Large-scale recoding of an arbovirus genome to rebalance its insect versus mammalian preference

Shen, Sam H.; Stauft, Charles B.; Gorbatsevych, Oleksandr; Song, Yinglin; Ward, Charles B.; Yurovsky, Alisa; Mueller, Steffen; Futcher, Bruce; Wimmer, Eckard

doi:10.1073/pnas.1502864112

Cited by 95 publications

(130 citation statements)

References 43 publications

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“…1 i and C). By testing CPD of various mammalian viruses (16,(56)(57)(58)(59)(60) in tissue culture or in experimental animals, we have concluded that there is no single specific effect of CPD that can be related with certainty to observed deficiencies in viral proliferation of Min constructs.…”

Section: Discussionmentioning

confidence: 99%

“…The increase in CpG content (Table S4) may lead to an enhanced innate immune response in the infected cell (56,61); (ii) a robust ts phenotype (59,60); (iii) a reduction (degradation?) of the yield of viral mRNA (57) The experiments with A549 cells were revealing because an inhibitor (BX795) of the innate immune response (30,31) reversed to some extent the low si values in these cells of the three viruses tested.…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

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.

Self Cite

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“…Since deoptimization is due to the summation effect at each of hundreds or thousands of nucleotide mutations without changing amino acid sequences, the likelihood of reversion to virulence is expected to be reduced. Indeed, this technology has proven useful to derive attenuated strains of positive-strand RNA viruses such as poliovirus (16)(17)(18), porcine reproductive and respiratory syndrome virus (PRRSV) (29), and dengue virus (26), as well as those of negative-sense RNA viruses such as influenza virus (20), respiratory syncytial virus (24), and vesicular stomatitis virus (27).…”

Section: Discussionmentioning

confidence: 99%

“…However, alternative mechanisms involving the induction of innate immunity as a result of changing dinucleotide frequency have been proposed by other authors (23). Furthermore, codon pair bias deoptimization of other viruses, including respiratory syncytial virus (RSV) (24), human immunodeficiency virus type 1 (25), dengue virus (26), and vesicular stomatitis virus (27), have also led to attenuated strains. All these reports indicate that codon and codon pair bias deoptimization can also be a useful tool to develop attenuated vaccine candidates against RNA viruses.…”

Section: Foot-and-mouth Disease (Fmd) Ismentioning

confidence: 99%

Synonymous Deoptimization of Foot-and-Mouth Disease Virus Causes Attenuation In Vivo while Inducing a Strong Neutralizing Antibody Response

Segundo

Medina

Ramírez-Medina

et al. 2016

J Virol

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Codon bias deoptimization has been previously used to successfully attenuate human pathogens, including poliovirus, respiratory syncytial virus, and influenza virus. We have applied a similar technology to deoptimize the capsid-coding region (P1) of foot-andmouth disease virus (FMDV). Despite the introduction of 489 nucleotide changes (19%), synonymous deoptimization of the P1 region rendered a viable FMDV progeny. The resulting strain was stable and reached cell culture titers similar to those obtained for wild-type (WT) virus, but at reduced specific infectivity. Studies in mice showed that 100% of animals inoculated with the FMDV A12 P1 deoptimized mutant (A12-P1 deopt) survived, even when the animals were infected at doses 100 times higher than the dose required to cause death by WT virus. All mice inoculated with the A12-P1 deopt mutant developed a strong antibody response and were protected against subsequent lethal challenge with WT virus at 21 days postinoculation. Remarkably, the vaccine safety margin was at least 1,000-fold higher for A12-P1 deopt than for WT virus. Similar patterns of attenuation were observed in swine, in which animals inoculated with A12-P1 deopt virus did not develop clinical disease until doses reached 1,000 to 10,000 times the dose required to cause severe disease in 2 days with WT A12. Consistently, high levels of antibody titers were induced, even at the lowest dose tested. These results highlight the potential use of synonymous codon pair deoptimization as a strategy to safely attenuate FMDV and further develop live attenuated vaccine candidates to control such a feared livestock disease. Our work demonstrates that newly developed codon pair bias deoptimization technologies can be applied to FMD virus to obtain attenuated strains with potential for further development as novel live attenuated vaccine candidates that may rapidly control disease without reverting to virulence.

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“…The resulting mutant viruses could replicate at the same level as the WT virus in mosquito cells, but their replication was impaired in mammalian cells. They were also attenuated in a mouse model of virus pathogenicity [110] .…”

Section: Large-scale Reencoding To Produce Attenuated Virusesmentioning

confidence: 99%

Identifying Attenuating Mutations: Tools for a New Vaccine Design against <b><i>Flaviviruses</i></b>

Khou

Pardigon²

2017

Intervirology

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Emerging Flaviviruses pose an increasing threat to global human health. To date, human vaccines against yellow fever virus (YFV), Japanese encephalitis virus (JEV), dengue virus (DV), and tick-borne encephalitis virus (TBEV) exist. However, there is no human vaccine against other Flaviviruses such as Zika virus (ZIKV) and West Nile virus (WNV). In order to restrict their spread and to protect populations against the diseases they induce, vaccines against these emerging viruses must be designed. Obtaining new live attenuated Flavivirus vaccines using molecular biology methods is now possible. Molecular infectious clones of the parental viruses are relatively easy to generate. Key mutations present in live attenuated vaccines or mutations known to have a key role in the Flavivirus life cycle and/or interactions with their hosts can be identified by sequencing, and are then inserted in infectious clones by site-directed mutagenesis. More recently, the use of chimeric viruses and large-scale reencoding and introduction of microRNA target sequences have also been tested. Indeed, a combination of these methods will help in designing new generations of vaccines against emerging and reemerging Flaviviruses.

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Large-scale recoding of an arbovirus genome to rebalance its insect versus mammalian preference

Cited by 95 publications

References 43 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

Synonymous Deoptimization of Foot-and-Mouth Disease Virus Causes Attenuation In Vivo while Inducing a Strong Neutralizing Antibody Response

Identifying Attenuating Mutations: Tools for a New Vaccine Design against <b><i>Flaviviruses</i></b>

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