Inhibition of the Secretory Pathway by Foot-and-Mouth Disease Virus 2BC Protein Is Reproduced by Coexpression of 2B with 2C, and the Site of Inhibition Is Determined by the Subcellular Location of 2C

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The family Picornaviridae consists of a large group of plus-strand RNA viruses that share a similar genome organization. The nomenclature of the picornavirus proteins is based on their position in the viral RNA genome but does not necessarily imply a conserved function of proteins of different genera. The enterovirus 2B protein is a small hydrophobic protein that, upon individual expression, is localized to the endoplasmic reticulum (ER) and the Golgi complex, reduces ER and Golgi complex Ca 2؉ levels, most likely by forming transmembrane pores, and inhibits protein trafficking through the Golgi complex. At present, little is known about the function of the other picornavirus 2B proteins. Here we show that rhinovirus 2B, which is phylogenetically closely related to enterovirus 2B, shows a similar subcellular localization and function to those of enterovirus 2B. In contrast, 2B proteins of hepatitis A virus, foot-and-mouth disease virus, and encephalomyocarditis virus, all of which are more distantly related to enteroviruses, show a different localization and have little, if any, effects on Ca 2؉ homeostasis and intracellular protein trafficking. Our data suggest that the 2B proteins of enterovirus and rhinovirus share the same function in virus replication, while the other picornavirus 2B proteins support the viral life cycle in a different manner. Moreover, we show that an enterovirus 2B protein that is retained in the ER is unable to modify Ca 2؉ homeostasis and inhibit protein trafficking, demonstrating the importance of Golgi complex localization for its functioning.The family Picornaviridae is a group of small, nonenveloped cytolytic viruses that include a number of important human and animal pathogens. The picornavirus family consists of nine genera, including enterovirus (e.g., coxsackievirus [CBV] and poliovirus [PV]), rhinovirus (e.g., human rhinovirus [HRV]), cardiovirus (e.g., encephalomyocarditis virus [EMCV]), aphthovirus (e.g., foot-and-mouth disease virus [FMDV]), hepatovirus (hepatitis A virus [HAV]), teschovirus (e.g., porcine teschovirus), erbovirus (e.g., equine rhinitis B virus), parechovirus (e.g., parechovirus 2), and kobuvirus (e.g., aichivirus). In addition, the picornavirus family contains a number of unassigned viruses. All picornaviruses have a similar genome organization. The viral genome typically consists of a positivestranded RNA molecule of approximately 7,500 to 8,000 nucleotides that contains one single large open reading frame preceded by a long 5Ј-untranslated region and followed by a much smaller 3Ј-untranslated region and a genetically encoded poly(A) tail. A small viral protein, VPg, is covalently linked to the 5Ј end of the viral genome. Translation of the RNA genome yields a polyprotein of approximately 2,200 amino acids (aa) that is divided into the P1, P2, and P3 regions. The polyprotein is processed by virus-encoded proteases to generate the individual structural and nonstructural proteins. Processing of the P1 region yields the structural capsid proteins 1A (VP4), 1B...

Section: Discussionmentioning

confidence: 87%

Functional Analysis of Picornavirus 2B Proteins: Effects on Calcium Homeostasis and Intracellular Protein Trafficking

Jong

Mattia²,

Dommelen³

et al. 2008

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124

“…In FMDV replication, the 2BC polypeptide, rather than 3A or 2B, is responsible for the blockage of ER-to-Golgi apparatus traffic (43,44). In view of the observed heterogeneity in protein structure of these small picornavirus proteins, it has been concluded that the mechanisms by which they exert their inhibition of protein secretion must be different (1,12).…”

Section: Discussionmentioning

confidence: 99%

“…For example, infection of COS-1 cells with enteroviruses such as poliovirus or coxsackie B virus type 3 (CVB3) or the individual expression of enterovirus proteins in target cells (e.g., 3A or 2B/2BC) blocks endoplasmic reticulum (ER)-to-Golgi apparatus traffic and results in the inhibition of cellular protein secretion (23,24,64). Interestingly, it was shown that CVB3 3A inhibits transport by binding to cellular factor GBF-1 and inhibiting GBF-1 dependent COP-1 recruitment to membranes (43,44). Polypeptides 3A and 2B and their precursors, 3AB and 2BC, contain hydrophobic transmembrane regions that play a role in their effect on secretion (2,26,62).…”

mentioning

confidence: 99%

“…Polypeptides 3A and 2B and their precursors, 3AB and 2BC, contain hydrophobic transmembrane regions that play a role in their effect on secretion (2,26,62). In the case of foot-and-mouth disease virus (FMDV), a picornavirus of the genus Aphthovirus, the 2BC precursor polypeptide rather than 3A or 2B is responsible for the block in the secretory pathway (43,44).…”

mentioning

confidence: 99%

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Evolution of Poliovirus Defective Interfering Particles Expressing Gaussia Luciferase

Song

Paul

Wimmer

2012

Polioviruses (PVs) carrying a reporter gene are useful tools for studies of virus replication, particularly if the viral chimeras contain the polyprotein that provides all of the proteins necessary for a complete replication cycle. Replication in HeLa cells of a previously constructed poliovirus expressing the gene for Renilla luciferase (RLuc) fused to the N terminus of the polyprotein H 2 NRLuc-P1-P2-P3-COOH (P1, structural domain; P2 and P3, nonstructural domains) led to the deletion of RLuc after only one passage. Here we describe a novel poliovirus chimera that expresses Gaussia luciferase (GLuc) inserted into the polyprotein between P1 and P2 (N 2 H-P1-GLuc-P2-P3-COOH). This chimera, termed PV-GLuc, replicated to 10% of wild-type yield. The reporter signal was fully retained for three passages and then gradually lost. After six passages the signal was barely detectable. On further passages, however, the GLuc signal reappeared, and after eight passages it had reached the same levels observed with the original PV-GLuc at the first passage. We demonstrated that this surprising observation was due to coevolution of defective interfering (DI) particles that had lost part or all of the capsid coding sequence (⌬P1-GLuc-P2-P3) and wild-type-like viruses that had lost the GLuc sequence (P1-P2-P3). When used at low passage, PV-GLuc is an excellent tool for studying aspects of genome replication and morphogenesis. The GLuc protein was secreted from mammalian cells but, in agreement with published data, was not secreted from PV-GLuc-infected cells due to poliovirus-induced inhibition of cellular protein secretion. Published evidence indicates that individual expression of enterovirus polypeptide 3A, 2B, or 2BC in COS-1 cells strongly inhibits host protein secretion. In HeLa cells, however, expression of none of the poliovirus polypeptides, either singly or in pairs, inhibited GLuc secretion. Thus, inhibition of GLuc secretion in PV-infected HeLa cells is likely a result of the interaction between several viral and cellular proteins that are different from those in COS-1 cells.

“…Many other positive-strand RNA viruses also initiate production of replication complexes upon infection of a cell (3,4,11,38,39). FMDV 2C, a 318-amino-acid protein, is the largest membranebinding component of the virus RNA replication complex (30). FMDV 2C binds ssRNA nonspecifically, has ATPase activity (44), and is involved in the RNA replication complex (25).…”

mentioning

confidence: 99%

Foot-and-Mouth Disease Virus Nonstructural Protein 2C Interacts with Beclin1, Modulating Virus Replication

Gladue

O’Donnell

Baker-Branstetter

et al. 2012

Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease, is an Apthovirus within the Picornaviridae family. Replication of the virus occurs in association with replication complexes that are formed by host cell membrane rearrangements. The largest viral protein in the replication complex, 2C, is thought to have multiple roles during virus replication. However, studies examining the function of FMDV 2C have been rather limited. To better understand the role of 2C in the process of virus replication, we used a yeast two-hybrid approach to identify host proteins that interact with 2C. We report here that cellular Beclin1 is a specific host binding partner for 2C. Beclin1 is a regulator of the autophagy pathway, a metabolic pathway required for efficient FMDV replication. The 2C-Beclin1 interaction was further confirmed by coimmunoprecipitation and confocal microscopy to actually occur in FMDV-infected cells. Overexpression of either Beclin1 or Bcl-2, another important autophagy factor, strongly affects virus yield in cell culture. The fusion of lysosomes to autophagosomes containing viral proteins is not seen during FMDV infection, a process that is stimulated by Beclin1; however, in FMDV-infected cells overexpressing Beclin1 this fusion occurs, suggesting that 2C would bind to Beclin1 to prevent the fusion of lysosomes to autophagosomes, allowing for virus survival. Using reverse genetics, we demonstrate here that modifications to the amino acids in 2C that are critical for interaction with Beclin1 are also critical for virus growth. These results suggest that interaction between FMDV 2C and host protein Beclin1 could be essential for virus replication.F oot-and-mouth disease virus (FMDV), a single-stranded positive-sense RNA virus, is the causative agent of foot-andmouth disease (FMD), a highly contagious viral disease of domestic and wild cloven-hoofed animals. Seven serotypes of FMDV exist (A, O, C, Asia, SAT1, SAT2, and SAT3), and recovery from one serotype does not provide immunity against the others (7,22). The infectious virion is a nonenveloped icosahedron composed of four structural proteins: VP1, VP2, VP3, and VP4. The genome of approximately 8,400 nucleotides has a single open reading frame (ORF) that is translated into a polyprotein, which is processed by the three viral proteases Lpro, 2A, and 3C into the polypeptide products P1 (VP1 to VP4), P2 (2A, 2B, and 2C), and P3 (3A, 3B, 3Cpro, and 3Dpol). Further cleavage of these regions yields 14 mature virus proteins, along with several protein intermediates, that are needed for viral replication (18,19).During replication, FMDV forms a replication complex produced by the rearrangement of intracellular membranes into vesicular structures containing viral nonstructural proteins (2, 31). Many other positive-strand RNA viruses also initiate production of replication complexes upon infection of a cell (3,4,11,38,39). FMDV 2C, a 318-amino-acid protein, is the largest membranebinding component of the virus RNA replication complex (30)....