The Making of a Picornavirus Genome

Wimmer, Eckard; Paul, Aniko V.

doi:10.1128/isbn978-1-55581-603-2.3

Cited by 14 publications

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“…There is evidence that a 20% reduction in genome size may represent the largest deletion compatible with DI virion stability, and Nomoto and his colleagues (27,36) made the important observation that deletions in the P1 region must be in frame with the initiation codon at nt 743 in order for DI genome replication to occur (15). This has led to the discovery of the secondary proofreading mechanism in picornavirus proliferation: correct translation and processing of the polyprotein are required for RNA replication, which, in turn, is required for encapsidation (45,49,50,71).…”

Section: Discussionmentioning

confidence: 99%

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Evolution of Poliovirus Defective Interfering Particles Expressing Gaussia Luciferase

Song

Paul

Wimmer

2012

J Virol

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Polioviruses (PVs) carrying a reporter gene are useful tools for studies of virus replication, particularly if the viral chimeras contain the polyprotein that provides all of the proteins necessary for a complete replication cycle. Replication in HeLa cells of a previously constructed poliovirus expressing the gene for Renilla luciferase (RLuc) fused to the N terminus of the polyprotein H 2 NRLuc-P1-P2-P3-COOH (P1, structural domain; P2 and P3, nonstructural domains) led to the deletion of RLuc after only one passage. Here we describe a novel poliovirus chimera that expresses Gaussia luciferase (GLuc) inserted into the polyprotein between P1 and P2 (N 2 H-P1-GLuc-P2-P3-COOH). This chimera, termed PV-GLuc, replicated to 10% of wild-type yield. The reporter signal was fully retained for three passages and then gradually lost. After six passages the signal was barely detectable. On further passages, however, the GLuc signal reappeared, and after eight passages it had reached the same levels observed with the original PV-GLuc at the first passage. We demonstrated that this surprising observation was due to coevolution of defective interfering (DI) particles that had lost part or all of the capsid coding sequence (⌬P1-GLuc-P2-P3) and wild-type-like viruses that had lost the GLuc sequence (P1-P2-P3). When used at low passage, PV-GLuc is an excellent tool for studying aspects of genome replication and morphogenesis. The GLuc protein was secreted from mammalian cells but, in agreement with published data, was not secreted from PV-GLuc-infected cells due to poliovirus-induced inhibition of cellular protein secretion. Published evidence indicates that individual expression of enterovirus polypeptide 3A, 2B, or 2BC in COS-1 cells strongly inhibits host protein secretion. In HeLa cells, however, expression of none of the poliovirus polypeptides, either singly or in pairs, inhibited GLuc secretion. Thus, inhibition of GLuc secretion in PV-infected HeLa cells is likely a result of the interaction between several viral and cellular proteins that are different from those in COS-1 cells.

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Section: Discussionmentioning

confidence: 99%

“…Since in vivo poliovirus RNA replication is stringently dependent upon translation in cis (33,49) and because encapsidation, in turn, requires RNA replication in cis (1,33,34,45,71), it is difficult to design reliable experiments that distinguish between defects in viral replication and morphogenesis.…”

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Evolution of Poliovirus Defective Interfering Particles Expressing Gaussia Luciferase

Song

Paul

Wimmer

2012

J Virol

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“…We extensively tested DEmARC on the proteome of the Picornaviridae (29), one of the most diverse and well-studied RNA virus families (23,59) with numerous species that has been developed by one of the most active SGs (36,37,64). The picornavirus genome is a singlestranded positive-sense RNA (ssRNAϩ) with a single open reading frame that encodes a polyprotein (46,50) flanked by two untranslated regions, 5=-UTR and 3=-UTR (69). The consistency and stability of the obtained results were evaluated by analyzing various data set derivatives which were compiled by varying the amount and/or the diversity of the input data, the alignment construction method, the measure of pairwise similarity, or a combination of parameters.…”

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Partitioning the Genetic Diversity of a Virus Family: Approach and Evaluation through a Case Study of Picornaviruses

Lauber

Gorbalenya

2012

J Virol

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The recent advent of genome sequences as the only source available to classify many newly discovered viruses challenges the development of virus taxonomy by expert virologists who traditionally rely on extensive virus characterization. In this proof-ofprinciple study, we address this issue by presenting a computational approach (DEmARC) to classify viruses of a family into groups at hierarchical levels using a sole criterion-intervirus genetic divergence. To quantify genetic divergence, we used pairwise evolutionary distances (PEDs) estimated by maximum likelihood inference on a multiple alignment of family-wide conserved proteins. PEDs were calculated for all virus pairs, and the resulting distribution was modeled via a mixture of probability density functions. The model enables the quantitative inference of regions of distance discontinuity in the family-wide PED distribution, which define the levels of hierarchy. For each level, a limit on genetic divergence, below which two viruses join the same group, was objectively selected among a set of candidates by minimizing violations of intragroup PEDs to the limit. In a case study, we applied the procedure to hundreds of genome sequences of picornaviruses and extensively evaluated it by modulating four key parameters. It was found that the genetics-based classification largely tolerates variations in virus sampling and multiple alignment construction but is affected by the choice of protein and the measure of genetic divergence. In an accompanying paper (C. Lauber and A. E. Gorbalenya, J. Virol. 86:3905-3915, 2012), we analyze the substantial insight gained with the genetics-based classification approach by comparing it with the expert-based picornavirus taxonomy.

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“…The PP (7) is an active molecule that cleaves itself into ∼29 polypeptides by two viral proteinases (2A pro and 3C pro /3CD pro ) and an enzyme-independent maturation cleavage (Fig. 1A) (5,6,8).Capsid domain P1 controls the identity of PV by determining virion structure (9), serotype identity (10), and interaction with the cellular receptor CD155 (10). Since PV replicates as quasispecies at an error rate of ∼10 −4 (11), the following questions arise: How conserved is its synonymous sequence given the astronomical number of alternative possibilities?…”

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Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

Song¹,

Gorbatsevych²,

Liu³

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Computer design and chemical synthesis generated viable variants of poliovirus type 1 (PV1), whose ORF (6,189 nucleotides) carried up to 1,297 "Max" mutations (excess of overrepresented synonymous codon pairs) or up to 2,104 "SD" mutations (randomly scrambled synonymous codons). "Min" variants (excess of underrepresented synonymous codon pairs) are nonviable except for P2 Min , a variant temperature-sensitive at 33 and 39.5 • C. Compared with WT PV1, P2 Min displayed a vastly reduced specific infectivity (si) (WT, 1 PFU/118 particles vs. P2 Min , 1 PFU/35,000 particles), a phenotype that will be discussed broadly. Si of haploid PV presents cellular infectivity of a single genotype. We performed a comprehensive analysis of sequence and structures of the PV genome to determine if evolutionary conserved cis-acting packaging signal(s) were preserved after recoding. We showed that conserved synonymous sites and/or local secondary structures that might play a role in determining packaging specificity do not survive codon pair recoding. This makes it unlikely that numerous "cryptic, poliovirus | genome recoding | packaging signal | specific infectivity T he capsid precursor P1 (881 amino acids) of type 1 poliovirus (PV), mapping to the N terminus of the polyprotein (PP) (Fig. 1A), can be encoded in 10 442 ways (1) due to the degenerate genetic code. The tiniest fraction of these possible sequences defines PV, the cause of poliomyelitis. PV occurs in three serotypes, of which the most neurovirulent type 1 Mahoney [PV1(M)], the main viral species analyzed in this study, was isolated in 1941 from pooled feces of three healthy children (2).Genome sequence (3, 4) and gene organization (3) of PV1(M) revealed highly complex structures in its 5'-terminal nontranslated region (5'-NTR), followed by a single ORF encoding the PP, followed by a complex 3'-heteropolymeric region and poly(A) tail (Fig. 1A) (5, 6). The PP (7) is an active molecule that cleaves itself into ∼29 polypeptides by two viral proteinases (2A pro and 3C pro /3CD pro ) and an enzyme-independent maturation cleavage (Fig. 1A) (5,6,8).Capsid domain P1 controls the identity of PV by determining virion structure (9), serotype identity (10), and interaction with the cellular receptor CD155 (10). Since PV replicates as quasispecies at an error rate of ∼10 −4 (11), the following questions arise: How conserved is its synonymous sequence given the astronomical number of alternative possibilities? What encoding could have coevolved that would be optimal to specify 881 capsid residues?If PV, a member of the genus Enterovirus of Picornaviridae, is an evolutionary descendant of C-cluster Coxsackie viruses (C-CAVs) (12), the evolution of PV nucleotide sequences was constrained as it adhered to the basic architecture of C-CAVs, its evolutionary parents (13). A second well-known restriction of sequence variability in ORFs is "codon bias" (14), the unequal use of synonymous codons. Encoding the PV1 P1 domain with an excess of "rarely used" (human) synonymous codons results in a ...

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The Making of a Picornavirus Genome

Cited by 14 publications

References 0 publications

Evolution of Poliovirus Defective Interfering Particles Expressing Gaussia Luciferase

Evolution of Poliovirus Defective Interfering Particles Expressing Gaussia Luciferase

Partitioning the Genetic Diversity of a Virus Family: Approach and Evaluation through a Case Study of Picornaviruses

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

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