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Review
, 27 (12), 1163-72

Synthetic Viruses: A New Opportunity to Understand and Prevent Viral Disease

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Review

Synthetic Viruses: A New Opportunity to Understand and Prevent Viral Disease

Eckard Wimmer et al. Nat Biotechnol.

Abstract

Rapid progress in DNA synthesis and sequencing is spearheading the deliberate, large-scale genetic alteration of organisms. These new advances in DNA manipulation have been extended to the level of whole-genome synthesis, as evident from the synthesis of poliovirus, from the resurrection of the extinct 1918 strain of influenza virus and of human endogenous retroviruses and from the restructuring of the phage T7 genome. The largest DNA synthesized so far is the 582,970 base pair genome of Mycoplasma genitalium, although, as yet, this synthetic DNA has not been 'booted' to life. As genome synthesis is independent of a natural template, it allows modification of the structure and function of a virus's genetic information to an extent that was hitherto impossible. The common goal of this new strategy is to further our understanding of an organism's properties, particularly its pathogenic armory if it causes disease in humans, and to make use of this new information to protect from, or treat, human viral disease. Although only a few applications of virus synthesis have been described as yet, key recent findings have been the resurrection of the 1918 influenza virus and the generation of codon- and codon pair-deoptimized polioviruses.

Figures

Figure 1
Figure 1
Comparison of lethality in mice infected with select 1918 and modern human H1N1 influenza A (Tx/91) reassortant viruses. BALB/c mice were inoculated intranasally with 105 PFU of virus to determine which virus genes of the 1918 virus contributed to virulence. Among all eight gene segments tested individually, the HA gene was the only 1918 virus gene able to confer a virulent phenotype when rescued on the genetic background of Tx/91 H1N1 virus. In the reciprocal experiments, the exchange of most of the individual 1918 influenza virus genes with seasonal influenza Tx/91 virus genes did not alter the virulence of the lethal 1918 virus; however, substitution of the HA, NA or PB1 genes substantially affected the ability of this virus to cause severe disease in mice. Illustration by J. Archer (Centers for Disease Control).
Figure 2
Figure 2
The poliovirus genome and the effect of codon bias. (a) Poliovirus genomic RNA, is of plus-strand polarity (that is, it functions as mRNA in viral replication). It is covalently linked at the 5′ end to the small viral protein VPg (3B of the polyprotein), followed by a long 5′ nontranslated region (5′ NTR), a continuous open reading frame (ORF), a 3′ NTR and poly(A). The 5′ NTR consists of structural elements that control RNA replication (cloverleaf) and translation (the internal ribosomal entry site (IRES)). The ORF encodes the polyprotein, the single translation product of the viral mRNA. The polyprotein is proteolytically processed by viral proteinases 2Apro and 3C/3CDpro into functional proteins, which have been divided into the structural region (P1 and the capsid precursors) and the nonstructural regions P2 and P3 (replication proteins). The ORF is followed by a 3′ NTR, which contributes to the control of RNA synthesis, and poly(A). The P1 coding region has been a target for codon and codon pair deoptimization. (b) Codon use statistics in synthetic P1 capsid designs. PV-SD maintains nearly identical codon frequencies compared to wild-type PV1(M), while maximizing codon positional changes within the sequence. In PV-AB capsids, the use of nonpreferred codons was maximized. The length of the bars and the numbers behind each bar indicate the occurrence of each codon in the sequence. As a reference, the normal human synonymous codon frequencies for each amino acid are given. Adapted from S.M. et a..
Figure 3
Figure 3
The codon pair bias (CPB) score for each of the 14,795 annotated human genes was calculated. Each dot represents the calculated CPB score of one gene plotted against its amino acid (aa) length. Predominant use of underrepresented codon pairs yields negative CPB scores. Various poliovirus constructs are plotted according to the CPB score of their P1 capsid precursor protein. As the CPB decreases, translatability decreases and the attenuation effect on the virus increases. PV-Min is nonviable; PV-Max expresses replication and virulence phenotypes similar to those of wild-type PV. Adapted from ref. .

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