Foot-and-mouth disease virus utilizes an autophagic pathway during viral replication

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)....

Section: Discussionmentioning

confidence: 99%

“…The virus was grown in baby hamster kidney-21 (BHK-21) cells, and the titer was determined by plaque assay on BHK-21 cells according to standard techniques (35).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 96%

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

Gladue

O’Donnell

Baker-Branstetter

et al. 2012

“…Many positive-sense RNA viruses, including hepatitis C virus, encephalomyocarditis virus, dengue virus, and foot-andmouth disease virus, subvert parts of the cellular autophagy machinery to promote their own replication (3,7,13,17,20). Certain picornaviruses, including poliovirus (PV), coxsackievirus, and enterovirus 71 subvert the autophagic pathway to generate double-membraned vesicles, which are thought to serve as substrates for viral genome replication (9,10,12,28).…”

mentioning

confidence: 99%

Human Rhinovirus 2 Induces the Autophagic Pathway and Replicates More Efficiently in Autophagic Cells

Klein

Jackson

2011

“…Interestingly, we also found that the extracellular vesicles preferentially contain LC3-I rather than LC3-II. The limited detection of LC3-II lipidated form, which is known to associate with membranes upon the induction of autophagy (47,48) and during infection with several different viruses, including poliovirus, rhinovirus, enterovirus 71, CVB3, and foot-and-mouth disease virus, among others (49)(50)(51), suggests that these structures are not derived from the autophagy pathway. Further, the shedding mechanism is distinct from the previously described autophagosome-mediated exit without lysis (AWOL) model for poliovirus release and also differs from a similar model described recently for CVB3 (33,52).…”

Section: Discussionmentioning

confidence: 99%

Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus

Silvas

Popov

Paulucci-Holthauzen

et al. 2016

Severe fever with thrombocytopenia syndrome (SFTS) virus is a newly recognized member of the genus Phlebovirus in the family Bunyaviridae. The virus was isolated from patients presenting with hemorrhagic manifestations and an initial case fatality rate of 12 to 30% was reported. Due to the recent emergence of this pathogen, there is limited knowledge on the molecular virology of SFTS virus. Recently, we reported that the SFTS virus NSs protein inhibited the activation of the beta interferon (IFN-␤) promoter. Furthermore, we also found that SFTS virus NSs relocalizes key components of the IFN response into NSs-induced cytoplasmic structures. Due to the important role these structures play during SFTS virus replication, we conducted live cell imaging studies to gain further insight into the role and trafficking of these cytoplasmic structures during virus infection. We found that some of the SFTS virus NSs-positive cytoplasmic structures were secreted to the extracellular space and endocytosed by neighboring cells. We also found that these secreted structures isolated from NSs-expressing cells and SFTS virus-infected cells were positive for the viral protein NSs and the host protein CD63, a protein associated with extracellular vesicles. Electron microscopy studies also revealed that the isolated CD63-immunoprecipitated extracellular vesicles produced during SFTS virus infection contained virions. The virions harbored within these structures were efficiently delivered to uninfected cells and were able to sustain SFTS virus replication. Altogether, these results suggest that SFTS virus exploits extracellular vesicles to mediate virus receptor-independent transmission to host cells and open the avenue for novel therapeutic strategies against SFTS virus and related pathogens. IMPORTANCESFTS virus is novel bunyavirus associated with hemorrhagic fever illness. Currently, limited information is available about SFTS virus. In the present study, we demonstrated that extracellular vesicles produced by SFTS virus-infected cells harbor infectious virions. We sought to determine whether these "infectious" extracellular vesicles can mediate transmission of the virus and confirmed that the SFTS virions were efficiently transported by these secreted structures into uninfected cells and were able to sustain efficient replication of SFTS virus. These results have significant impact on our understanding of how the novel tick-borne phleboviruses hijack cellular machineries to establish infection and point toward a novel mechanism for virus replication among arthropod-borne viruses. The Bunyaviridae family comprises five genera, including Orthobunyavirus, Phlebovirus, Nairovirus, Hantavirus, and Tospovirus. The majority of viruses within this family (with the exception of Hantaviruses) are considered arthropod-borne viruses and are important causes of morbidity and mortality around the world. These viruses are associated with a range of clinical symptoms characterized by febrile illness and in the most severe cases fatal hepa...