triphosphate (IP3) activates IP3R, triggers Ca2+ release from stores, and further increases IP3R's sensitivity to Ca2+. Calcium pumps (the plasma membrane Ca2<sup>+</sup>-ATPase (PMCA), sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)) and the Na+/Ca2<sup>+</sup> exchanger (NCX) are responsible for transporting Ca2+ from the cytosol to external medium or into cellular calcium stores (Figure 1). The normal function of these calcium channels and pump is important for cells to maintain intracellular Ca2+ homeostasis.

**Figure 1.** Schematics of host cell elevated cytosolic calcium concentration induced by a virus. Calcium channels (voltage-gated calcium channels (VGCCs), receptor-operated channels (ROC), store-operated Ca2+ (SOC), channels and transient receptor potential (TRP) channels) mediate the entry of Ca2+ from extracellular medium (black arrows). The IP3 receptor (IP3R) and the ryanodine receptors (RyR) on the endoplasmic reticulum (ER) mediate the release of Ca2+ from internal stores (black arrows). Calcium pumps (the plasma membrane Ca2<sup>+</sup>-ATPase (PMCA), sarcoplasmic/endoplasmic reticulum Ca2<sup>+</sup>-ATPase (SERCA)) and the Na+/Ca2<sup>+</sup> exchanger (NCX) are responsible for transporting Ca2+ from the cytosol to external medium or into cellular calcium stores (red arrows). Viruses utilize these calcium components to elevate cytosolic calcium concentration to activate Ca2<sup>+</sup>-dependent/sensitive enzymes and transcriptional factors to promote virus replication (right panel).

These channels and pumps are activated in a flexible and precise manner to generate specific Ca2+ signaling, satisfying various spatiotemporal requirements. During the viral infections, host cells modulate these calcium-signaling components in response to the infection. On the other hand, viruses utilize these components to create a cellular environment that benefits their own lifecycles [4]. Viruses induce elevated cytosolic calcium concentration to activate Ca2+ dependent/sensitive enzymes and transcriptional factors to promote virus replication. Mitochondria could take the Ca2+ to generate more energy to support continuous virus replication. Moreover, regulating the calcium concentration in ER or Golgi may inhibit host proteins tra fficking and promote virus protein maturation. The inhibition of host proteins frustrates host antiviral immune responses, while the promotion of virus protein maturation benefits virus propagation (Figure 1). The journey begins when a virus encounters the host cell and attaches to the cell surface. The virus particle penetrates the cytoplasm via direct membrane fusion or receptor-mediated endocytosis followed by exposing its genome to cellular machinery for replication. When the viral proteins and viral genomes are accumulated, they are assembled to form a progeny virion particle and then released [5]. During the viral lifecycles, they utilize various calcium channels and pumps to obviate the cell membrane barriers, enter the host cell, complete replication, acquire infection ability, and release.

### **3. Viruses Control Host Voltage-Gated Calcium Channels (VGCCs) and Two-Pore Channels (TPCs)**

VGCCs are widely found in the membrane of excitable cells [6] and one of the most well studied viral targets because of the availability of specific channel blockers. There are several di fferent types of VGCCs: L-type (Long-conducting) channels are mostly found in skeletal and smooth myocytes, bone (osteoblasts), and ventricular myocytes; N-type (Non-L or Neuronal), P/Q-type (Purkinje and Granular), and R-type (dihydropyridine- Resistant) channels display longer-lasting conductance and are expressed throughout the nervous system; T-type (Transient) channels produce short-term conductance and are found in neurons and cells that have pacemaker activity [6]. The activation of the channels results in a Ca2+ cascade, which is associated with numerous host physiological functions including excitation–contraction–relaxation coupling of muscles, synaptic transmission, immunoprotection, etc.

Since 1984, it has been known that verapamil, the blocker of VGCCs, inhibits influenza A virus (IAV) infection [7]. Moreover, IAV infection induces Ca2+ influx, and the elevated intracellular Ca2+ promotes endocytic uptake of IAV [8]. Until recently, the underlying mechanism was revealed by Fujioka et al. [9]. They reported that several VGCC blockers and siRNA against the CaV1.2 channel (L-type channel) inhibited H1N1 and H3N2 IAV infection in multiple cell lines. IAV attaches to the target cells by the viral hemagglutinin protein (HA), which is a type I transmembrane protein embedded in the viral envelope, binding to sialic acids [10]. Fujioka et al. showed that the virus HA binds to domain IV of CaV1.2, which contains two potential sialylated asparagine residues (N1436 and N1487) [9,11]. When CaV1.2 mutants in one or both these residues were replaced with glutamine (N1436Q, N1487Q, and N1436Q + N1487Q), the interaction between HA and the Cav1.2 was attenuated compared with the wild-type fragment. They demonstrated that a VGCC blocker, diltiazem, significantly prolonged the survival of IAV-infected mice and allowed the recovery of the survivors. Therefore, CaV1.2 may serve as a host cell surface receptor that binds IAV and is critical for IAV entry (Figure 2A). New world hemorrhagic fever arenaviruses (NWA) are another virus that is reportedly sensitive to the VGCC blocker club member [12,13]. An siRNA screen with Junín virus glycoprotein-pseudotyped viruses identified that VGCCs are involved in NWA entry. Treatment with the channel blocker gabapentin, an FDA (U.S. Food and Drug Administration)-approved analgesic that targets the channel α2δ2 subunit, protects against NWA infection [13]. This research work demonstrated that the interaction between a virus and VGCCs promotes virus entry at the virus-cell fusion step.

**Figure 2.** Examples of viruses interplaying with host calcium channels or pumps to achieve viral entry (**A**) and release (**B**). VGCCs are important for influenza A virus (IAV) and severe fever with thrombocytopenia syndrome virus (SFTSV) entry into the host cell as well as TPC1/2 for EBOV (**A**). RSV, Zika virus (ZIKA), dengue virus (DENV) and West Nile virus (WNV) hijack SPCA1 to facilitate their release as well as Ebola virus (EBOV), MARV, LASV, JUNV, HIV-1 and DENV manipulates STIM1/ORAI1 (**B**). For a complete list of definitions, see Table 1.

The effect on VGCCs is not only restricted to virus entry. A high-throughput screening of an FDA-approved drug library for inhibitors of Japanese encephalitis virus (JEV) identified five hit drugs, three of which are VGCCs blockers (manidipine, cilnidipine, and benidipine hydrochloride) [14]. Recombinant viral particles (RVPs) with the luciferase-reporting replicon were used to quantify the efficiency of JEV replication, which confirmed that these drugs inhibited JEV infection at the stage of replication. These drugs were subsequently validated for their antiviral activities against other flavivirus, such as Zika virus (ZIKV), dengue virus (DENV), and West Nile virus (WNV). Similarly, another research group screened the FDA-approved drug library and found that nifedipine and benidipine hydrochloride inhibited severe fever with thrombocytopenia syndrome virus (SFTSV) replication in vitro [12]. Moreover, the retrospective clinical investigation on SFTS patients showed that nifedipine can significantly inhibit SFTSV infection. These studies indicate that the VGCCs blockers are excellent candidates for broad-spectrum anti-virus treatment. Most of these FDA-approved VGCC blockers are clinically used to treat cardiovascular diseases. Similar to a double-edged sword, the cardiovascular effect of VGCC blockers may limit their antiviral application. Therefore, repurposing these drugs requires more analysis before clinical trials.

Further examples of virus-regulating VGCCs to service their replication can be found in HIV-1 and herpes simplex virus (HSV)-1. Two HIV-1 proteins, the membrane glycoprotein gp120 [15,16] and the transcriptical transactivator Tat [17,18], have been identified to induce the elevation of host intracellular Ca2+ in various cell types, including neuronal, immune, and epithelial cells, via targeting the activity of VGCCs. The detailed information about the dysregulation of the L-type calcium channel by Tat can be found in the review [16,17].

Taking together, the infections of viruses could increase the host's intracellular calcium to facilitate viral entry and replication by manipulating host VGCCs. In addition, herpes simplex virus (HSV)-1 could downregulate the VGCCs on infected neuronal cells to escape detection by host cells. T-type Ca2+ channels were reported as the targets of HSV-1 in sensory-like ND7-23 cells [19,20]. HSV-1 infection of differentiated ND7-23 cells causes a significant loss of T-type Ca2+ channels from the membrane, which depends on viral replication and protein synthesis. This downregulation of T-type Ca2+ channel expression may alter the ability of sensory neurons to transmit pain information. Thus, the lower expression of VGCCs may diminish the detection for viral infection by the host, which benefits virus survival and further infection.

Ebola virus (EBOV) used to be considered a VGCC blocker-sensitive virus, and several research groups independently reported that compounds blocking L-type channels (such as verapamil, nimodipine, and diltiazem) inhibited EBOV infection in vivo [21–23]. However, gabapentin, representing a fifth distinct class of L-type channel inhibitor, had no effect even at high concentrations. It has been shown that verapamil, nimodipine, and diltiazem also inhibit the activity of two-pore channels (TPCs) [24]. TPCs are intracellular voltage-gated and receptor-operated calcium permeable channels, playing an integral role in membrane trafficking pathways [25,26]. Mouse embryonic fibroblasts (MEFs) lacking TPC1 or TPC2 expression (Tpcn1−/<sup>−</sup>, Tpcn2−/−) resisted EBOV infection [27]. It turns out that the target of EBOV may not be classical L-type calcium channels but rather endosomal calcium channels termed TPCs (Figure 2A). The calcium channels inhibitors prevented virus–endosome membrane fusion and virus capsid releasing into the cell cytoplasm, which is a late entry step. EBOV acts on TPCs, which control the movement of endosomes containing virus particles, and thereby facilitate its intracellular trafficking [27,28]. The channel inhibitor, tetrandrine, significantly enhanced the survival of mice challenged with mouse-adapted EBOV without any detectable side effects. This indicates that tetrandrine is highly effective against EBOV disease in mice.

### **4. Store-Operated Calcium (SOC) Channel in Viral Assembly and Egress**

SOC channels are the major Ca2+ entry pathways in non-excitable cells. The protein Orai1 on the plasma membrane and STIM1 (stromal interaction molecule) on ER are the molecular identities that are responsible for SOC channels activation. The depletion of ER Ca2+ stores promotes STIM1 proteins aggregation and interaction with Orai1 to open the channel, mediating Ca2+ entry [29,30].

Most enveloped viruses are released extracellularly via exocytosis, also called budding, as an analogy of buds in plants [31,32]. The budding process of the enveloped viruses is triggered by a peptide motif (termed late (L) domains), which was discovered in the Gag polyproteins of retroviruses and M (matrix) proteins of rhabdoviruses [33]. These L domains interact with cellular proteins to promote the formation of virus vesicles that bud away from the cytoplasm [32,34]. Research works established that this essential final step of related viruses depends on the host Ca2+ signal mediated by SOC channels (STIM1/Orai1).

The research work done on four distinct hemorrhagic fever viruses (Ebolavirus (EBOV), Marburgvirus (MARV), Lassa Virus (LASV), and Junín Virus (JUNV)) demonstrated that EBOV, MARV VP40, and JUNV Z proteins trigger host cell Ca2+ signals by activating the ER Ca2+ sensing protein STIM1 and the plasma membrane ORAI1 channel. The STIM1/Orai1-mediated Ca2+ signal is critical for EBOV and related viruses budding from host cells [35]. The suppression of STIM1 expression

and genetic inactivation or the pharmacological blockade of ORAI1 inhibits infections of EBOV, MARV, and JUNV in cultured cells (Figure 2B). Obviously, the matrix proteins or live virus activates STIM1 and the ORAI channel. Similarly to hemorrhagic fever viruses, the HIV-1 matrix protein Gag, directing HIV-1 budding, and mediating VLP (Virus-like Particle) formation also exhibit dependence on Ca2+ regulation [36,37]. Therefore, further study is needed to validate the role for STIM1 and the ORAI channel in HIV- 1 budding.

Infections of other enveloped RNA viruses that buds in similar mechanisms may also be inhibited by STIM1 and ORAI1 inhibitors. Indeed, SOC channel antagonists significantly reduced DENV yield [38]. When the human hepatic HepG2 and Huh-7 cells are infected by DENV, STIM1 and ORAI1 were shown to be co-localized in infected cells, indicating activation of the SOC channel [38,39]. Therefore, DENV infection alters cell Ca2+ homeostasis possibly via promoting the interaction between STIM1 and ORAI1.

It is possible that the viral proteins trigger the Ca2+ depletion in ER or prevent ER refilled with Ca2+ to maintain resting ER Ca2+ levels [40–42]. Alternately, these viral proteins may directly modify STIM1 and uncouple the activation of STIM1 from Ca2+ levels [35,43]. Thus, STIM1/ORAI might represent a conserved target to regulating the budding of enveloped RNA viruses and possibly DNA viruses that rely on similar host cellular proteins for budding. The example is that the hepatitis B virus X (HBx) protein upregulates the activity of the STIM1–ORAI1 channel complex [44]. The mechanisms by which these viruses activate STIM1 and ORAI1 represent novel therapeutic targets for controlling budding.

Calcium influx through the SOC channel also contributes to the elevated cytosolic calcium concentration induced by rotavirus (RV) infection. It has been well established that the dramatically increased cellular Ca2+ is the hallmark of rotavirus (RV) infection [45,46]. Rotavirus nonstructural protein 4 (NSP4) is an ER-localized viroporin that functionally depletes ER calcium. Thus, in rotavirus-infected cells, STIM1 is constitutively active and colocalizes with the ORAI1 channel. The knockdown of STIM1 or the pharmacological inhibition of SOC channels significantly reduced rotavirus yield, indicating that the SOC channel plays a critical role in the RV lifecycle [47,48].

### **5. Host Transient Receptor Potential (TRP) Channels and Receptor-Operated Calcium (ROC) Channels**

### *5.1. TRP Channels*

The TRP channel is a non-selective cation channel predominately permeable for Ca2+ [49,50]. It is divided into six sub-families according to their amino acid sequence: TRP canonical or classical (TRPC), TRP vanilloid (TRPV), TRP melastatin (TRPM), TRP ankyrin (TRPA), TRP polycystin (TRPP), and TRP mucolipin (TRPML) [50]. They are ubiquitously expressed in different tissues and cell types and are a key player in the regulation of intracellular calcium. Nevertheless, reports about virus control TRP channels are not numerous.

TRPV4 mediates intracellular Ca2+ signals in response to several stimuli, including hypotonic cell swelling, mechanical forces, moderating heat, and UVB radiation [51]. When cells are exposed to the Zika virus or the purified viral envelope protein, TRPV4 mediates Ca2+ influx and drives the nuclear translocation of DEAD-box RNA helicase (DDX3X) [52]. DDX3X is an ATP-dependent RNA helicase from the DEAD-box helicase family and is involved in multiple stages of the RNA metabolism, from transcription to translation [53,54]. Moreover, diverse RNA viruses hijack DDX3X to multiply efficiently. Targeting TRPV4 reduces the infectivity of dengue, hepatitis C, and Zika viruses [52]. Overall, the research work demonstrated the role of TRPV4 in the regulation of DDX3X-dependent control of the RNA metabolism and viral infectivity [52].

The TRPV1, TRPA1, and TRPM8 channels are directly activated by chemical, thermal, and mechanical stimuli. They are potentially associated with respiratory virus-induced airway hypersensitivity [55]. The infection of respiratory viruses, such as respiratory syncytial virus (RSV), measles virus (MV), and human rhinovirus (HRV), was reported to increase the expression of these channels in sensory neurons and human bronchial epithelium cells [56,57]. The increase in TRPA1 and

TRPV1 levels can be mediated by soluble factors induced by infection, whereas TRPM8 requires virus replication [56,57]. These reports may explain the possible mechanism by which respiratory viruses induce cough. Alternatively, the up-regulated TRP channels may be utilized by the respiratory viruses to create a favorable calcium environment. Further investigation is required to determine the possible pathway by which this happens.

### *5.2. N-Methyl-D-Aspartate (NMDA) Receptors*

NMDArs and IP3Rs are the two receptor-operated calcium (ROC) channels reported to be involved in virus–host interactions. NMDAr, which is activated by the endogenously synthesized excitant amino acid, glutamate, is widely expressed throughout the mammalian central nervous system and is particularly enriched in the cerebral cortex and hippocampus.

The Zika virus infection is a "neurodegenerative" disease. Costa and colleagues showed that blockage of the NMDAr channel activity with FDA-approved memantine or other antagonists prevents neuronal cell death and microgliosis induced by Zika in vitro and in vivo, without a ffecting the ability of Zika to replicate in the host [58,59]. It seems that NMDAr mainly contributes to Zika, triggering the neuronal cell death progress. Anyway, the blockade of NMDAr may be a viable treatment for patients at risk for Zika infection-induced neurodegeneration [59]. NMDAr blockade also significantly abrogated neuronal cell death and inflammatory response triggered by JEV infection [60]. Therefore, NMDAr are probably common attack targets of flavivirus, inducing host neuron cell death.
