Poliovirus 2A Pro Increases Viral mRNA and Polysome Stability Coordinately in Time with Cleavage of eIF4G

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A 3 poly(A) tail is a common feature of picornavirus RNA genomes and the RNA genomes of many other positive-strand RNA viruses. We examined the manner in which the homopolymeric poly(A) and poly(U) portions of poliovirus (PV) positive-and negative-strand RNAs were used as reciprocal templates during RNA replication. Poly(A) sequences at the 3 end of viral positive-strand RNA were transcribed into VPg-linked poly(U) products at the 5 end of negative-strand RNA during PV RNA replication. Subsequently, VPg-linked poly(U) sequences at the 5 ends of negative-strand RNA templates were transcribed into poly(A) sequences at the 3 ends of positive-strand RNAs. The homopolymeric poly(A) and poly(U) portions of PV RNA products of replication were heterogeneous in length and frequently longer than the corresponding homopolymeric sequences of the respective viral RNA templates. The data support a model of PV RNA replication wherein reiterative transcription of homopolymeric templates ensures the synthesis of long 3 poly(A) tails on progeny RNA genomes.Many positive-strand RNA viruses (e.g., members of the Picornavirales, Nidovirales, Togaviridae, Caliciviridae, and Astroviridae) have long poly(A) sequences at the 3Ј termini of their RNA genomes (16,25), yet the mechanisms by which 3Ј poly(A) sequences are derived during viral replication are unclear. Picornavirus RNA genomes, including that of poliovirus (PV), have a covalently linked 5Ј-terminal protein called VPg (viral protein, genome linked), a 5Ј untranslated region (UTR), a single large open reading frame, a 3Ј UTR, and a poly(A) tail of variable length (ϳ20 to 150 adenosine residues) (1). RNA polymerases encoded by viruses in the order Picornavirales utilize viral proteins (and their nucleotidylylated intermediates) to prime the initiation of RNA replication at the 3Ј termini of viral RNA templates (28,29,31,34). In this mechanism, the viral protein VPg becomes covalently linked to the 5Ј ends of both positive-and negative-strand RNAs during viral RNA replication (30, 32). PV, which is commonly studied to elucidate mechanisms of picornavirus replication, is viable when the 3Ј UTR of the genome is deleted (12, 44); however, the 3Ј poly(A) tail is essential for RNA replication (33, 39). The length of the 3Ј poly(A) tail required for virus viability and for efficient negative-strand RNA synthesis has been examined in some detail (35,45). PV RNAs with 3Ј poly(A) tails less than 9 bases long support less than 1% of wild-type negative-strand RNA synthesis, whereas poly(A) tails Ն20 bases long support wild-type levels of negative-strand RNA synthesis (35).In this investigation, we programmed PV RNAs with defined 3Ј 84-, 51-, and 32-base poly(A) sequences [designated poly(A) (84) , poly(A) (51) , and poly(A) (32) , respectively] into cell-free reactions that faithfully reconstitute all of the metabolic steps of viral mRNA translation (11,22,23) and viral RNA replication (5, 7, 27). A significant advantage of this experimental system is the ability to study one cycle of seq...

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Poly(A) at the 3′ End of Positive-Strand RNA and VPg-Linked Poly(U) at the 5′ End of Negative-Strand RNA Are Reciprocal Templates during Replication of Poliovirus RNA

Steil¹,

Kempf²,

Barton

2010

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“…It catalyzes an essential cleavage in the polyprotein in virus replication (5). 2A pro also cleaves several host cellular proteins thus facilitating viral replication and viral pathogenesis (30). Cys 110 is essential for the protease activity of 2A pro (57,62).…”

Section: Fig 5 Identification 2amentioning

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...

“…Enterovirus 2A pro processes the viral polyprotein (62), and it also cleaves a variety of host proteins, including the translation proteins eIF4GI (36), eIF4GII (43), and poly(A)-binding protein (27). Proteinase 2A pro has been linked to the inhibition of host translation that occurs during enterovirus infection (1,63) and is also involved in processes as varied as viral RNA replication (42), viral translation (33), and viral RNA stability (30). Interferons (IFNs) are pleiotropic cytokines that are well known for their role in the cellular antiviral response (4,35,57).…”

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confidence: 99%

Proteinase 2A^proIs Essential for Enterovirus Replication in Type I Interferon-Treated Cells

Morrison

Racaniello

2009

The Picornaviridae family comprises a diverse group of small RNA viruses that cause a variety of human and animal diseases. Some of these viruses are known to induce cleavage of components of the innate immune system and to inhibit steps in the interferon pathway that lead to the production of type I interferon. There has been no study of the effect of picornaviral infection on the events that occur after interferons have been produced. To determine whether members of the Enterovirus genus can antagonize the antiviral activity of interferon-stimulated genes (ISGs), we pretreated cells with alpha interferon (IFN-␣) and then infected the cells with poliovirus type 1, 2, or 3; enterovirus type 70; or human rhinovirus type 16. We found that these viruses were able to replicate in IFN- ␣