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

Abstract

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 ...

Extensive recoding of dengue virus type 2 specifically reduces replication in primate cells without gain-of-function in Aedes aegypti mosquitoes

“…This is intriguing as the ribosome travels through the entire ORF thereby encountering individual segments of codon pair deoptimized sequences. Unlike the loss of protein expression observed in CPD Influenza viruses [24,29] or reduced protein expression in a codon pair deoptimized poliovirus variant [49]we do not have evidence of specific loss of recoded protein expression in DENV2 syn infected cells. Importantly, neither decreased temperature (Fig.…”

Emergency Services of Viral RNAs: Repair and Remodeling

“…Many nucleotide point mutations in the protein-encoding regions are synonymous, and synonymous mutations are often neutral. Remarkably, simultaneous introduction of 1,297 synonymous nucleotide changes into the poliovirus genome did not appreciably change the viral phenotype (53). However, the biological equality of synonymous codons has several limitations.…”

“…First, synonymous mutations are not necessarily neutral because of the phenotypic significance of codon (and codon pair) biases, as shown for picornaviruses (50,51,53,(209)(210)(211)(212), other positive-strand RNA viruses (213)(214)(215)(216)(217), and negative-strand RNA viruses (217)(218)(219)(220)(221)(222)(223). These biases may be due mainly to the effects on kinetics of translation (e.g., owing to uneven representation of tRNA species corresponding to different synonymous codons and to some features of the ribosome machinery [224]).…”

“…Hence, only a minute minority of the potentially infectious genomes invading target cells appears to have the chance to initiate viral reproduction. Very little is known about the nature and factors affecting this phenomenon, except that the specific infectivity of viruses or viral RNA (number of virions or RNA molecules able to start productive infection) may vary by several orders of magnitude, depending on the primary structure of the genome (50)(51)(52)(53), and also varies for different host cells. The number of physical particles needed to avoid a fitness decrease due to a natural bottleneck is generally unknown, although these values may be quite relevant.…”

Coronavirus genomic RNA packaging

“…In particular, for poliovirus no clearly defined PS has ever been uncovered, despite extensive searches (Jiang et al, 2014). Moreover, exhaustive recoding experiments appear to rule out the existence of cryptic PSs in the poliovirus genome (Song et al, 2017). This has led to the hypothesis that polio capsid protein-replicase interactions are sufficient to ensure the specificity of genome packaging.…”