In vitro Inhibition of Infectivity of Polio Viruses by Guanidine

Due to their small genome size, picornaviruses must utilize host proteins to mediate cap-independent translation and viral RNA replication. The host RNA-binding protein poly(rC) binding protein 2 (PCBP2) is involved in both processes in poliovirus infected cells. It has been shown that the viral proteinase 3CD cleaves PCBP2 and contributes to viral translation inhibition. However, cleaved PCBP2 remains active in viral RNA replication. This would suggest that both cleaved and intact forms of PCBP2 have a role in the viral RNA replication cycle. The picornavirus genome must act as a template for both translation and RNA replication. However, a template that is actively being translated cannot function as a template for RNA replication, suggesting that there is a switch in template usage from translation to RNA replication. We demonstrate that the cleavage of PCBP2 by the poliovirus 3CD proteinase is a necessary step for efficient viral RNA replication and, as such, may be important for mediating a switch in template usage from translation to RNA replication. IMPORTANCEPoliovirus, like all positive-strand RNA viruses that replicate in the cytoplasm of eukaryotic cells, uses its genomic RNA as a template for both viral protein synthesis and RNA replication. Given that these processes cannot occur simultaneously on the same template, poliovirus has evolved a mechanism(s) to facilitate the switch from using templates for translation to using them for RNA synthesis. This study explores one possible scenario for how the virus alters the functions of a host cell RNA binding protein to mediate, in part, this important transition.T he picornavirus family is made up of small, single-stranded, positive-sense RNA viruses that cause a range of diseases. Poliovirus, coxsackievirus, and human rhinovirus (HRV) are some of the more well-studied members of this family, causing poliomyelitis, myocarditis, and the common cold, respectively. All picornaviruses have a similar genomic RNA structure that lacks a methyl guanosine cap on the 5= terminus. Additionally, the 5= noncoding region (NCR) is highly structured, with six RNA stem-loop structures that preclude canonical ribosome scanning from the 5= end of the template to the initiating start codon (1). Therefore, translation of the picornavirus genome is initiated in a cap-independent manner via an internal ribosome entry site (IRES). For poliovirus, coxsackievirus, and HRV, the IRES is comprised of stem-loop structures II to VI of the 5= NCR (2, 3). Ribosomes must be recruited to the IRES by a mechanism distinct from the canonical cap-dependent recruitment, although the mechanism is not yet understood. Canonical and noncanonical cellular translation factors aid in ribosome recruitment and translation initiation to translate the picornavirus genome into a single polyprotein that is subsequently processed to produce mature viral proteins. Among those proteins produced are the RNA-dependent RNA polymerase 3D and the viral proteinases 2A and 3C, as well as the 3C precursor 3CD,...

Section: Cleavage Of Pcbp2 During Poliovirus and Coxsackievirus Infecmentioning

confidence: 78%

Section: Methodsmentioning

confidence: 99%

Inhibition of Poliovirus-Induced Cleavage of Cellular Protein PCBP2 Reduces the Levels of Viral RNA Replication

Chase

Daijogo

Semler

2014

“…To differentiate between the luciferase signals due to translation from the input viral RNA and from newly replicated RNA, one portion of the transfected cells received 2 mM GnHCl (Fig. 4), a potent inhibitor of PV RNA replication without any toxic effect on cellular processes or viral translation (3,23,33). Translation and replication mediated by the HRV2 IRES in case of the PV1(RIPO)-luc replicon were similar to those of the PV1(M)-luc replicon in HeLa R19 cells, although the kinetics of PV1(RIPO)-luc replication was somewhat delayed at 39.5°C (Fig.…”

Section: Resultsmentioning

confidence: 99%

A Host-Specific, Temperature-Sensitive Translation Defect Determines the Attenuation Phenotype of a Human Rhinovirus/Poliovirus Chimera, PV1(RIPO)

Jahan¹,

Wimmer²,

Mueller³

2011

By using a rhinosvirus/poliovirus type 1 chimera, PV1(RIPO), with the cognate internal ribosome entry site (IRES) of human rhinovirus type 2 (HRV2), we set out to shed light on the mechanism by which this variant expresses its attenuated phenotype in poliovirus-sensitive, CD155 transgenic (tg) mice and cynomolgus monkeys. Here we report that replication of PV1(RIPO) is restricted not only in human cells of neuronal origin, as was reported previously, but also in cells of murine origin at physiological temperature. This block in replication was enhanced at 39.5°C but, remarkably, it was absent at 33°C. PV1(RIPO) variants that overcame the replication block were derived by serial passage under restrictive conditions in either mouse cells or human neuronal cells. All adapting mutations mapped to the 5-nontranslated region of PV1(RIPO). Variants selected in mouse cells, but not in human neuronal cells, exhibited increased mouse neurovirulence in vivo. The observed strong mouse-specific defect of PV1(RIPO) at nonpermissive temperature correlated with the translational activity of the HRV2 IRES in this chimeric virus. These unexpected results must be kept in mind when poliovirus variants are tested in CD155 tg mice for their neurovirulent potential, particularly in assays of live attenuated oral poliovirus vaccine lots. Virulence may be masked by adverse species-specific conditions in mouse cells that may not allow accurate prediction of neurovirulence in the human host. Thus, novel poliovirus variants in line for possible development of human vaccines must be tested in nonhuman primates.Virulence is the most intriguing phenotype of a virus. It is also the most difficult phenotype to understand, because it involves numerous facilities of the virus and the host organism that work together or against each other. These processes can be studied in tissue culture and in animal models, if available. The virulence phenotype of poliovirus (PV), an enterovirus of Picornaviridae, has been studied for over 100 years, yet many of the fundamental steps in infection and disease progression remain unknown.The host range of poliovirus is restricted to humans and nonhuman primates. This is principally related to the expression of CD155 (pvr), its only cellular receptor. CD155, whose gene expresses four splice variants, is a glycoprotein and cell adhesion protein carrying multiple functions in the host organism (29,37,41). The isolation and characterization of the CD155 gene made it possible to construct CD155 transgenic (tg) mice which, on injection with PV, show symptoms of paralysis similar to those of human poliomyelitis (28, 50). The occurrence of classical poliomyelitis in the CD155 tg mice implies that the distribution and function of the receptor in neuronal tissues in the animals are similar to those in humans (28, 50). However, CD155 tg mice cannot be infected orally, the natural route of poliovirus entry in humans, regardless of how the receptor is expressed (different promoters, different splice variants), a phenomenon that...

“…The most extensively studied inhibitor of 2C is probably GuaHCl (14,17,42,56). Several studies with guanidine-resistant poliovirus (52)(53)(54)66) led to the conclusion that guanidine resistance can be attributed mainly to two mutations in a loop adjacent to motif B, designated "class N" or "class M" mutations.…”

Section: Discussionmentioning

confidence: 99%

The Thiazolobenzimidazole TBZE-029 Inhibits Enterovirus Replication by Targeting a Short Region Immediately Downstream from Motif C in the Nonstructural Protein 2C

Palma

Heggermont

Lanke

et al. 2008

TBZE-029 {1-(2,6-difluorophenyl)-6-trifluoromethyl-1H,3H-thiazolo[3,4-a]benzimidazole} is a novel selec-Entero-and rhinoviruses are involved in a wide range of infections in humans and animals. Among the species in the enterovirus genus are the coxsackieviruses, which have been reported to be associated with various clinical manifestations, including myocarditis, pancreatitis, meningitis, and encephalitis. Other clinically relevant enteroviruses are poliovirus, which can cause paralytic poliomyelitis, and echovirus, causing aseptic meningitis or encephalomyelitis (33,36,43). Rhinoviruses are the main pathogens associated with the common cold, and, although usually mild and self-limiting, rhinovirus infections have an enormous socioeconomical impact (47,59).Enteroviruses belong to the family Picornaviridae, which consists of small, nonenveloped viruses containing a singlestranded positive-strand RNA [(ϩ)RNA] genome of 7.5 kb, which is covalently linked to a small viral protein (VPg) at its 5Ј end and polyadenylated at its 3Ј end (49). The genomic RNA has a long, highly structured 5Ј noncoding region, which contains the internal ribosome entry site, necessary for translation initiation, and a shorter 3Ј noncoding region preceding the poly(A) tract, which are both thought to be involved in RNA replication and translation (9). The coding region encodes a single polyprotein that will eventually be cleaved to generate 4 structural and 10 nonstructural proteins (either mature or in their precursor form). The icosahedral capsid of the virus is formed by 60 protomers, each one assembled by the four structural proteins, designated VP1 to -4. The nonstructural region comprises two proteases, the viral RNA-dependent RNA polymerase and seven other proteins that are involved in viral replication (8).One of the most conserved nonstructural viral proteins among picornaviruses is 2C. Although this protein is an indispensable component of the replication complex, its exact role in viral replication has remained elusive. Protein 2C appears to be multifunctional, and this entails multiple interactions (23). The amino acid sequence of 2C contains three conserved motifs which are typically found in NTP-binding proteins (motifs A and B) or in members of helicase superfamily III (motif C) (28)(29)(30)(31)69). In fact, ATPase activity has been demonstrated for several picornavirus 2C proteins (37, 57, 60) whereas so far, every attempt to demonstrate in vitro RNA helicase activity has failed. Protein 2C contains two regions involved in RNA binding (58). It has been suggested that, in poliovirus, this binding occurs at the 3Ј cloverleaf of (Ϫ)RNA (3-5); for echovirus, RNA binding was reported to occur in a nonspecific way