Computer-aided codon-pairs deoptimization of the major envelope GP5 gene attenuates porcine reproductive and respiratory syndrome virus

Ni, Yanyan; Zhao, Zhao; Opriessnig, Tanja; Subramaniam, Sakthivel; Zhou, Lei; Cao, Dianjun; Cao, Qian M.; Yang, Hanchun; Meng, Xiang‐Jin

doi:10.1016/j.virol.2013.12.009

Cited by 63 publications

(53 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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%

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.

View full text Add to dashboard Cite

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 ...

show abstract

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.

View full text Add to dashboard Cite

show abstract

“…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%

“…Wild-type FMDV A12 (FMDV A12-WT) was generated from the full-length serotype A12 infectious clone pRMC35 (28). FMDV A12-P1 deopt was derived by modifying the codon pair bias in the 2,210-nucleotide (nt) complete P1-coding region using SAVE technology (19,29). The deoptimized fragment and 355-nt flanking viral sequences were synthesized by GenScript (Piscataway, NJ).…”

Section: Cellsmentioning

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

View full text Add to dashboard Cite

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.

show abstract

“…In principle, on the background of thousands of attenuating mutations, any single-site reversion should yield only a minuscule selective advantage. The most likely path to reversion imaginable under this model is the progressive accumulation of many individual mutations, providing for a slow progression of deattenuation (12)(13)(14).To date, genetic stability studies of large-scale deoptimized viruses have shown that deattenuation, indeed, seems to be low, suggesting that these viruses are genetically stable (6,8,(15)(16)(17)(18)(19)(20)(21). However, an important limitation of these studies is that the deoptimized viruses generally have not been subjected to strong selective pressure that…”

mentioning

confidence: 99%

“…To date, genetic stability studies of large-scale deoptimized viruses have shown that deattenuation, indeed, seems to be low, suggesting that these viruses are genetically stable (6,8,(15)(16)(17)(18)(19)(20)(21). However, an important limitation of these studies is that the deoptimized viruses generally have not been subjected to strong selective pressure that…”

mentioning

confidence: 99%

Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure

Nouën

McCarty

Brown

et al. 2017

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

View full text Add to dashboard Cite

Recoding viral genomes by numerous synonymous but suboptimal substitutions provides live attenuated vaccine candidates. These vaccine candidates should have a low risk of deattenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. We evaluated phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure. Codon pair deoptimized (CPD) versions of RSV were attenuated and temperature-sensitive. During serial passage at progressively increasing temperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs did not lose temperature sensitivity, remained genetically stable, and was restricted at temperatures of 34°C/35°C and above. However, a CPD RSV containing 1,378 synonymous mutations solely in the polymerase L ORF quickly lost substantial attenuation. Comprehensive sequence analysis of virus populations identified many different potentially deattenuating mutations in the L ORF as well as, surprisingly, many appearing in other ORFs. Phenotypic analysis revealed that either of two competing mutations in the virus transcription antitermination factor M2-1, outside of the CPD area, substantially reversed defective transcription of the CPD L gene and substantially restored virus fitness in vitro and in case of one of these two mutations, also in vivo. Paradoxically, the introduction into Min L of one mutation each in the M2-1, N, P, and L proteins resulted in a virus with increased attenuation in vivo but increased immunogenicity. Thus, in addition to providing insights on the adaptability of genome-scale deoptimized RNA viruses, stability studies can yield improved synthetic RNA virus vaccine candidates.negative-strand RNA virus | respiratory syncytial virus | live attenuated vaccine | codon pair deoptimization | genetic stability T he availability and affordability of large-scale custom DNA synthesis opened the new field of synthetic biology (1). The combined approach of sequence design and synthetic biology allows the generation of DNA molecules with extensive targeted modifications. Synonymous genome recoding, in which one or more ORFs of a microbial pathogen are modified at the nucleotide level without affecting amino acid coding, currently is being widely evaluated to reduce pathogen fitness and create potential live attenuated vaccines, particularly for RNA viruses (reviewed in ref.2) (3-7). The main strategies for attenuation by synonymous genome recoding are codon deoptimization (CD), codon pair deoptimization (CPD), and increasing the dinucleotide CpG and UpA content (which is usually the result of CD and CPD) (2).The mechanisms of attenuation by these strategies are currently under intensive research. It has been suggested that the primary effect of CD and CPD is to reduce translation efficiency of pathogen mRNAs, thereby providing attenuation (8). Effects on mRNA stability also can be a factor (9). In addition, recent studies suggested that codon pa...

show abstract

Computer-aided codon-pairs deoptimization of the major envelope GP5 gene attenuates porcine reproductive and respiratory syndrome virus

Cited by 63 publications

References 29 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

Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure

Contact Info

Product

Resources

About