Involvement of membrane traffic in the replication of poliovirus genomes: Effects of brefeldin A

Previously, we identified a cellular kinase inhibitor, GW5074, that inhibits poliovirus (PV) and enterovirus 71 replication strongly, although its target has remained unknown. To identify the target of GW5074, we searched for cellular kinase inhibitors that have anti-enterovirus activity similar or related to that of GW5074. With this aim, we performed screenings to identify cellular kinase inhibitors that could inhibit PV replication cooperatively with GW5074 or synthetically in the absence of GW5074. We identified MEK1/2 inhibitors (SL327 and U0126), an EGFR inhibitor (AG1478) and a phosphatidylinositol 3-kinase inhibitor (wortmannin) as compounds with a cooperative inhibitory effect with GW5074, and an Akt1/2 inhibitor (Akt inhibitor VIII) as a compound with a synthetic inhibitory effect with MEK1/2 inhibitors and AG1478. Individual treatment with the identified kinase inhibitors did not affect PV replication significantly, but combined treatment with MEK1/2 inhibitor, AG1478 and Akt1/2 inhibitor suppressed the replication synthetically. The effect of AG1478 in this synthetic inhibition was compensated by other receptor tyrosine kinase inhibitors (IGF-1R inhibitor II and Flt3 inhibitor II). We isolated mutants resistant to Flt3 inhibitor II and GW5074 and found that these mutants had crossresistance to each treatment. These mutants had a common mutation in viral protein 3A that results in an amino acid change at position 70 (Ala to Thr), a mutation that was previously identified in mutants resistant to a potent anti-enterovirus compound, enviroxime. These results suggest that cellular kinase inhibitors and enviroxime have a conserved target in viral protein 3A to suppress enterovirus replication. INTRODUCTIONThe genus Enterovirus consists of at least ten species of non-enveloped viruses with a single-stranded, positivesense genomic RNA that belong to the family Picornaviridae. Enterovirus infection is mostly asymptomatic, but sometimes causes severe neurological symptoms exemplified by poliomyelitis. In infection by poliovirus (PV), which is the causative agent of poliomyelitis and belongs to the species Human enterovirus C, motor neurons are the major target for neurovirulence (Bodian, 1949). Enterovirus 71 (EV71), another neurotropic enterovirus belonging to the species Human enterovirus A, is a causative agent of hand, foot and mouth disease and herpangina, but sometimes causes severe neurological diseases, such as brainstem encephalitis and poliomyelitis-like paralysis (Chumakov et al., 1979;McMinn, 2002;Wang et al., 2003). EV71 causes fatal pulmonary oedema and pulmonary haemorrhage in young children by destruction of the vasomotor and respiratory centres in the brainstem Ho et al., 1999;Huang et al., 1999;Komatsu et al., 1999;Lum et al., 1998;Wang et al., 1999). For PV, live-attenuated and inactivated vaccines have been established and are currently being used for global eradication of poliomyelitis (Sabin, 1965;Salk et al., 1954). However, there is no effective therapeutic means for vaccine-associated...

Section: Introductionmentioning

confidence: 99%

Cellular kinase inhibitors that suppress enterovirus replication have a conserved target in viral protein 3A similar to that of enviroxime

Arita

Wakita

Shimizu

2009

“…In contrast, emergence of virus mutant resistant to inhibitors against cellular factors was limited, depending in part on the capacity of viral protein activities in the target step. A PV mutant resistant to brefeldin A, which blocks membrane traffic between the cis-and trans-Golgi compartments and inhibits PV replication (Irurzun et al, 1992;Maynell et al, 1992), required two mutations and was isolated after five passages under a stepwise selection pressure by raising the concentration of the inhibitor (Crotty et al, 2004). The emergence of a PV mutant resistant to geldanamycin treatment, which targets Hsp90 and interferes with the folding of PV capsid probably in cooperation with Hsp70 (Macejak & Sarnow, 1992), was not observed (Geller et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Characterization of pharmacologically active compounds that inhibit poliovirus and enterovirus 71 infectivity

Arita

Wakita

Shimizu

2008

Poliovirus (PV) and enterovirus 71 (EV71) cause severe neurological symptoms in their infections of the central nervous system. To identify compounds with anti-PV and anti-EV71 activities that would not allow the emergence of resistant mutants, we performed drug screening by utilizing a pharmacologically active compound library targeting cellular factors with PV and EV71 pseudoviruses that encapsidated luciferase-encoding replicons. We have found that metrifudil (N-[2-methylphenyl]methyl)-adenosine) (an A2 adenosine receptor agonist), N 6 -benzyladenosine (an A1 adenosine receptor agonist) and NF449 (4,49,40,409-[carbonylbis[imino-5,1,3-benzenetriyl bis(carbonyl-imino)]] tetrakis (benzene-1,3-disulfonic acid) octasodium salt) (a Gs-a inhibitor) have anti-EV71 activity, and that GW5074 (3-(3, 5-dibromo-4-hydroxybenzylidine-5-iodo-1,3-dihydro-indol-2-one)) (a Raf-1 inhibitor) has both anti-PV and anti-EV71 activities. EV71 mutants resistant to metrifudil, N 6 -benzyladenosine and NF449 were isolated after passages in the presence of these compounds, but mutants resistant to GW5074 were not isolated for both PV and EV71. The inhibitory effect of GW5074 was not observed in Sendai virus infection and the treatment did not induce the expression of OAS1 and STAT1 mRNA. Small interfering RNA treatment against putative cellular targets of GW5074, including Raf-1, B-Raf, Pim-1, -2, and -3, HIPK2, GAK, MST2 and ATF-3, did not consistently suppress PV replication. Moreover, downregulation of Raf-1 and B-Raf did not affect the sensitivity of RD cells to the inhibitory effect of GW5074. These results suggest that GW5074 has strong and selective inhibitory effect against the replication of PV and EV71 by inhibiting conserved targets in the infection independently of the interferon response.

“…Amongst the picornaviruses, rhinoviruses and poliovirus are sensitive to the antibiotic, whereas encephalomyocarditis virus (EMCV) is resistant. A negative-stranded RNA virus, such as vesicular stomatitis virus, is blocked by BFA whereas vaccinia virus, a cytoplasmic DNA virus, is resistant (Irurzun et al, 1992). These findings suggest that even viruses that do not have a lipid envelope and do not synthesize glycoproteins may require newly made membranes to allow the synthesis of new genomes (Irurzun et al, 1992;Maynell et al, 1992).…”

Section: Introductionmentioning

confidence: 97%

“…A negative-stranded RNA virus, such as vesicular stomatitis virus, is blocked by BFA whereas vaccinia virus, a cytoplasmic DNA virus, is resistant (Irurzun et al, 1992). These findings suggest that even viruses that do not have a lipid envelope and do not synthesize glycoproteins may require newly made membranes to allow the synthesis of new genomes (Irurzun et al, 1992;Maynell et al, 1992). Indeed, when this macrolide antibiotic is added late during infection it has no effect on poliovirus protein synthesis, whereas the replication of virus genomes is strongly blocked (Irurzun et al, 1992).…”

Section: Introductionmentioning

confidence: 97%

“…In this manner, enveloped viruses that mature from the plasma membrane or mature intracellularly are inhibited by this macrolide antibiotic (Ulmer & Palade, 1990;Chen et al, 1991;Whealy et al, 1991;Cheung et al, 1991). The effects of BFA on late protein synthesis by animal viruses vary according to the virus species examined (Irurzun et al, 1992). Amongst the picornaviruses, rhinoviruses and poliovirus are sensitive to the antibiotic, whereas encephalomyocarditis virus (EMCV) is resistant.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Action of brefeldin A on translation in Semliki Forest virus-infected HeLa cells and cells doubly infected with poliovirus

Pérez

Irurzun

Carrasco

1994

Brefeldin A (BFA) is a macrolide antibiotic that blocks membrane traffic through the vesicular system and affects the glycosylation of viral glycoproteins. Treatment of HeLa cells infected with Semliki Forest virus (SFV) with BFA enhances the synthesis of late viral proteins. Proteolytic cleavage of p107 is partially blocked and viral glycoproteins accumulate in BFAtreated cells. This enhanced synthesis of late SFV proteins is due, at least in part, to an increase in the formation of the subgenomic SFV 26S mRNA. Since BFA blocks the replication of poliovirus genomes without affecting the cleavage of the translation initiation factor p220, protein synthesis was analysed in doubly infected cells. HeLa cells infected with SFV and poliovirus at the same multiplicity predominantly synthesize poliovirus proteins. But if these cells are treated with BFA they synthesize significant amounts of SFV capsid protein C for several hours, despite the fact that p220 has been degraded.