The Picornaviruses Replication Complex

Published Date: 02 Nov 2017

Many viral replication sites are associated with cytoplasmic membranes. Many organelles are involved in the formation of replication complex of various viruses. These organelles include ER (eg. picornovirus), endosomes and lysosomes (eg.togaviruses), peroxisomes and chloroplasts (eg.tombusviruses), and mitochondria (eg.nodaviruses). The replication also includes virus nonstructural proteins. These proteins help the replication complex association with membrane of the respective targeted organelle (Salonen et al., 2005; Burgyan et al., 1996; Carette et al., 2000).

The PV replication complex consists of a cluster of vesicles 40-200 nm in diameter. Studies reveal that the COP1 plays important role in the replication of PV. Inhibiting COP1 coat production results in the inhibition of PV replication. Brefeldin A, an inhibitor of the secretory pathway, reduces PV replication by preventing the COP1 vesicle formation by inhibiting the COP1 association with membranes and the GTPase ARF1, which regulates the COP1 coat vesicle (Gazina et al., 2002). Brefeldin A inhibits the catalytic Sec7 domain of GBF1 and inhibits ARF1 activity. (Belov et al., 2011).

Protein trafficking

Cell homeostasis requires communication between different organelles and any alteration in this communication can have serious consequences for the cell. There have been several detailed genetic and biochemical studies on intracellular trafficking by the means of vesicles. Protein synthesis and transportation depend upon two major organelles of the cell, ER and the Golgi apparatus which make up the secretory pathway. Proteins that are targeted to ER, Golgi, lysosomes, integral plasma membrane proteins, or proteins meant to be secreted out of the cell are transported by this pathway. Other than ER and Golgi, some other cellular compartments involved in and affected by vesicular transport are ERGIC, endosomes, lysosomes and plasma membrane. ERGIC is essentially the group of vesicles present between ER and Golgi. It is the first compartment to get affected when either anterograde or retrograde transport is blocked. Endosomes are vesicles that may arise either from Golgi or from plasma membrane. They are usually transporting proteins to lysosomes. Sorting of cargo and carrier proteins also occurs in endosomes. Lysosomes are the degradation organelle of the cell. Lysosomal resident proteins are targeted to lysosome via ER Golgi pathway. Other proteins that need to be digested in lysosomes reach there via endosomes. While soluble proteins can be carried simply within the lumen of endosomes but membrane proteins targeted for destruction are sent via multi-vesicular bodies. Plasma membrane is the point where both exocytosis (fusion of vesicle) and endocytosis (formation of vesicle) occur (Lodish et al., 2003).

Protein transport is mediated by vesicles and occurs in two directions – Anterograde (from ER to Golgi) and Retrograde (from Golgi to ER). These vesicles can be differentiated from each other on the basis of coat proteins they are made up of. They involve two sets of cytoslic coat protein, namely COP1 for retrograde transport and COP2 for anterograde transport (Lee et al., 2004). Another type of coated vesicles are clathrin coated vesicles that transport proteins from plasma membrane to Trans Golgi network (TGN) and from TGN to late endosomes (Figure 3).

Figure 3. Membrane traffic between the ER and the Golgi. (Krogerus dissertation, 2007).

Protein coats are made up of several different components that come together to initiate vesicle formation as well as target proteins to these vesicles. COP2 coats are formed at specialized exit sites present on the ER. Three abundant cytosolic components are involved in its formation, one is a small GTPase Sar1 and others are two heterodimeric complexes, Sec23–Sec24 and Sec13–Sec31 and Sec16. The small GTPase Sar1 initiates the assembling process by hydrolyzing GTP to GDP and thereby going from cytosolic form to membrane anchored form. It anchors to the membrane via Sec12. The membrane bound Sar1 then recruits the heterodimeric complex Sec23–Sec24 to the site. Sec23–Sec24 complex directs the selection of cargo which includes both soluble and membrane proteins. The Sec13–Sec31 protein complexes recruited to the site are responsible for the formation of a flexible cage that can accommodate the selected cargo of various shapes and size (Sharpe et al., 2011).

COP1 vesicles on the other hand use Arf1 as the monomeric GTPase. Their protein coat is made of proteins called Coatomers. COP1 assembly takes place on Coatomer-containing membranes likely to be pre-Golgi and Golgi structures. It is initiated by recruitment of the Arf protein and its activation on the Coatomer containing membrane mediated by the GEF GBF1. The complex of Coatomer-Arf1-Arf1GAP polymerizes into coat lattice on the membrane, which deformed into a coated bud and released as the coated vesicle. The Arf1 GAP regulates the dissembling of the COP1 through hydrolyzing the Arf1-GTP into Arf1-GDP (Lippincott-S et al., 2006). Coatomers contain seven different COP subunits and these are grouped together in two sub-complexes. The F- sub complex consists of β, δ, γ and ζ sub units, while the B- sub complex consists of ε, β’ and α sub units. The αCOP, also called Ret1p, has a WD40 domain and recruits cargo proteins accessory proteins. The βCOP is the one that binds Arf1-GTP and thereby assembles the remaining coatomer subunits. It is known to bind to di-acidic cargo motifs. It also has an appendage domain that helps recruit cargo. β’COP or Sec26p also contains WD40 domain that binds to KxKxx motif. γCOP or Sec21p binds to proteins of p24 family and is responsible for recruitment of ArfGAP. δCOP or Ret2p is known to recruit the accessory protein Dsl1p via binding to WxxxW motif. ζCOP is responsible for stabilizing interaction between βCOP and γCOP while εCOP is essential for stabilization of the entire coatomer complex (McMahon and Mills, 2004).

Clathrin coated vesicles also use Arf1 as the GTPase. The coat proteins depend upon the direction of transport mediated by the vesicles. Vesicles moving from trans Golgi to endosomes have either clathrin + AP1 or clathrin + GGA complex. Vesicles moving from plasma membrane to endosomes are made up of clathrin + AP2 cmplexes. Vesicles moving from Golgi to lysosomes, melanosomes or platelet vesicles are coated with AP3 complexes (Lodish et al., 2003). Clathrin is a trimer with a central hub domain and 3 WD40 domains on each of its terminals. When clathrin is concentrated on a membrane it assembles itself to form a triskelia. Cargo proteins are recruited to these vesicles via adaptor proteins present in the coats. The classical adaptor protein families are AP1, AP2, AP3 and AP4 complexes. Some other cargo adaptors are also present such as GGA and Hrs (McMahon and Mills, 2004).

Arf1 and Sar1 both belong to the small GTPase superfamily. They act as switches which cycle between active GTP bound and inactive GDP forms. GTPase - activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) mediate the cycling of Arf1 and Sar1 proteins between active GTP-bound and inactive GDP-bound forms (Behnia and Munro, 2005). GEFs are responsible for removing the GDP and replacing it with GTP, while GAPs are responsible for activating the GTPase to hydrolyse the GTP into GDP. Every GTPase is known to have its own GAP and GEF.

Once the vesicle buds off from the organelle membrane, the protein coat around it dissociates. This dissociation is initiated by GAPs. GAPs convert Arf1/Sar1-GTP to Arf1/Sar1-GDP and therefore bring about their dissociation from the rest of the protein coat complex. This leads to dissociation of all the coat components and thus they are now available as cytosolic forms to form vesicles again.

These vesicles next need to dock onto their target membranes. This is brought about by Rab GTPases. Different types of Rab GTPases exist and they have affinity to different types of vesicles. This affinity is defined by the lipid profile of the vesicle membrane. Rab GTPases also attach to vesicles in the GTP bound form and then bind to their effectors on the target membranes effectively docking the vesicles.

Fusion of vesicles with the target membrane is mediated by proteins called SNAREs. Both membranes contain a SNARE protein. The vesicle contains v-SNARE while its target membrane contains the cognate t-SNARE. Fusion between the membranes is brought about the binding of these two SNAREs along with a SNAP molecule which results in bringing the membranes so close together that fuse into each other (Lodish et al., 2003).

Brefeldin A (BFA) is an inhibitor of the GEF of Arf1 (GBF1) and it functions to block the secretion of proteins from cells by inhibiting the regeneration of Arf1-GTP from Arf1-GDP (Peyroche et al., 1999). BFA treated cells show characteristic lack of COPI vesicles. BFA treated cells show coalescence of cis and median Golgi with ER and loss of ERGIC (ER Golgi Intermediate Compartment) due to apparent excessive retrograde transport but this effect is reversed upon removal of the inhibitor. These two facts are at odds with each. The phenomenon of Golgi merging with ER is explained by a temporary increase in the number of v-SNAREs and tethering factors present on the Golgi membrane. This results in direct and uncontrolled fusion of Golgi membranes with ER (Nebenführ et al., 2002).

Role of lipids in protein trafficking

Other than proteins, the lipid compositions of the vesicle and target membrane also play a major role in vesicular transport. The major regulators are phosphoinositides. Different types of phosphoinositides(PI) present in the cell’s membranes on the basis of phosphate groups attached to the inositide backbone. These are PI, PI - 3P, PI - 4P, PI - 5P, PI - 4,5P, PI - 3,4P, PI - 3,5P and PI - 3,4,5P.

Out of these one of the major phoshoinositides is PI-4P. It is present mostly in the Golgi complex and it is essential for reception of COPII vesicles coming from ER (Lorente-Rodríguez and Barlowe, 2011). PI-4P is also essential for anterograde vesicle trafficking from TGN to plasma membrane or endosomes. Formation of exocytic vesicles is blocked in the absence of Pi-4P. Recent reports suggest that PI-4P may regulate retrograde transport of resident proteins of Golgi up to some extent as well (Tu et al., 2008).

Phosphatidylinositol – 4 kinases are the proteins that produce PI – 4P. They regulate transport from Golgi via effectors that bind to PI – 4P present in the Golgi membrane. There are 4 different types of PI – 4 Kinases that are found in humans that produce PI-4P from phosphatidylinositol, two of which are TypeII PI-4Ks (PI-4KIIα and PI-4KIIβ) and other two are TypeIII PI-4Ks (PI-4KIIIα and PI-4KIIIβ). Out of these the main ones found on Golgi are PI-4KIIα and PI-4KIIIβ. Other than this, a phosphatase, OCRL, also present in Golgi is responsible for generating PI-4P from PI-4,5P (Graham and Burd, 2011).

Localization of PI-4KIIIβ to Golgi is mediated by its binding to GTP bound form of Arf1. PI-4KIIIβ is also known to bind a calcium binding protein neuronal calcium sensor – 1 (NCS-1). NCS-1 on the other hand is recruited to Golgi via its binding to Arf1. Therefore, Arf1 seems to regulate positioning of PI-4K to Golgi. PI-4K is known to get strongly activated by protein kinase D (PKD) and Arf1 is the protein that binds PKD and localizes it to Golgi. Therefore, Arf1 regulates PI -4K activity and thus induces the formation of PI-4P. This PI-4P is known to bind various effector proteins such as Kes1, FAPP1 & 2, AP1, OSBP, GPBP and EPSINR in the Golgi complex (De Matteis and Godi, 2004). PI-4P is also known to recruit proteins that bring about curvature of the membrane and thus help in vesicle formation.

Other important lipids required for vesicle transport especially in Golgi are DAG and PA. Phospholipase D is responsible for production of PA and this PA can be modified to form DAG. Phospholipase D activity is again activated by membrane bound Arf1 protein. PA and Dag are responsible for recruiting fission proteins that bring about the scission of the growing vesicle buds so that they can be released into the cytoplasm and may find their target membrane (De Matteis and Godi, 2004).

Membrane trafficking proteins such as Arf1 and a few Rabs have also been found to adorn the surface of lipid droplets. Though lipid droplets function majorly as lipid stores but they have been known to ‘hold’ hydrophobic proteins to either prevent them from aggregation and at other times to target them for degradation (Renue, 2011).

The role of transport protein from endoplasmic reticulum to Golgi in infected cell

Role and function of non-structural proteins on protein trafficking

Viruses are known to subvert host metabolism systems to facilitate their own survival and propagation. One of the cells processes affected by various viruses is protein trafficking. Different viruses belonging to the picornaviridae family are able to bring about changes in the protein trafficking pattern of the cell. Individual non-structural proteins have been implicated in these roles. The different non-structural proteins involved in replication process are 2A, 2B, 2C, 3A, 3B, 3C, 3D as well as intermediates such as 2BC, 3AB and 3CD. The functions of these proteins are not fixed and are found to vary within different genera of picornaviridae family.

2B

Study of Enterovirus 2B proteins have shown that they modify intracellular membrane structures and functions, as a result of which they affect intracellular protein trafficking. 2B is a small sized hydrophobic protein that is found to localize at the Golgi and ER membranes. Many 2B proteins congregate at these sites to form homomultimers. These homomultimers form pores in the membranes of ER and Golgi. Such pores make these organelles permeable to free movement of ions. Such movement is shown to cause a reduction in the intra-organellar concentrations of Ca2+ and H+ in the lumen of ER and Golgi. The cleavage precursor of 2B, that is 2BC, also shows similar effect on calcium homeostasis. A major consequence of the disruption of Ca2+ levels and change in pH of the Golgi lumen is inhibition of trafficking of proteins via Golgi complex. Cells expressing only Coxsakievirus 2B proteins were found to accumulate secretory proteins in the Golgi complex. It is believed that change in the pH of Golgi results in alteration of the activity of various Golgi resident sugar – modifying enzymes. This causes aberrations in the glycosylation patterns of proteins as they try to pass through Golgi and therefore, they are retained in Golgi itself. Changes in calcium levels inside Golgi also affect protein trafficking as they are involved in correct protein folding. Incorrect protein folding due to lack of calcium may result in retention of secretory proteins within Golgi (de Jong et al., 2008, 2006). Analysis of poliovirus 2B viroporin has shown that the protein has two clusters and the first one of them forms a amphipathic α-Helix. This structure is essential for the pore forming capability of 2B and mutations in this part of the protein result in the host cell not showing a block in protein trafficking (Martínez-Gil et al., 2011). Study of parechovirus 2B protein also showed results similar to PV 2B and CV 2B (Krogerus et al., 2007). Unlike 2B proteins from Enterovirus species such as Poliovirus, Coxsakievirus etc. 2B proteins from other genera such as Hepatitis A virus, Foot and mouth disease virus or Encephalomyocarditis virus have no effect on protein trafficking or calcium homeostasis. While FMDV 2B protein doesn’t interfere with protein trafficking or calcium homeostasis but it does localize on ER like Enterovirus 2B and is found to relegate ER resident proteins to vesicular structures around the nucleus. It is also known to block the delivery of proteins to the cell surface (Carrilo, 2012). Similarly HAV 2B protein also remodels ER but doesn’t block delivery of proteins cell surface. Theiler's Murine Encephalomyelitis virus (TMEV) 2B protein shows punctate distribution throughout the host cells which is more concentrated in the perinuclear region. The ER in cells expressing TMEV 2B show loss of reticulation similar to those observed in cells expressing FMDV and HAV 2B proteins but do not show colocalization with ER markers. Unlike Enterovirus 2B, they don’t show distribution similar to β-COP proteins present on Golgi either (Murray et al., 2009).

2BC

Another non – structural protein, that is the precursor of 2B, 2BC is also shown to affect membranes as well as inhibit protein trafficking. In virus infected cells, 2BC is found exclusively in the virus replication complex that is present in the virus induced vesicles (Porter, 1993). Poliovirus 2BC protein when expressed in S. cerevisiae led to induction of small membranous vesicle proliferation. These vesicles seemed to fill the complete cytoplasm of the cells. Furthermore, expression of 2BC also inhibited the transport and processing of various yeast proteins such as Ape1, vacuolar carboxypeptidase Y amongst others. This again suggests inhibition of protein trafficking via Golgi complex (Barco and Carrasco, 1995). Analysis of the regions of PV 2BC essential for the membrane permeabilization activity which results in inhibition of protein trafficking has been done using deletion mutants. Deletion of most of the region that codes for 2C does not affect the transport or permeability significantly and neither does the deletion of intermediate region between B and C. Deletion of N terminal amino acids of 2BC on the other hand abolish the membrane permeabilizing effect but surprisingly the same deletion in 2B proteins doesn’t do that to the same level. This suggests that 2BC have more affinity to bind to the membrane via the N terminal region than 2B and thus bring about membrane permeabilization (Aldabe et al., 1996). It is believed to contribute to the accumulation of the vesicles derived from secretory pathway in the cytoplasm (de Jong et al., 2008). When FMDV 2BC protein is expressed alone in host cells, it is found in the membrane fractions, showing that it is capable of attaching to membranes without the requirement of other viral proteins. Fluorescence microscopy showed that FMDV 2BC is localized on two types of structures in the cell when it is expressed alone. First are small punctate forms that are spread throughout the cytoplasm, the other are comparatively large punctate forms that are mostly located close to the nuclear periphery. The 2BC protein was found to inhibit transfer of G protein to Golgi suggesting that it blocks ER to Golgi transport. The G protein was found to accumulate in the punctate structures that contain 2BC and ER markers (Moffat et al., 2005). Similarly in cells infected with Coxsakievirus, expression of 2BC is found to result in accumulation of VSV-G protein in the juxtanuclear Golgi region (de Jong et al., 2006). TMEV 2BC is found to work differently from the PV 2BC and FMDV 2BC. First of all, unlike FMDV 2BC, TMEV 2BC does not cause the ER to lose its original structure and breakdown into numerous small vesicles that are spread around the cytoplasm. Secondly, TMEV 2BC is found to co-localize with β-COP proteins on large perinuclear vesicular structures to a certain extent suggesting that TMEV 2BC might be associated with Golgi membranes. On the other hand, unlike PV 2BC proteins, TMEV 2BC proteins show loss of their apparent localization and function upon deletion of the 2C part of the protein rather than the 2B part of the protein suggesting a completely different mode of action for membrane localization. This might also explain the inability of TMEV 2BC to cause membrane permeabilization (Murray et al., 2009).

2C

One of the cleavage products of 2BC is 2C and its role is also varying between different genera of picornaviruses. In cells expressing FMDV 2C individually it has been observed that it is unable to block protein synthesis even though as mentioned above 2BC is capable of blocking the secretory pathway. However, when 2C is co-expressed with 2B it is able to bring about a block in protein transport. Under these conditions 2C is found to partially co-localize with the β-COP protein present on the Golgi and is found to block transport at that step itself. However, when 2C is directed to ER by genetic maneuvering, it blocks protein transport at ER itself. This shows that the site of transport block is determined by 2C even though co-expression of both 2B and 2C is required to mediate this block (Moffat et al., 2007). In parechovirus 2C was found to localize neither to proper ER or Golgi but instead it was found on lipid droplets and small vesicles that had been derived from ER membrane. Its expression alone in cell lines did not affect protein secretion or induce vesicle formation either (Krogerus et al., 2007). In the attempts to figure out which membrane does Poliovirus 2C bind, it was observed that PV 2C didn’t co-localize with any organelle membrane marker completely but was found to partially co-localize with markers on membranes with multiple origins such as Golgi, ER, lysosomes, nuclear membrane etc. (Schlegel et al., 1996). PV 2C when expressed individually, is also able to bring about intracellular membrane network rearrangement. It was found that 2C expression led to the formation of membranous tubular structures arranged like myelin in the lumen of ER and 2C was found to be associated with them as well. These structures are not found when other viral proteins are expressed along with PV 2C or when the cell is infected with poliovirus but a novel property of 2C alone (Cho et al., 1994; Aldabe and Carrasco, 1995). The intracellular membrane rearrangement by 2C completely disrupted the presence of Golgi stacks. PV 2C activity to bring about membrane rearrangement and form tubular structures was retained when 2C proteins with just N terminal domain were used, suggesting that the determinants of membrane binding and reorganization lie within these regions. Further analysis showed that its forms an amphipathic helix, a structure similar to the one responsible for 2B interaction with the membranes (Teterina et al., 1997). 2C protein of Human Rhinovirus 16 and 39 is also found to have similar membrane reorganizing effects on host cells as poliovirus 2C protein (Harris and Racaniello, 2003). For TMEV 2C protein, two different distribution patterns were obtained when it was expressed in human host cells. Under low levels of expression 2C stained reticular structures that co-localized with ER markers. On the other hand, during high levels of expression 2C was found to be localized to nuclear envelope and large vesicular perinuclear structures that co-localized with β-COP. This suggests that 2C localization is time – dependent, starting off with a diffused pattern and then concentrating around the nucleus. Even for TMEV 2C protein, the N terminal region containing the amphipathic helix alone is sufficient to bring about the same distribution pattern and change in the membrane networks as PV 2C does (Murray et al., 2009).

3A

3A is another non – structural protein that is found to block protein transport in the host cell by most of the picornavirus’s. It is obtained after the processing of 3AB. Studies with ectopic expression of Coxsackievirus B 3A protein showed block in the transport of both membrane bound secretory proteins such VSV-G protein as well as luminal secretory proteins such as alpha – 1 protease inhibitor. The inhibition was at the step of transfer from ER to Golgi. This ability of the protein was attributed to a proline rich region present at the N terminal of the protein. Mutation in the proline residues to alanine disrupted the ability to block transport of proteins but didn’t overall inhibit the ability of the virus to replicate. Furthermore it was observed that the proline mutations in 3C caused the accumulation of VSV-G protein in the plasma membrane instead of secreting them completely or blocking them at the ER itself. These results question the necessity of protein transport block for replication of the virus. The membrane anchoring hydrophobic patch of the protein lies on the C terminal end. So far there is no knowledge of the mechanism by which CSVB 3A blocks transport of proteins but since proline rich patches are usually used for protein – protein interactions, it might recruit a number of proteins that help bring about the block in transport (Wessels et al., 2005). Similar to CSVB 3A, 3A of another enterovirus, PV is also a very potent inhibitor of secretory pathway. The block is again at the ER to Golgi step. A noticeable feature of cells expressing PV 3A is a swollen up and bulbous ER. Localization of 3A to these bulbous vesicles has shown that 3A localizes to ER and blocks transport at the step of vesicle formation from ER itself resulting in the swelling (Doedens et al., 1997; Doedens and Kirkegaard, 1995). For both CSVB and PV 3A proteins it has been shown that they interact with GBF-1 and sequester it onto the membranes, stopping it from recycling. GBF-1 is required for Arf1 activation and its unavailability reduces the amount of Arf1 activated. Since Arf1 is the GTPase that initiates COP-I vesicle formation, therefore expression of both CSVB and PV 3A resulted in cytosolic redistribution of COP-I instead of their usual localization to Golgi in uninfected cells (Wessels et al., 2006a). The ability to bind GBF-1 is retained in the N terminal region of the protein. Enteroviruses are the only picornaviruses to inhibit recruitment of COP-I to membranes. A chimeric protein containing the N terminal region of CSVB 3A and remaining portion of human rhinovirus 3A also resulted in similar activity as CSVB and PV 3A (Wessels et al., 2006b). PV 3A was also found to interact with Lis1 and this interaction is believed to dis-regulate ER to Golgi transport (Kondratova et al., 2005). 3A of HPEV – 1 when expressed solely in the host cell was found to be accumulated in the perinuclear region and co-localized with trans Golgi marker p230. It didn’t bring about any change in the organelle morphology and neither was it capable of blocking protein transport on its own like PV 3A even though HPEV 1 as a whole blocks protein secretion (Krogerus et al., 2007). TMEV 3A expression in cells showed similar bulbous morphology of ER as found for PV 3A but there haven’t been any reports on its interaction with COP-I or its role in inhibition of protein trafficking (Murray et al., 2009). FMDV 3A protein is found equally in the cytosol as well as membrane fractions and localizes on ER. Unlike FMDV 2C protein, 3A is unable to bring about any change in the membrane morphology or stop transport of proteins (Moffat et al., 2005).

Role of whole virus and its interaction with host proteins on protein trafficking

Most picornaviruses are known to inhibit protein trafficking in the host cells they infect. Since picornaviruses are not enveloped viruses therefore they do not require the vesicular system of secretion. It has been observed that no single non-structural protein can bring about the exact change in membranous structures and block of protein trafficking as can be brought about by infection with the intact virus.

Study of poliovirus infected cells has shown that protein trafficking between ER and Golgi is stopped. Going deeper into this phenomenon it has been found that COPII vesicles are able to form at the ER and move towards Golgi but the block lies at the ERGIC compartment from where the vesicles are unable to fuse with cis Golgi. It has also been found that poliovirus disintegrates the Golgi compartment into multiple small vesicles diffused throughout the cytoplasm. While nocodazole (a microtubule depolymerizing agent) treatment is able to prevent the disruption of Golgi but it doesn’t affect inhibition of protein transport. On the other hand, a PV mutant that showed extremely reduced block of protein transport still showed same vigor in disrupting Golgi, suggesting that both of these phenomena occur by different mechanisms. It is known that GBF-1 and Arf1 are the targets of PV 3A that cause block in ERGIC to Golgi transport and overexpression of GBF-1 and Arf1 is shown to overcome the protein transport block caused by PV. Dissociation of COP-I from Golgi membrane that is observed during 3A expression is not found when PV infected cells are treated with nocodazole suggesting that COP-I presence on Golgi doesn’t have anything to do with the block in protein trafficking (Beske et al., 2007). COP-I has been found to be involved in viral replication complexes of PV though. BFA treatment of PV infected cells results in loss of the replication capability of the virus. This is attributed to loss of Arf1 activation and hence loss of formation of COP-I vesicles. This phenomenon is at odds with the fact that PV itself also blocks Arf1 activation which causes block in protein transport. Thus PV blocks Arf1 during protein trafficking but recruits Arf1 for formation of replication complexes (Gazina et al., 2002). Similarly Human rhinovirus and echovirus are also sensitive to BFA while Parechovirus is only partially sensitive. FMDV, HAV and EMCV on the other hand are not affected by BFA action (Monaghan et al., 2003). However, FMDV still shows disruption of Golgi and block in protein trafficking suggesting that it acts via a method different form PV’s inhibition of Arf1 activation (O’Donnell et al., 2001). Similarly EMCV also shows membrane proliferation, rearrangement and formation of vesicles and but it is yet to be established with confirmed proof whether EMCV is capable of blocking protein transport at the ER to Golgi step (Carocci and Bakkali-Kassimi, 2012).

Other than COP-I, even COP-II is found to be necessary for picornavirus replication. It has been found that Poliovirus replication complex is located in vesicles that are generated from the ER via COP-II coat formation. This may suggest that part of the protein transport block from ER to Golgi may be due to redirection of COP-II vesicles meant for protein transport to formation of viral replication complexes (Rust et al., 2001).

PI4-K IIIβ is major regulator of transport via Golgi and is known to interact with activated Arf1 protein. It has been found that PI-4K IIIβ is essential for replication of picornaviruses. It is presumed that enhanced recruitment of this kinase on membranes that are sites of viral replication leads to a decrease in the amount of total anterograde transport (Sasaki et al., 2011; Hsu et al., 2010).

The mechanism of protein transport inhibition and membrane rearrangement by different picornavirus is still not well characterized and quite poorly understood. Furthermore, it is full of discrepancies regarding the roles of various host proteins in virus replication and opposing roles in blocking of protein transport in the host.