Viral and host proteins involved in picornavirus life cycle

Stress granules (SGs) are cytosolic ribonucleoprotein aggregates that are induced during cellular stress. Several viruses modulate SG formation, suggesting that SGs have an impact on virus infection. However, the mechanisms and impact of modulating SG assembly in infected cells are not completely understood. In this study, we identify the dicistrovirus cricket paralysis virus 1A (CrPV-1A) protein that functions to inhibit SG assembly during infection. Moreover, besides inhibiting RNA interference, CrPV-1A also inhibits host transcription, which indirectly modulates SG assembly. Thus, CrPV-1A is a multifunctional protein. We identify a key R146A residue that is responsible for these effects, and mutant CrPV(R146A) virus infection is attenuated in Drosophila melanogaster S2 cells and adult fruit flies and results in increased SG formation. Treatment of CrPV(R146A)-infected cells with actinomycin D, which represses transcription, restores SG assembly suppression and viral yield. In summary, CrPV-1A modulates several cellular processes to generate a cellular environment that promotes viral translation and replication.IMPORTANCE RNA viruses encode a limited set of viral proteins to modulate an array of cellular processes in order to facilitate viral replication and inhibit antiviral defenses. In this study, we identified a viral protein, called CrPV-1A, within the dicistrovirus cricket paralysis virus that can inhibit host transcription, modulate viral translation, and block a cellular process called stress granule assembly. We also identified a specific amino acid within CrPV-1A that is important for these cellular processes and that mutant viruses containing mutations of CrPV-1A attenuate virus infection. We also demonstrate that the CrPV-1A protein can also modulate cellular processes in human cells, suggesting that the mode of action of CrPV-1A is conserved. We propose that CrPV-1A is a multifunctional, versatile protein that creates a cellular environment in virus-infected cells that permits productive virus infection.

Section: Discussionsupporting

confidence: 72%

Disruption of Stress Granule Formation by the Multifunctional Cricket Paralysis Virus 1A Protein

Khong

Kerr

Yeung

et al. 2017

“…IRESdependent translation depends on both canonical translation initiation factors and IRES-specific trans-acting factors (ITAFs) which interact with various IRES elements to regulate their activities by affecting ribosome recruitment or modifying the structure of the IRES itself (39,47,52). Sam68 can now be added to the list of noncanonical host factors already reported that interact with the IRES elements of various picornaviruses (13,53), such as FMDV. In order to investigate regulation of the EV71 IRES-dependent translation by Sam68, the FLuc reporter plasmid was used to evaluate the EV71 IRES activity.…”

Section: Discussionmentioning

confidence: 99%

“…The latter are involved mainly in the replication and translation of the viral RNA (12)(13)(14)(15). During virus replication, the genomic RNA not only directs the synthesis of the viral polyprotein but also serves as the template for RNA synthesis as well as packing into virions.…”

mentioning

confidence: 99%

Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation

Zhang

Song

Cong

et al. 2015

Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5= untranslated region (5=UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation. IMPORTANCEThe nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially enhance the translation of virus protein. To our knowledge, this is the first report that describes Sam68 actively participating in the life cycle of EV71 at a molecular level. These studies will not only improve our understanding of the replication of EV71 but also have the potential for aiding in developing a therapeutic strategy against EV71 infection. Hand-foot-and-mouth disease (HFMD) is a common viral illness in infants and children. The disease causes fever and skin rash or blister-like eruptions (1). HFMD is caused by viruses that belong to the Enterovirus genus of the Picornavirus family, which mainly includes coxsackieviruses and echoviruses (1, 2). Enteroviruses, especially enterovirus 71 (EV71), have been associated with HFMD and may lead to disease with severe effects on morbidity and mortality (3) and a high rate of neurological complications, such as aseptic meningitis, acute flaccid paralysis, and fatal neurogenic pulmonary edema (4, 5). The first EV71 strain (BrCr-CA-70) was isolated in 1970 in California and reported ...

“…pro and 3C pro , to form four structural proteins (VP1, VP2, VP3, and VP4) and seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D) (39). EV71 infection was a major cause of outbreaks of hand, foot, and mouth disease (HFMD) in infants and young children (19,22).…”

mentioning

confidence: 99%

Enterovirus 71 Disrupts Interferon Signaling by Reducing the Level of Interferon Receptor 1

Liu

Zhao

et al. 2012

135

130

The recent outbreak of enterovirus 71 (EV71) infected millions of children and caused over 1,000 deaths. To date, neither an effective vaccine nor antiviral treatment is available for EV71 infection. Interferons (IFNs) have been successfully applied to treat patients with hepatitis B and C viral infections for decades but have failed to treat EV71 infections. Here, we provide the evidence that EV71 antagonizes type I IFN signaling by reducing the level of interferon receptor 1 (IFNAR1). We show that the host cells could sense EV71 infection and stimulate IFN-␤ production. However, the induction of downstream IFN-stimulated genes is inhibited by EV71. Also, only a slight interferon response and antiviral effects could be detected in cells treated with recombinant type I IFNs after EV71 infection. Further studies reveal that EV71 blocks the IFN-mediated phosphorylation of STAT1, STAT2, Jak1, and Tyk2 by reducing IFNAR1. Finally, we identified the 2A protease encoded by EV71 as an antagonist of IFNs and show that the protease activity is required for reducing IFNAR1 levels. Taken together, our study for the first time uncovers a mechanism used by EV71 to antagonize type I IFN signaling and provides new targets for future antiviral strategies. E nterovirus 71 (EV71) is a typical positive-strand RNA virus which belongs to the Picornaviridae family (44). The genome of EV71 is approximately 7.5 kb in length and contains a single open reading frame encoding a polyprotein precursor. This polyprotein is cleaved by two viral proteases, 2Apro and 3C pro , to form four structural proteins (VP1, VP2, VP3, and VP4) and seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D) (39). EV71 infection was a major cause of outbreaks of hand, foot, and mouth disease (HFMD) in infants and young children (19,22). As a typical neurotropic virus, EV71 has a propensity to cause neurological disease during acute infection and may lead to permanent paralysis and even death (1,42,45). The outbreaks and severity of EV71 infection have been frequently reported worldwide (60), and recently EV71 has become a major threat to public health in China (56). The Chinese government reported that there were ϳ1,770,000 cases of HFMD and herpangina with over 900 deaths in 2010 (http://www.moh.gov.cn/publicfiles/business/htmlfiles/zwgkzt /pyq/list.htm). However, to date, no effective vaccine or therapy can be applied to prevent or treat EV71 infection.Type I interferon (IFN), as the first line of host immune response, is critical in mediating antiviral defense. The host recognizes viral invasion and activates the type I IFN response through the recognition of pathogen-associated molecular patterns (PAMPs) by patternrecognition receptors (PRRs) (61). Binding with the corresponding receptors, IFN receptor 1 (IFNAR1) and IFNAR2 (11, 50), the secreted IFNs activate the Janus kinases Jak1 and Tyk2 and then phosphorylate signal transducers and activators of transcription STAT1 and STAT2 (23). The phosphorylated STAT1 and STAT2 form heterodimers and bind with...