Virus attenuation by genome-scale changes

Published online Aug Collins , a Ursula J. Buchholz , a, 3 and Joshua M. DiNapoli a, 3, 4. Linda G. Peter L. Ursula J. Joshua M. Author information Copyright and License information Disclaimer. Email: vog. Contributed by Eckard Wimmer, June 18, sent for review February 14, Copyright notice. This article has been cited by other articles in PMC.

Significance Human respiratory syncytial virus RSV is the most important viral agent of serious pediatric respiratory-tract disease. Keywords: negative strand RNA virus, pneumovirus, live attenuated vaccine.

Abstract Human respiratory syncytial virus RSV is the most important viral agent of serious pediatric respiratory-tract disease worldwide. Open in a separate window. Table 1. For viruses with a ts phenotype, the shut-off temperatures T SH are listed in boldface at the right. Effects on Specific Viral Infectivity. Table 2. The number of animals shedding virus is indicated in parentheses. A value of 0. Based on this assumption, NP shedding results from this AGM were excluded from calculation of means and from statistical analysis.

Discussion We investigated the recently described strategy of CPD 5 , 10 as a means to develop new attenuated versions of RSV as potential live vaccine candidates.

Virus Growth and Titration. Supplementary Material Supplementary File Click here to view. Footnotes Conflict of interest statement: C. References 1. Respiratory syncytial virus and metapneumovirus. Viral and host factors in human respiratory syncytial virus pathogenesis. J Virol. Hall CB, et al. The burden of respiratory syncytial virus infection in young children.

N Engl J Med. Progress in understanding and controlling respiratory syncytial virus: Still crazy after all these years. Virus Res. Coleman JR, et al. Virus attenuation by genome-scale changes in codon pair bias. Folley LS, Yarus M. Codon contexts from weakly expressed genes reduce expression in vivo. E-mail: ude. Copyright notice. The publisher's final edited version of this article is available at Science. See other articles in PMC that cite the published article.

Abstract As a result of the redundancy of the genetic code, adjacent pairs of amino acids can be encoded by as many as 36 different pairs of synonymous codons. Open in a separate window. Table 1 Poliovirus specific infectivity and attenuation.

Supplementary Material pdf Supporting Online Material: www. References and Notes 1. See supporting material on Science Online. Buchan J. Nucleic Acids Res. Burns CC, et al. Zhao WD, Wimmer E. Koike S. In: Cellular Receptors for Animal Viruses.

Wimmer E, editor. Landsteiner K, Popper E. Z Immun, Forsch. Moura G, et al. Jiang P, et al. Kew O, et al. Cello, Cancer Res. We thank H Toyoda for lending his expertise to the mouse experiments, W. McCaig for performing serial passages of viral variants, and J. Cello and W. Karzai for comments on the manuscript. All authors declare that they have a patent pending relating to certain aspects of this work.

Support Center Support Center. External link. The observed fitness evolution of T7:LME offers mixed support for the prediction that fitness will continue increasing indefinitely toward the wild-type value.

Although molecular and fitness evolution continued beyond generations, the rate of fitness increase was small, and extrapolation of the current trajectory would suggest a fitness limit well below that of wild-type. Even at its current fitness, this genome is the least attenuated of the 3 genomes studied here Figure 7. Evolutionary stable attenuation levels of T7 genomes with different types of gene rearrangements.

All maximum fitness values vertical axis are substantially below that of the evolved wild-type Error bars represent 1 std err and are barely visible in all cases. Note that fitness maxima are expressed as log 2 , so major fitness differences are only slightly different visually. This study addressed several aspects of viral attenuation by genome rearrangement, a method of attenuation that was first developed for the eukaryotic virus VSV Ball et al. The fact that genome rearrangement also attenuates the larger, double-stranded DNA virus T7 suggests that it may be a broadly applicable mechanism in viruses.

Not all rearrangements of VSV exhibited strongly reduced fitness Novella et al. Our specific focus is the evolutionary stability of attenuation—whether the attenuated virus will readily evolve to greater fitness across tens to hundreds of generations of adaptation.

We equated the degree of attenuation with a quantitative reduction in viral fitness, a measure suitable for many purposes. Only one previous study has evaluated the evolutionary stability of attenuation by genome rearrangement Springman et al.

The virus used here was the bacteriophage T7. Following attachment to the host, the rate-limiting step in the T7 life cycle is expression of the phage RNAP gene. The rearranged genome with RNAP gene in its wild-type location but with an exchange of other early-region genes with some late-region genes was considered to be the most likely to recover high fitness because there was no apparent delay in genome entry and expression of the phage RNAP gene.

This a priori expectation does not neglect the imbalance in gene expression that ensues from the atypical gene order, but it does assume that those imbalances can be corrected by compensatory evolution. Despite this expectation, the genome realized only a small fitness increase during generations, and the final fitness was substantially lower than that of wild type. The implication is that many modifications of genome order can achieve attenuation for long periods of evolution, even gene orders that retain the natural order of the elements controlling entry and timing of gene expression.

Furthermore, the fact that gene order has major effects on growth rate even when genome entry is normal suggests that the consequence of altered gene order probably extends to fitness measures that are not tied to a short generation time. The ability to predict the evolutionary stability of attenuation and to predict the final fitness attained by an attenuated genome relies on understanding how fitness can be restored.

The main basis for prediction in two experiments here was displacement of the RNAP gene downstream of its wild-type location. The phage RNAP is central to the phage life cycle. The main effects attributed to RNAP gene displacement are 1 a delay in its expression due to delayed entry, and 2 abnormal levels of expression due to autoamplification the RNAP gene expresses itself when it is placed downstream of phage promoters.

The former effect is easy to quantify with existing data, the latter more difficult. Furthermore, it is to be expected that displacement of the RNAP gene will have other effects; the question is how successful are predictions based on this limited perspective.

Yet even the predicted benefit of an early entry mutation was itself merely an observation from other studies extended to this study, not a theoretically or computationally derived result. Another experiment relied on predictions from the virtual life cycle model of T7 that short displacements of the RNAP gene should have little fitness effect. The observed life cycle delay of such a construct was considerable, implying a serious fitness defect. Consequently, this genome was considered to be a good candidate for recovery of greater fitness because of the contrasting observations and virtual model prediction.

Our results suggest that prediction of the evolutionary response to genome rearrangement is challenging, even in well-studied systems, but not without some success. The evolutionary stability of rearranged T7 genomes may be contrasted with an alternative means of attenuation, the use of rare codons that do not alter protein sequence to maintain lower levels of protein production Burns et al.

Work on poliovirus and T7 have both shown that different forms of engineered suboptimal codon usage can attenuate quantitatively to arbitrary levels. Recovery of attenuated poliovirus was also observed within bounds Burns et al. If the T7 results generalize, genome rearrangement may be far superior to codon de-optimization as a lasting means of attenuation.

One drawback of rearranged genomes, however, is that they carry all the genetic information to restore the wild-type virus through recombination. It must therefore be assured that the probability of such recombination is miniscule. We thank S. Stolte for fitness assays of two phages and reviewers for helpful comments, especially on improving figures. Google Scholar. Nature : — Heredity Edinb : — Virology : — Science : — USA 97 : — Evolution N Y 4 : — Evolution 61 : — BMC Bioinformatics 8 : Google Preview.

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Close mobile search navigation Article Navigation. Volume 3. Article Contents Abstract. Literature Cited. Nicole Cecchini , Nicole Cecchini. Department of Integrative Biology. Oxford Academic. Matthew Schmerer. F Plaque phenotypes of viruses after 72 hours of incubation, stained with crystal violet plate diameter, 35 mm. We developed a computer algorithm that can recode a given amino acid sequence, but using different codon pairs, while controlling other features of the sequence such as the codon bias and the folding free energy of the RNA 2 figs.

S1 and S2. The P1 region is suitable for such experiments because it can be deleted or substituted without affecting genome replication 7.

Virus PV-Min was recoded to use codon pairs that are underrepresented relative to the human genome, and it contains synonymous mutations. Virus PV-Max was recoded to use overrepresented codon pairs, and it contains synonymous mutations. Both PV-Min and PV-Max encode precisely the same amino acid sequences as the wild type, but they use different pairwise arrangements of synonymous codons [ Fig. These P1 fragments were synthesized, sequenced, and incorporated into a full-length cDNA construct of poliovirus Fig.

The PV-Max virus generated plaques identical in size to the wild type Fig. In contrast, the PV-Min RNA produced no visible cytopathic effect after 96 hours, and no viable virus could be isolated even after four blind passages of the supernatant from transfected cells. We subcloned portions of the PV-Min P1 region into an otherwise wild-type virus to reduce the number of underrepresented codon pairs Fig. These subclones yielded viruses with varying degrees of attenuation Fig. Viruses containing P1 fragments X and Y were each slightly attenuated; however, when added together they yielded virus PV-MinXY, which was substantially attenuated Fig.

We conclude that the in-viability of PV-Min was due to the sum of defects in the various subportions. One-step growth kinetics were examined. Like the wild-type virus, the chimeric viruses had an eclipse phase followed by exponential growth. However, as measured by plaque-forming units PFUs , the final titer of PV-Min constructs was decreased by up to a factor of with re-spect to wild-type viruses Fig.

This low plaque titer could have resulted from lower production of virions i. We examined both possibilities 2. When the number of viral particles produced per infected cell was measured, we found that cells infected with PV-MinXY or PV-MinZ produced fewer viral particles than did the wild type, but the effect was only about a factor of 10 or slightly less Fig. For wild-type virus, the number of virions applied per plaque generated was about , whereas for PV-MinZ, the number of virions applied per plaque generated was 13,; hence, the main defect was reduced specific infectivity of the virions.

Poliovirus specific infectivity and attenuation. This observation suggests that their low specific infectivity is not a result of gross defects in the capsid 2 fig. To measure the possible effect of codon pair bias on translation, we used a dicistronic reporter encoding both R-Luc and F-Luc 2 , 5 Fig. Because the F-Luc reporter is translated as a fusion protein with the proteins of the P1 region, the translatability of the P1 region directly affects the amount of F-Luc protein produced.

Thus, the ratio of F-Luc luminescence to R-Luc luminescence is a measure of the translatability of the various P1 encodings. Effect of altered codon pair bias on translation. A Structure of a dicistronic reporter 5. This first cistron provides an internal control to normalize the amount of input RNA.

The region labeled P1 was replaced by the recoded, synthetic P1 regions of the indicated viruses. The variously encoded P1 regions were tested Fig. The reduced translation is probably sufficient to explain the attenuated phenotype, because smaller reductions in translation caused by other methods have been observed to attenuate poliovirus; apparently, poliovirus has a fairly high threshold requirement for translation 5 , 9.

If the attenuation of these viruses were due to hundreds of small defects, it should be difficult for these viruses to revert to wild-type virulence. Alternatively, if most mutations are neutral, with a small minority contributing to attenuation, then reversion should occur.

The titer was monitored for phenotypic reversion, and the passaged virus was sequenced. We next tested whether the synthetic viruses were also attenuated in animals. Viruses were administered to CD transgenic tg mice which express the poliovirus receptor via intracerebral injection 10 , allowing direct exposure to the central nervous system, the ultimate target of poliovirus pathogenesis PV-Max virulence was identical to that of the wild type.

Alternatively, the relatively poor translation of the mutant mRNAs might prevent such a response. Ten days after the final injection, the protective antibodies of the seven surviving mice in each group were measured via microneutralization assay, and a robust immune response was detected fig.

Subsequent challenge of the immunized mice with an otherwise lethal dose of wild-type poliovirus via intramuscular injection did not lead to death or signs of paralysis or paresia; in contrast, all mock-immunized mice died. Technology for the synthesis of large DNAs and for the redesign of living systems 5 , 6 , 12 allows the reengineering of viruses for specific purposes such as vaccines.

We have used these approaches to generate polioviruses that use a large proportion of under- or overrepresented codon pairs. Although it has been known for many years that codon pair usage is biased 1 , this phenomenon has previously been studied primarily by informatics 13 , We now find that underrepresented codon pairs cause poor translation and attenuation in poliovirus. One theory for the existence of codon pair bias is that certain tRNAs interact poorly on the ribosome 3 , and so the codon pairs causing the juxtaposition of such tRNAs are underrepresented; our translation data are consistent with this theory.

We note that attenuation is not caused by random changes in synonymous codons if those changes do not systematically reduce codon bias or codon pair bias. In PV-SD, neither codon bias nor codon pair bias was changed, and that virus was not attenuated 2 , 5 table S2.

Here, we created virus PV-Max, which similarly contained mutations in synonymous codons, and it was also not attenuated Fig. It is noteworthy that even though PV-Max contains overrepresented codons, it is not more virulent than the wild type Table 1 , possibly because evolution has already effectively optimized encoding.

The correlation between the degree of codon pair deoptimization and the degree of viral attenuation, as well as the lack of viral reversion upon passaging, are consistent with the idea that many of the mutations in PV-Min cause small, additive defects.