For infection to occur, the virus must first bind to and penetrate the host plasma membrane to deliver genetic material to the cytoplasm for replication. Enveloped and non-enveloped viruses employ different strategies to achieve the same goal. Viroporins, which are a structural component in enveloped viruses, actively facilitate the viral entry process, such as the influenza virus M2 protein. The influenza virus binds to the cell surface and is internalized into endosomes, and its encoded viroporin M2 is located in the viral lipid bilayer and is critical for infection. M2 acts as a proton-conducting channel in the viral envelope, supporting acidification of the viral interior in the endosome (208, 209). This process drives a series of conformational changes in the viral hemagglutinin (HA) protein, leading to the fusion of the viral envelope with the endosomal membrane and the delivery of nucleoprotein complexes into the cytoplasm (210). Acidification of M2 is also thought to promote the structural rearrangement of viral particles, which is required for the efficient uncoating of viral RNAs in the cytoplasm (45). Relevant studies have shown that the HMPV SH protein also has viroporin activity, which can regulate the fusion protein function during virus infection and may play a role in HMPV entering cells (67).

Non-envelope virions lack viroporins as structural components, and the involvement of such proteins in the process of virus entry into cells has been less well reported. A study found that antiretroviral (ARV) p10 is shown to play a key role in its viral fusion process, and the expression of p10 induces extensive cell-cell fusion in transfected cells. Thus, p10 is not only the first non-enveloped viral protein capable of promoting fusion from within, but also the first non-structural viral protein capable of inducing cell-cell fusion (75). Non-enveloped simian virus 40 (SV40) encodes three proteins with viroporin activity, VP2, VP3, and VP4, which play an important role in the life cycle of SV40. During infection, SV40 binds to the cell surface and is endocytosed into caveolin-coated vesicles (211) and subsequently translocated to the ER, where capsid reorganization and entry of viral particles into the cytoplasm occurs. VP2 and VP3 can integrate into the ER membrane and play an important role in the uncoating stage of the viral genome (78, 79). In addition to triggering viral particle infection, several viroporins encoded by influenza virus and SV40 play other roles in the viral life cycle, as will be discussed later.

After the release of the viral genome, the virus controls cellular proteins and organelles to achieve replication. Studies have shown that rearranged membranes are utilized during viral replication (104), and picornaviruses induce rearrangement of the host cell inner membrane to create structures that serve as functional scaffolds for genome replication (212). One of the most striking morphological changes that can be observed in enterovirus-infected cells is the massive accumulation of small ER and Golgi membrane vesicles in the cytoplasm (213). These vesicles are the site where viral RNA replication occurs. The 2BC protein has been implicated in the accumulation of these vesicles, where mutations that interfere with the pore formation capacity of 2B lead to defects in the early stages of viral RNA replication (213). Suhy et al. found that the poliovirus (PV) 2B protein alters cell membrane permeability and together with the 3A protein induces significant rearrangement of the intracellular membrane (104). PV 2B and 3A viroporins can also inhibit the cellular protein secretion pathway by disassembling the Golgi complex or blocking ER-Golgi transport, leading to the accumulation of membrane vesicles in the cytoplasm (101, 214). In addition, polioviruses harboring mutations in the 3A protein result in a marked reduction in positive-strand RNA synthesis (8). Picornavirus 2B protein also facilitates viral release by increasing the permeability of the plasma membrane, which will be discussed in the next section.

Throughout the replication cycle of coronaviruses, viruses use significant rearrangements of the host membrane for replication, protein expression, assembly, and release. The coronavirus E protein may also promote membrane rearrangement. When expressed alone in BHK-21 cells, mouse hepatitis virus (MHV) E can drive the intracellular formation of ERGIC-derived electron-dense membranes (19). The higher oligomers of infectious bronchitis virus (IBV) E are required for the production of virus-like particles (VLPs), suggesting that this form of protein is involved in the assembly of viral particles (215). Studies have shown that the MHV E protein is palmitoylated after translation, which contributes to the assembly of virions (216). The M, E, and N structural proteins of SARS-CoV are required for efficient assembly, transport, and release of virus-like particles (23). Recombinant CoVs (rCoVs) lacking the E gene (ΔE) exhibit abnormal morphology (217), suggesting that the E protein is involved in the assembly process. The function of the E protein is not to coordinate viral assembly but to induce bending of the viral envelope membrane so that the CoV particles acquire their characteristic spherical shape and morphology. Another important role of the coronavirus E protein is to regulate the pH within the lumen of intracellular organelles (e.g., the Golgi apparatus). For example, expression of the E protein of infectious bronchitis virus increases the pH within the organelle, induces neutralization of the Golgi pH, which results in a protective effect, allows isolation of the IBV spike-in protein from protein hydrolysis, and promotes viral assembly and release (32). Furthermore, the colocalization of ORF 3a with the M and E proteins essential for viral assembly suggests that ORF3a is important in the assembly or budding of SARS-CoV (25).

The rotavirus (RV) NSP4 protein also plays an important role in the viral assembly process. NSP4 manipulates the autophagic membrane trafficking process to take viral protein-containing membranes and transport them to viral replication sites for infectious particle assembly (191). The increase in cytoplasmic Ca²⁺ concentration mediated by NSP4 viroporin activates specific Ca²⁺-inducible signaling pathways to initiate autophagy, which allows the transport of the NSP4 and VP7 proteins to the virion for subsequent viral assembly (192). NSP4 binds to immature particles in the virion and triggers particle budding through these membranes to facilitate the assembly of capsid proteins on the particles to form mature infectious viral particles. Lopez et al. showed that small interfering RNAs that inhibit NSP4 expression in rotavirus-infected cells affect the distribution of other viral proteins, mRNA synthesis, and virion formation for viral RNA replication, suggesting a previously unrecognized function for NSP4 in RV replication (72).

The M2 protein is required for influenza virus assembly and budding. When viral budding begins, M2 is located at the edge of the budozone and induces a negative membrane curvature (218), thereby stabilizing the HA and M-induced positive membrane curvature in the budozone. Experimental results suggest that targeting M2 away from the apical plasma membrane may disrupt influenza virus assembly, budding, and replication (219). Phosphorylation of influenza virus CM2 promotes efficient virus replication (50). Deletion of CM2 results in impaired packaging and uncoating of virus-like particles (VLPs) and recombinant influenza viruses (49). The cytoplasmic structural domain of the BM2 protein performs the function of M1 binding to the viral ribonucleoprotein complex alone at the viral particle budding site (44, 220) and plays an important role in viral assembly. IAV M2 ubiquitination plays an important role in the production of infectious viruses by coordinating the efficient packaging of the viral genome into viral particles and the timing of virus-induced cell death (221). In addition, HCV p7 protein and HIV Vpu protein have also been shown to be involved in their viral assembly and release processes (52, 55, 62–64). The positively charged residue R84 in the dengue virus NS2A protein is essential for both viral RNA synthesis and intracellular viral particle assembly and maturation (60). Lulla et al. explored the role of the human astrovirus X protein in the viral life cycle by knocking it out and showed that the X protein may play a role in viral particle formation and viral release (89).

After assembly into infectious viral particles, viruses are released from host cells by budding (e.g., IAV, coronaviruses) or lysis (e.g., picornavirus, SV40). Alterations in plasma membrane permeability are important for cell lysis and the release of viral progeny (203). Enteroviruses are non-enveloped viruses that require the lysis of host cells to release newly formed virions. The expression of Enterovirus 2B protein disrupts intracellular Ca²⁺ homeostasis by progressively enhancing membrane permeability, leading to increased plasma membrane permeability and ultimately to membrane damage and release of the virus (6). For other viruses that are non-enveloped or are enveloped only by a protein coat, viral release often involves cell membrane perforation. For example, SV40 VP4 facilitates virus release by forming pores of approximately 3 nm in diameter in the host cell membrane to penetrate the membrane (82).

For enveloped viruses, budding and division are typically used to release virus from infected cells, and the mechanisms involved in the release process vary from virus to virus. During influenza virus release, the M2 protein is located at the neck of the budding virion, and the amphiphilic helix inserts into the membrane, inducing bending of the positive membrane, and finally pinching off the budding virion (218, 222). Mutations or deletions in the M2 cytoplasmic tail structural domain (CTD) significantly impair the assembly of viral proteins and genomic fragments into viral particles and viral particle release (218, 223, 224). A recent study found that the reduced hydrophobicity of the 91-94 motif of the IAV M2 protein significantly affected the budding ability of the M2 protein and compromised the bilayer membrane integrity of mutant viruses. It was suggested that the hydrophobic residues of the intracellular domain of the M2 protein play an important role in the release and membrane integrity of influenza virus (225). Several studies have shown that the coronavirus E protein is important for viral particle release. IBV E proteins have been shown to function in the secretory pathway, altering the luminal environment and rearranging secretory organelles, ultimately facilitating the efficient transport of viral particles (29–32). The E protein targets the Golgi apparatus and directs the release of virus-like particles (28). Mutations introduced into the HD of MHV or IBV affect the release of infectious particles from the cell (20, 29, 31). These findings suggest that IBV and MHV E viroporins may mediate viral release through their ion channel activity. In addition, Lu et al. found a significant reduction in viral release in cells transfected with ORF3a-specific siRNA (24), suggesting that the 3a protein may act as an ion channel promoting the viral release, but the mechanism by which the 3a protein affects virus release requires further study.

Unlike other viruses that mature in the lumen of the ER, RV is not released via the classical cytosolic pathway. Instead, recent data suggest that this virus may be transported from the ER directly through vesicles to the apical plasma membrane of polarized epithelial cells without passing through the Golgi complex. RV NSP4 interacts with immature particles to trigger outgrowth and synthesis into ER transmembrane proteins, and this rotavirus “budding” maturation process occurs through autophagy-hijacked COPII vesicle membranes (226). BTV NS3 protein of the same family as rotaviruses may function as the membrane protein of enveloped viruses, responsible for intracellular trafficking and budding of viral particles. Therefore, the NS3 protein is considered to act as a bridge between mature virions and cellular proteins during viral shedding (227). Recent studies have found that filovirus delta peptides function as viroporin to enhance the release of viral particles across host cell membranes (88). In addition, other viroporins such as alphavirus 6K protein, HCV p7 protein, HIV Vpu protein also play an important role in the virus release process (36, 55, 228) ( Table 1 and Figure 2 ).

Induction of changes in cell membrane permeability is a key function of viroporins (57, 83, 206, 207, 232). In addition to this, many virus-encoded viroporins can allow different ions (e.g., H⁺ and K⁺) across the cell membrane, affecting the concentration of ions inside and outside the cell as well as the membrane permeability to these ions. After infection of cells by picornavirus, increased membrane permeability leads to disruption of the extracellular ion gradient and thus disrupts the host intracellular ion concentration balance, in which 2B viroporin plays an irreplaceable role. 2B proteins such as foot-and-mouth disease virus (FMDV), PV, and Coxsackievirus B (CVB) can increase intracellular Ca2⁺ ion concentration, but by different mechanisms. The expression of CVB3 and PV 2B proteins leads to a decrease in Ca²⁺ concentration in the ER and Golgi complex as well as in Ca²⁺ uptake by mitochondria. At the same time, an increased influx of extracellular Ca²⁺ leads to increased cytoplasmic Ca²⁺ levels (9, 12). Similarly, the expression of human rhinovirus (HRV) 2B protein showed a decrease in Ca²⁺ concentration in the ER and Golgi apparatus, while encephalomyocarditis virus (EMCV) 2B protein only significantly decreased Ca²⁺ concentration in the ER (12). In contrast, other studies have shown that the expression of hepatitis A virus (HAV) and FMDV 2B proteins increased cytoplasmic Ca²⁺ levels but did not alter the levels of Ca²⁺ stored in organelles (3, 12). Specifically, the experimental results of Xie et al. suggest that the enterovirus (EV) 71 2B protein may mediate Cl^- dependent currents in African Xenopus oocytes (14). The reason for this 2B-induced ion concentration variation among different small ribonucleic acid viruses may stem from differences in the proteins themselves and experimental settings and systems, and no studies are showing the permeability of other 2B proteins to Cl^-. Therefore, further analysis of 2B protein-mediated ion permeability under viral infection and separate expression is required. Also significant for Ca²⁺ homeostasis is the RV NSP4 protein, whose effect on Ca²⁺ levels, along with that of the enterovirus 2B protein, has been characterized using fluorescent Ca²⁺ imaging (233). NSP4 is an ER transmembrane glycoprotein that activates ER calcium sensor matrix interaction molecule 1 (STIM1), leading to the plasma membrane (PM) Ca²⁺ inward flow (190). NSP4 also elevates cellular Ca²⁺ levels through a phospholipase C (PLC)-independent pathway, suggesting that it disrupts Ca²⁺ homeostasis and releases Ca²⁺ channels (189).

Experimental results suggest that viroporin E protein may play an important role in regulating ion homeostasis and the microenvironment of host cells (234, 235). Westerbeck et al. found that IBV E protein transient overexpression alters Golgi pH, providing the first evidence of a coronavirus-mediated alteration of the secretory pathway tubular microenvironment to facilitate infectious virus production (32). A recent study showed that the intracellular expression of SARS-CoV-2 E protein increases intra-Golgi PH (21). IAV M2 also downregulates the expression and function of two host ion channels, the amiloride-sensitive epithelial sodium channel (ENaC) (236) and the cystic fibrosis transmembrane conductance regulator (CFTR) (237) chloride channel, which promotes influenza pathogenesis.

In addition to altering the permeability of the invading cells themselves, cell permeabilization by PV 2B viroporin triggers bystander permeabilization in neighboring cells through a mechanism involving gap junctions, and proteins from MHV E, sindbis virus (SINV) 6K, and HCV NS4A are also able to penetrate neighboring cells to varying degrees (238). Some viroporins (e.g., PV 2B (5)and 3A (103, 104), IBV E (29)) are also able to target intracellular compartments that affect pH or Ca²⁺ homeostasis, thereby blocking protein secretion.

The inflammasome is a molecular platform activated upon cellular infection or stress, triggering the maturation of pro-inflammatory cytokines such as IL-1β, and participating in innate immune defense. The NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome is an oligomeric complex composed of the NOD-like receptor NLRP3, the adaptor protein ASC, and Caspase-1 (229). This complex is critical in the host antiviral immune response as it promotes IL-1β and IL-18 secretion and induces pyroptosis (239). Increasing evidence suggests that the effect of viroporins on membrane permeability and the subsequent disruption of ion homeostasis in cellular compartments may be the activation signal required to activate the NLRP3 inflammasome and produce IL-1β and IL-18 ( Figure 3 ). This highlights the important role of ion concentration imbalance caused by the ion channel activity of viroporin in activating the NLRP3 inflammasome. Viroporins such as FMDV and HRV 2B protein (93, 114), respiratory syncytial virus (RSV) SH protein (66), IAV M2 protein (140), EMCV 2B protein (109), SARS-CoV E protein (122), SARS 3a protein (126), and HCV p7 protein (168) was reported to activate the NLRP3 inflammasome by disturbing intracellular ion concentrations (K⁺, Ca²⁺, Na⁺). The 2B protein from a variety of picornaviruses (including EMCV, PV, and EV 71), the DNEV NS2A protein and the SARS-Cov E protein were shown to induce NLRP3 cytoplasmic relocalization and inflammasome activation in an intracellular Ca²⁺-mediated manner (58, 109, 122). Other experimental results have shown that influenza virus M2 proton channel activates the NLRP3 inflammasome pathway by regulating intracellular K⁺, Na⁺, and Ca²⁺ concentrations (140).

In addition to disrupting intracellular ion concentrations, viroporins activate the inflammasome in other ways, resulting in an inflammatory response. DNEV NS2A and NS2B protein expression increase apoptosis-associated speck-like protein-containing caspase recruitment domain (ASC) oligomerization and secretion of IL-1β through caspase 1 activation (58). SARS 3a activates NLRP3 inflammasome by inducing disruption of intracellular ion concentration, mitochondrial damage, and TNF receptor-associated factor 3 (TRAF3)-mediated ubiquitination of ASC (126, 128, 131). Paradoxically, Siu et al. found that the oligomerization of ORF 3a was dispensable for the activation of NF-κB or NLRP3 inflammasome, indicating how the viral ion channel protein works in an ion channel-independent manner (128). In contrast, results from another related study showed that SARS-CoV 3a activates the NLRP3 inflammasome in an ion channel-dependent manner (126). This discrepancy may require further studies in the same experimental system to resolve. In addition to this, 3a upregulates the production of pro-inflammatory cytokines and chemokines by activating C-Jun N-terminal kinase (JNK) and the transcription factor nuclear factor-kappa B (NF-kappaB) (129). In contrast, human metapneumovirus (hMPV) SH has been reported to inhibit NF-κB transcriptional activity in airway epithelial cells (184). Other studies have shown that RSV SH viroporin accumulates in the Golgi apparatus within lipid raft structures and may form ion channels that trigger NLRP3 translocation from the cytoplasm to the Golgi apparatus and activate NLRP3 inflammasome (66). HCV-p7 induces the molecular mechanism of SOCS3 through STAT3 and ERK activation and has been shown to inhibit p7 in response to TNF-α pro-inflammatory response (165).

The IAV PB1-F2 protein has a special performance in regulating inflammatory response, exhibiting two distinct roles in promoting and inhibiting NLRP3 inflammasome. On the one hand, PB1-F2 can be integrated into the phagocytic lysosomal compartment triggering NLRP3 inflammasome activation, inducing the secretion of the IL-1β, which leads to severe pathophysiology (153). Several experimental results have shown that PB1-F2 activates NLRP3 inflammasomes and NLRP3-dependent cell recruitment (154), inducing lung inflammation (152). On the other hand, the PB1-F2 protein inhibits the activation of the RLRP3 inflammasome under certain conditions (155). The highly pathogenic H7N9 PB1-F2 protein selectively inhibits RNA-induced NLRP3 inflammasome activation by inhibiting MAVS-NLRP3 interactions in infected cells, but intracellular PB1-F2 does not affect extracellular PB1-F2-induced NLRP3 inflammasome maturation (151). H5N1 and H3N2 PB1-F2 expression reduced IL-1β levels secreted by infected macrophages (150). The intrinsic link between the activation and inhibition of the RLRP3 inflammasome by viroporin PB1-F2 is unclear, but ultimately it is for the better survival and reproduction of the virus in the host cell.

Autophagy is a physiological catabolic process in which cells degrade internalized pathogens or worn organelles by forming membrane-enclosed autophagosomes. Although viruses must escape autophagic destruction, some viruses can also disrupt autophagy for their benefit. Studies have shown that viroporins can manipulate autophagy ( Figure 4 ) and thus promote viral replication, and among the viroporins that have a strong effect on cellular autophagy are mainly the small ribonucleic acid virus 2B protein, coronavirus 3a protein, influenza virus M2 protein, and rotavirus NSP4 protein. FMDV and CVB3 2B proteins induce robust autophagy in host cells (3, 108). Simultaneous expression of PV 2BC and 3A proteins induces the co-localization of LC3 and LAMP1, which induces autophagy to promote viral replication (106). According to reports, NSP4 can induce autophagy by activating the Ca²⁺/calmodulin-dependent kinase kinase-β (CaMKK-β) signaling pathway or targeting IGF1R (191, 192), and further studies revealed that COPII vesicle transport plays an important role in this (226). Furthermore, early in viral infection, the miRNA encoded by the NSP4 gene targets IGF1R, blocking the PI3K/Akt pathway and triggering autophagy, but ultimately inhibiting autophagic maturation (193).

SARS-CoV 3a proteins can both induce autophagy by inserting into the lysosomal membrane, causing lysosomal damage and dysfunction (Yuan 131), and inhibit autophagosome degradation and thus cellular autophagy by preventing the assembly of the SNARE complex required for HOPS complex-mediated autolysosome formation (132). By comparison, it is found that 3a needs its viroporin activity to promote autophagy, while whether preventing autophagy also needs to be further studied. Similarly, the influenza virus M2 protein also exhibits both autophagy-promoting and autophagy-inhibiting aspects. IAV M2 inhibits autophagy by directly interacting with the autophagy proteins LC3 or Beclin-1, blocking the fusion of autophagosomes and lysosomes (141–143). In contrast, Wang et al. showed that M2 triggers extracellular Ca²⁺ influx-dependent ROS production, which subsequently leads to activation of ATG5 and inhibition of AKT/PKB and MTOR activities via the class I phosphatidylinositol 3-kinase (PI3K)-AKT-MTOR signaling pathway, ultimately triggering activation of autophagy (145). The inhibition of autophagy by 3a and M2 proteins may be a strategy employed by viruses during the pre-infection phase to survive in the host cell while releasing daughter viral particles from the cell by promoting autophagy after the maturation of their viral particles. At present, further studies on the regulation of host cell autophagy by viroporins other than the above-mentioned proteins are underway.

Apoptosis is a genetically programmed mechanism used by the host to eliminate damaged or unwanted cells by activating the caspases cascade and can be activated through two major signaling pathways: the extrinsic or death receptor-mediated pathway and the intrinsic or mitochondrial pathway. In addition, excessive Ca²⁺ loading in mitochondria can induce apoptosis by causing the opening of permeability transition pores, permeability swelling of mitochondria, and rupture of the outer mitochondrial membrane, which leads to the release of pro-apoptotic factors such as cytochrome C (230). Some virus-encoded viroporins can promote their propagation by affecting host cell apoptosis ( Figure 5 ).

Accumulating evidence suggests that picornavirus 2B proteins can regulate host cell apoptotic responses in multiple ways. HRV16 2B protein activates PERK and ATF6 but not IRE1 to trigger ER stress (15). EV 71 2B protein induces apoptosis by recruiting and directly interacting with the pro-apoptotic protein Bax or by regulating the redistribution and activation of Bax (13, 111). Furthermore, PV 2B protein induces permeabilization of the plasma membrane, triggering apoptosis through the mitochondrial pathway (102). CVB 2B proteins play a major role in inhibiting apoptotic host cell responses by manipulating intracellular Ca²⁺ homeostasis (9). Other viroporins also regulate host cell apoptosis in different ways. Studies have shown that overexpression of the E protein of the family Coronaviridae can induce apoptosis (124, 137) and the SARS-CoV E protein can interact with Bcl-xL, making the anti-apoptotic protein Bcl-xL unable to function normally (124). SARS-CoV E activates ER stress and induces pro-inflammatory cytokines to promote apoptosis during late infection (137). In contrast, DeDiego et al. used a microarray-based approach to show that SARS-CoV E may also have anti-apoptotic effects during infection (125). The 3a and 8a proteins of SARS-CoV also induce apoptosis (27, 134). The 8a protein is located in mitochondria, where it can perturb mitochondrial membrane potential and induce apoptosis through a caspase-3-dependent pathway (27).

In addition, the influenza virus M2 protein induces host cell apoptosis by blocking autophagosome maturation (148). It has been reported that Hsp40 acts as a regulator of PKR signaling by interacting with the PKR cytostatic p58^IPK. Guan et al. found that M2 protein interacts with Hsp40 both in vitro and in vivo, speculating that it may enhance PKR autophosphorylation by forming a stable complex with Hsp40 and P58^IPK, thereby inducing cell death (149). Another viroporin PB1-F2 encoded by the influenza virus interacts with two mitochondrial proteins, adenine nucleotide transporter (ANT3) and voltage-dependent anion channel 1 (VDAC1), which are present in the inner and outer mitochondrial membranes, respectively, leading to dissipation of mitochondrial membrane potential, inducing cell death (156). In addition, the PB1-F2 protein can also translocate to the inner mitochondrial membrane space via the TOMM40 channel, resulting in a decrease in mitochondrial membrane potential (MMP) and induction of apoptosis (155). In contrast, the BM2 protein interacts with p53 and inhibits its transcriptional and apoptotic activities (164). Some other viroporins (e.g., human respiratory syncytial virus SH protein, HPV E5 protein) have also been reported to have anti-apoptotic activity (174, 176, 182, 200).

The innate immune response is the first line of defense against viral infection, and to break through this line of defense and replicate effectively in vivo, many virus-encoded viroporins disrupt host immune defenses in a variety of ways ( Figure 6 ). The regulation of host cell immune response by viroporins is mainly manifested as antagonism.