Enteroviruses, members of the Picornaviridae family Enterovirus genus, include poliovirus (PV), enterovirus (EV), coxsackievirus (CV), and human rhinovirus (HRV), which can cause poliomyelitis, meningitis, hand-foot-and-mouth disease, respiratory disease, and so on (Tapparel et al., 2013). Enteroviruses are non-enveloped particles, the capsid is a symmetric icosahedral capsid, and the genome is non-segmented single-stranded positive-strand RNA (about 7.5 kb). From the 5’end, the first element of the genome is VPg (3B), which is connected to the genome by covalent bond. After the VPg is the 5’non-translated region (NTR), where the “cloverleaf” and the internal ribosome entry site (IRES) are. This is followed by a single large open reading frame (ORF). The 3’end is a short 3’NTR, containing a poly (A) tail (Jiang et al., 2014; Peersen, 2017). The ORF encodes a polyprotein, about 2,300 amino acids. It is artificially separated into three sections, P1, P2, and P3. P1 encodes structural proteins, which are related to the assembly of viral capsids. P2 and P3 encode non-structural proteins and participate in virus replication and virus-host interaction (Belov et al., 2012; Cao et al., 2012; Wen et al., 2015).

Positive-strand RNA viruses from different families (such as Picornaviridae, Flaviviridae, Coronaviridae, and Togaviridae) rely on host cell machinery to replicate their genome and generate progeny viral particles. Upon infection, virus induce the rearrangements of cellular membranes to form specific intracellular compartment called virus factory or viroplasma, which contain unique platforms known as replication organelles (ROs) to replicate viral RNA (vRNA). Virus replication complexes include viral proteins needed for viral replication and coopted host factors, which constitute ROs with the sites of virus particle assemble (Novoa et al., 2005; Owino and Chu, 2019; Sachse et al., 2019). Like all positive-strand RNA viruses, enterovirus infection causes the cellular membranes modified to produce ROs for replication of vRNA. After years of efforts by scientists, the morphology, biogenesis, and related pathways of ROs biogenesis (including the secretory pathway, autophagy, and lipid metabolism) are discovered. In addition, finding potential targets for antiviral therapies during the ROs formation process is a very active field of research. In this review, we outline the research progress about the role, morphology, and biogenesis of enterovirus ROs, and briefly describe inhibitors that target ROs formation. It improves our understanding of enterovirus replication and provides reference for the development of anti-enterovirus drugs.

It is theorized that ROs promote virus replication by raising the concentrations of proteins involved in virus replication, providing platform to assemble multisubunit ROs, and effectively coordinating the different steps of the replication process to improve replication efficiency (McCloskey and Poo, 1986; Ravindran et al., 2016). The membrane lipids on the ROs can regulate virus polyprotein processing and enzyme activity. For example, cholesterol on ROs is vital for regulating the processing kinetics of viral 3CD^pro (Ilnytska et al., 2013). ROs also help viruses evade innate immunity by hiding double-stranded RNA (dsRNA), a replication intermediate of vRNA, from cellular sensors (den Boon and Ahlquist, 2010; Belov et al., 2012; Viktorova et al., 2018). In the case of PV, membranous scaffold of the ROs is essential to protect vRNA from innate immunity, especially in multicycle replication conditions (Viktorova et al., 2018). However, innate immunity do not increase when CVB3 ROs generation is delayed, suggesting that ROs are not indispensable for innate immune evasion (Melia et al., 2017).

Although the RO structures induced by different viruses have their own unique characteristics, their structures are very similar, suggesting that viruses have adopted a conserved mechanism to assemble ROs during long-term evolution (Zhang et al., 2015). There are two forms of ROs. The cell membrane of the ER or other donor organelles recess inward, forming a concealed structure containing all the elements necessary for replication (formed by negative curved membranes), which is adopted by dengue virus, Zika virus, and so on. Other viruses (hepatitis C virus, picornaviruses, arterivirus, and coronavirus) produce double-membrane vesicles (DMVs), which are formed by squeezing the donor membrane (formed by positive curved membranes). Enteroviruses induce membrane rearrangement by the second way (Bienz et al., 1992; Paul and Bartenschlager, 2013; Roulin et al., 2014; Blanchard and Roingeard, 2015; Ertel et al., 2017; Strating and van Kuppeveld, 2017). Viral proteins participate in the formation of ROs. PV 2BC and 3A proteins cause ER change, triggering the arising of ROs (Suhy et al., 2000). EV71 2C protein interacts with reticulon 3 (RTN3), which can change the curvature of the ER membrane (Tang et al., 2007).

For a long time, in the studies using two-dimensional electron microscopy, enterovirus ROs were regarded as vesicles with single or double membranes (Kallman et al., 1958; Bienz et al., 1980; Egger et al., 1996; Paul and Bartenschlager, 2013; Romero-Brey and Bartenschlager, 2014). In recent years, the three-dimensional structure of enteroviral ROs has been revealed. During infection, the structures of ROs are constantly changing, indicating that ROs are dynamic. They seem to evolve from each other with the process of viral infection. In early infection, enteroviruses ROs appear in cytoplasm with the form of single-membrane tubules (SMTs). With the progress of infection, SMTs transform into DMVs via membrane pairing and fission. In the late stage of infection, multilamellar vesicles (MVs) appear in the cytoplasm. The formation of DMVs and MVs is via membrane apposition, enwrapping, and fusion (Limpens et al., 2011; Belov et al., 2012; van der Schaar et al., 2016b; Oh et al., 2018; Wolff et al., 2020). DMVs are enwrapped by one or multiple cisternae to form MVs (Limpens et al., 2011). The membrane structures formed after enterovirus infection are discontinuous, and SMT and DMV are independent, separate structures (Figure 1). Enterovirus ROs are different from ROs of encephalomyocarditis virus, equine arteritis virus, and severe acute respiratory syndrome (SARS) coronavirus, which induce interconnected DMVs to comprise reticulovesicular networks (Knoops et al., 2008, 2012; Melia et al., 2018). Enterovirus ROs apparently lack of pores which let the vRNA enter ROs inside, suggesting vRNA replication takes place on the enterovirus ROs cytoplasmic leaflet (Limpens et al., 2011; Belov et al., 2012; Strating and van Kuppeveld, 2017).

Do the three structures (SMTs, DMVs, and MVs) all participate in genome replication? Researches on PV and CVB3 infection elucidated that there is a link between viral proteins, dsRNA, and both SMTs and DMVs. In the exponential phase of vRNA replication, the structure of ROs mainly forms SMTs, suggesting that the early and mid-term SMTs are related to the onset and most dense rates of RNA synthesis. DMVs appear in large numbers after the most active stage of vRNA synthesis, indicating that DMVs may contribute to other steps of viral life cycle, such as virus assembly (Limpens et al., 2011; Belov et al., 2012; Oh et al., 2018). This is different from HCV that DMVs of HCV is the place vRNA replication take place (Lee et al., 2019). DMVs of PV are postulated to mediate non-lytic release of progeny virus particles (Kirkegaard and Jackson, 2005; Chen et al., 2015). DMVs of SARS-coronavirus are suggested to conceal vRNA to escape the antiviral response triggered by dsRNA (Knoops et al., 2008).

Previous studies have shown that enteroviruses form ROs that are juxtaposed with exit sites of the ER, where viruses anchor the viral replication mechanism (Suhy et al., 2000; Hsu et al., 2010; Limpens et al., 2011). During the process of enteroviral infection, Golgi collapse and cannot be detected, suggesting that the membranes of the ROs may be derived from the Golgi (Hsu et al., 2010; Mousnier et al., 2014; van der Schaar et al., 2016b). The origin of CVB3 ROs was elucidated. Biogenesis of CVB3 ROs was found to occur first at the ER and then at Golgi membranes (Melia et al., 2019). In the early infection, when the level of viral proteins is low, PV and CVB3 make use of the Golgi compartment and trans-Golgi network (TGN) to synthesize vRNA (Hsu et al., 2010; van der Schaar et al., 2016b). The CVB3 3A-H57Y mutant, which can replicate under phosphatidylinositol 4-kinase beta (PI4KB) inhibition and can not form ROs, replicates at the Golgi (Melia et al., 2017).

Several studies have investigated whether the replication of PV and CVB3 requires guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor (GEF) of ADP ribosylation factor (Arf), involved in the secretory pathway. GBF1 recycles Arf from GDP-bound form to GTP-bound form (Belov et al., 2007a; Lanke et al., 2009). The replication of EV71 and PV depends on GBF1-mediated activation of Arf (Wang et al., 2014; Viktorova et al., 2019). Upon CVB3 infection, newly synthesized proteins, such as 3A protein, are located on secretory organelle membranes and recruit effector through GBF1/Arf1, thereby enhancing the accumulation of PI4KB over Coat protein complex I (COPI), causing the producing of uncoated phosphatidylinositol 4-phosphate (PI4P)-enriched structures adjacent to ER exit sites (Hsu et al., 2010). PV requires a little subset of normal GBF1 functions. GBF1 components are crucial to support virus replication under different situations. 3A-GBF1 interaction and membrane targeting function are mediated by the GBF1 C-terminal domain, which are vital to recruiting GBF1 to ROs. If either is damaged, functional ROs are still formed, but when both are inactivated, no RO is formed (Viktorova et al., 2019). Viral protein 3CD has also been demonstrated to be related to the GBF1, Arf1, and PI4KB pathways (Belov et al., 2007b; Banerjee et al., 2018). Other GEFs (BIG1 and BIG2) are also involved in the replication of enteroviruses. After PV infection, viral 3CD protein promotes the binding of Arf to the membrane by recruiting BIG1 and BIG2, which is linked to the ROs formation (Belov et al., 2007a, b).

COPI and Coat protein complex II (COPII) vesicles are important part of the transport between ER and Golgi. COPI vesicles assume the role of transporting goods to Golgi, limiting lipid storage and maintaining lipid homeostasis. COPII act a vital role in the transport of proteins and lipids from ER to Golgi (Lee et al., 2004; Beller et al., 2008). COPI and COPII both are involved in the formation of enterovirus RO. Brefeldin A (BFA), an inhibitor that targets COPI, shows a strong inhibitory action on the vRNA replication of PV. EV11, CVA-21, and CVB3 replication is also inhibited by BFA, indicating that COPI vesicle trafficking is instrumental to the replication of enteroviruses (Irurzun et al., 1992; Maynell et al., 1992; Gazina et al., 2002; van der Linden et al., 2010). Replication of EV-A71 requires COPI, which may be recruited to ROs through the interaction of the COPI coatomer and 2C protein of EV-A71 (Wang et al., 2012). The generation of PV ROs is associated in cellular COPII germination mechanism (Rust et al., 2001; Trahey et al., 2012).

UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1) is a crucial protein in ER involved in the unfolded protein response (UPR). UGGT1 and EV-A71 3D polymerase (3D^pol) coprecipitate with other factors related to ROs to encourage EV-A71 replication (Huang et al., 2017; Table 1).

Autophagy is a highly conservative lysosomal pathway unique to eukaryotic cells that degrades misfolded or redundant proteins in cells, damaged organelles, and intracellular pathogens. Autophagy is initiated by the generation of crescent isolation membrane vesicles called isolation membranes or phagophores. Subsequently, phagophores grow and fuse to form closed double-layer membrane autophagosomes. After elongation, autophagosomes have two directions. One is to form autolysosomes by fusing with lysosomes. The other is to fuse with late endosomes first to generate amphiphiles, and then fuse with lysosomes to produce autolysosomes (Lai et al., 2016; Pleet et al., 2018; Sun et al., 2019).

Multiple studies have reported that enteroviruses can induce and exploit autophagy to support the replication of virus. Studies find that the vesicles formed after enterovirus infection showed the features of autophagosomes, for example, the double-layer membrane structure of vesicles, the increase of esterified LC3, and the enrichment of cells with LC3 punctate location (Taylor and Kirkegaard, 2007; Wong et al., 2008; Yoon et al., 2008; Klein and Jackson, 2011; Alirezaei et al., 2012; Shi et al., 2015). Viral components of ROs (2B, 2BC, 3A, and 3AB) can trigger generation of esterified LC3 and/or double-membraned liposomes (Suhy et al., 2000; Wang et al., 2013; Wu et al., 2016; Lai et al., 2017). Induction of autophagy by rapamycin and nutrient deprivation leads to an increase in enteroviral replication. Contrary to the inhibition of autophagosomes formation by 3-methyladenine (3-MA), which can inhibit the formation of autophagosomes by inhibiting the activation of type III PI-3 kinase, and knockdown/knockout the genes vital for autophagosomes formation (ATG5, ATG7, ATG9, Beclin-1, VPS34, LC3, ULK1, and FIP2000) significantly reduced enterovirus replication (Sugden et al., 2005; Wong et al., 2008; Alirezaei et al., 2012; Abernathy et al., 2019). Using an antibody against dsRNA, dsRNA was found to colocalize with LC3 in late-infection of PV (Richards et al., 2014). The above results indicate that autophagy can promote virus replication and that autophagy may be involved in the ROs generation. Enteroviruses may use the autophagy pathway to provide membranous scaffolds for ROs generation after infection. CVB3 infection cause inhibition of the fusion of autophagosomes and lysosomes/late endosomes through CVB3 proteinase 3C cleave synaptosomal-associated protein 29 (SNAP29) and pleckstrin homology domain containing protein family member 1 (PLEKHM1), two vital proteins related to the autophagosomes fusion. This promotes autophagosomes accumulation to provide additional membrane scaffolds for ROs generation (Mohamud et al., 2018). SNAP29 was also found to be cleaved after EV-D68 infection (Corona et al., 2018). Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) was identified to be involved in CVB infection for it negatively regulated non-canonical form of autophagy. After silencing BPIFB3, CVB replication increased and CVB induced ROs morphology changed dramatically (Delorme-Axford et al., 2014; Table 1).

Other studies have shown that the autophagy pathway is associated with other aspects of the enterovirus life cycle, including entry, genome package, assembly, maturation, and release (Richards and Jackson, 2012; Chiramel et al., 2013; Mohamud and Luo, 2019).

The membrane is mainly composed of 5 kinds of phospholipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM) (Spector and Yorek, 1985). Phospholipids are amphiphilic and are divided into hydrophilic groups (head) and hydrophobic groups (tail) (De Craene et al., 2017). The head group is the main determinant of lipid properties (charge, shape, and interaction with proteins), and changes in fatty acids (FAs) length and saturation that make up the hydrophobic part of phospholipids also affect membrane properties (such as lipid accumulation and fluidity) (van Meer et al., 2008). Sterols are essential elements of membrane lipids and participate in regulating membrane fluidity, stability, and permeability. Cholesterol is the main sterol in mammalian cell membranes (De Craene et al., 2017). Compared with the organelle membrane in uninfected cells, the composition of enterovirus ROs is special. The ROs membrane is rich in PI4P, PC, and cholesterol (Ilnytska et al., 2013; Nchoutmboube et al., 2015; Altan-Bonnet, 2017).

Researches on enterovirus ROs give us a more comprehensive and in-depth understanding of the enterovirus life cycle. In this review, we summarized the current knowledge of enterovirus ROs, including their function, morphology, biogenesis, and generation mechanism. Enteroviruses share significantly conserved mechanisms to build ROs. The proteins involved in the secretory pathway, the autophagy pathway, and lipid metabolism are all vital for ROs formation. We have found many host factors related to ROs, but several other host proteins that have been proved to play a role in enterovirus replication are often poorly understood. More researches are needed to illuminate how the virus hijacks host factors to initiate the reorganization of the membrane and maintain such an unstable membrane. The current researches on the role of lipids in the formation of ROs are focused on PI4P, and study on other lipids is poor. The function of other lipids remains to be studied. In addition, there are more studies needed to be done to better comprehend the role of ROs in enterovirus life cycle, especially regarding how viruses utilize ROs compartments to consort different steps, such as replication, assembly, and release to achieve enterovirus efficient replication.

XL conceived, designed, and wrote the manuscript. MW and AC revised the manuscript. XW, XO, SM, QG, DS, RJ, QY, YW, DZ, XZ, SC, ML, SZ, YL, YY, LZ, BT, LP, and XC helped with the manuscript. All authors read and approved the final manuscript for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.