Encapsidation studies of poliovirus subgenomic replicons.

Barclay, Wendy S.; Li, Quanyi; Hutchinson, Gordon B.; Moon, David Henry; Richardson, Andrew J.; Percy, N; Almond, Jeffrey W.; Evans, David J.

doi:10.1099/0022-1317-79-7-1725

Cited by 40 publications

(43 citation statements)

References 49 publications

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“…3). The 5'-and 3'-terminal RNA segments do not determine assembly specificity, as was shown (35,52). Domain P1 has also been omitted because it does not play any role in morphogenesis based on properties of PV "defective interfering particles" (DI particles) (53,54).…”

Section: Resultsmentioning

confidence: 99%

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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Section: Resultsmentioning

confidence: 99%

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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“…2 Standard naming conventions: A pT7/prefix indicates a plasmid with a T7 promoter containing the virus or subgenomic replicon cDNA, pT7/ Rep3 was derived from pT7/FLC (Barclay et al 1998), a "full-length (infectious) clone" of poliovirus type 3 Leon. The resulting replicon or virus generated following transfection are designated Rep3 or FLC, respectively.…”

Section: Functional Analysis Of the Poliovirus Crementioning

confidence: 99%

“…Construction and use of a cassette system for CRE mutagenesis and duplication pT7/Rep3 is a plasmid containing a cDNA for a poliovirus subgenomic replicon 2 previously constructed by replacing the majority of the P1-encoding region (the virus capsid proteins) with a chloramphenicol acetyltransferase (CAT) reporter gene, engineered in-frame with the truncated polyprotein (Percy et al 1992;Barclay et al 1998).…”

Section: Functional Analysis Of the Poliovirus Crementioning

confidence: 99%

“…PCR mutagenesis was used to introduce a unique Sma I site adjacent to the Bss HII site near the in-frame fusion of the chloramphenicol acetyl transferase (CAT) gene and the region encoding the carboxyl-terminus of VP1 in the poliovirus type 3 (Leon)-derived replicon, pT7/Rep3 (described previously by Barclay et al 1998;Goodfellow et al 2000). Similar modifications were made to pT7/Rep3/SL3, a replication-incompetent version of pT7/Rep3 containing eight noncoding substitutions within the region of poliovirus 2C that forms the CRE (Goodfellow et al 2000).…”

Section: Construction Of Cat-encoding Replicon-based Cassette Vectorsmentioning

confidence: 99%

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Structure and function analysis of the poliovirus cis-acting replication element (CRE)

Goodfellow

Kerrigan²,

Evans³

2003

RNA

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The poliovirus cis-acting replication element (CRE) templates the uridylylation of VPg, the protein primer for genome replication. The CRE is a highly conserved structural RNA element in the enteroviruses and located within the polyprotein-coding region of the genome. We have determined the native structure of the CRE, defined the regions of the structure critical for activity, and investigated the influence of genomic location on function. Our results demonstrate that a 14-nucleotide unpaired terminal loop, presented on a suitably stable stem, is all that is required for function. These conclusions complement the recent analysis of the 14-nucleotide terminal loop in the CRE of human rhinovirus type 14. The CRE can be translocated to the 5 noncoding region of the genome, at least 3.7-kb distant from the native location, without adversely influencing activity, and CRE duplications do not adversely influence replication. We do not have evidence for a specific interaction between the CRE and the RNA-binding 3CD pro complex, an essential component of the uridylylation reaction, and the mechanism by which the CRE is coordinated and orientated during the reaction remains unclear. These studies provide a detailed overview of the structural determinants required for CRE function, and will facilitate a better understanding of the requirements for picornavirus replication.

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“…We therefore engineered the expression of a reporter gene to monitor replication and packaging of EDIbased replicons in a sensitive and convenient manner. A similar approach has been used successfully during molecular studies on the replication, packaging, and transcription of, e.g., coronavirus and poliovirus subgenomic replicons (1,20,27,29,53). Clearly, this kind of analysis is most straightforward when the assay is based directly on translation of the replicating RNA itself.…”

Section: Generation Of Eav DI Rnas During Serial Undiluted Virus Passmentioning

confidence: 99%

Isolation and Characterization of an Arterivirus Defective Interfering RNA Genome

Molenkamp

Rozier

Greve

et al. 2000

J Virol

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Equine arteritis virus (EAV), the type member of the family Arteriviridae, is a single-stranded RNA virus with a positive-stranded genome of approximately 13 kb. EAV uses a discontinuous transcription mechanism to produce a nested set of six subgenomic mRNAs from which its structural genes are expressed. We have generated the first documented arterivirus defective interfering (DI) RNAs by serial undiluted passaging of a wild-type EAV stock in BHK-21 cells. A cDNA copy of the smallest DI RNA (5.6 kb) was cloned. Upon transfection into EAV-infected BHK-21 cells, transcripts derived from this clone (pEDI) were replicated and packaged. Sequencing of pEDI revealed that the DI RNA was composed of three segments of the EAV genome (nucleotides 1 to 1057, 1388 to 1684, and 8530 to 12704) which were fused in frame with respect to the replicase reading frame. Remarkably, this DI RNA has retained all of the sequences encoding the structural proteins. By insertion of the chloramphenicol acetyltransferase reporter gene in the DI RNA genome, we were able to delimitate the sequences required for replication/DI-based transcription and packaging of EAV DI RNAs and to reduce the maximal size of a replication-competent EAV DI RNA to approximately 3 kb.Equine arteritis virus (EAV) is the type member of the family Arteriviridae (38), which was recently grouped together with the coronaviruses and the toroviruses in the newly established order of the Nidovirales (4, 13). Other members of the Arteriviridae are Porcine reproductive and respiratory syndrome virus, Lactate dehydrogenase-elevating virus, and Simian hemorrhagic fever virus.EAV is a spherical, enveloped virus with a diameter of 50 to 60 nm (18, 30) and a positive-stranded RNA genome of about 12,700 nucleotides (nt) (8). The virion envelope is derived from intracellular host cell membranes and contains two major and three or four minor structural proteins (12,38,39). The envelope surrounds an isometric nucleocapsid of about 35 nm (18), which is composed of the genomic RNA and multiple copies of the nucleocapsid protein (N).The EAV replicase is produced in the form of two large polyproteins: the open reading frame 1a (ORF1a) protein and the ORF1ab protein, the C-terminal part of which is expressed by ORF1a/1b ribosomal frameshifting (8). An apparently complete proteolytic processing scheme for the ORF1a and ORF1ab nonstructural polyproteins, which results in the generation of 12 end products (nsp1 to nsp12) and a large number of processing intermediates, was recently obtained (41,46,47,50).The EAV structural proteins are translated from a 3Ј-coterminal nested set of subgenomic (sg) mRNAs, which also carry a common 5Ј leader sequence derived from the 5Ј end of the genome (11). The mechanism of sg mRNA transcription, which resembles that of the coronaviruses in many aspects, involves a discontinuous step of which many details remain to be elucidated (4,24,38,48).Little is known about the genome replication of arteriviruses at the molecular level. The genomic 3Ј end is polyadenyla...

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Encapsidation studies of poliovirus subgenomic replicons.

Cited by 40 publications

References 49 publications

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

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

Structure and function analysis of the poliovirus cis-acting replication element (CRE)

Isolation and Characterization of an Arterivirus Defective Interfering RNA Genome

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