Mechanisms of Attenuation by Genetic Recoding of Viruses

Abstract

The development of safe and effective vaccines against viruses is central to disease control. With advancements in DNA synthesis technology, the production of synthetic viral genomes has fueled many research efforts that aim to generate attenuated viruses by introducing synonymous mutations. Elucidation of the mechanisms underlying virus attenuation through synonymous mutagenesis is revealing interesting new biology that can be exploited for vaccine development. Here, we review recent advancements in this field of synthetic virology and focus on the molecular mechanisms of attenuation by genetic recoding of viruses. We highlight the action of the zinc finger antiviral protein (ZAP) and RNase L, two proteins involved in the inhibition of viruses enriched for CpG and UpA dinucleotides, that are often the products of virus recoding algorithms. Additionally, we discuss current challenges in the field as well as studies that may illuminate how other host functions, such as translation, are potentially involved in the attenuation of recoded viruses.

Keywords: RNA; codon; translation; virus.

FIG 1

Codon substitution approaches used in genetic recoding of viruses. Several synonymous mutagenesis approaches may be applied to generate attenuated viruses. Codon deoptimization aims to replace highly frequent codons (such as GAG for glutamate and UGC for cysteine) with underrepresented codons (such as GAA and UGU, respectively). Codon substitution may also lead to increased CpG and/or UpA frequencies by introducing these dinucleotides either at the codon-codon boundary (e.g., replacing the GCA-GAG pair with GCG-GAG) or within a codon (e.g., AGA-to-CGU substitution). Replacing serine and leucine codons with “near-stop” codons (i.e., AGU and CUU to UCA and UUA) may also lead to viruses whose replication is aborted at unusually high frequencies through the frequent generation of mutants expressing truncated viral proteins.

FIG 2

Molecular mechanisms that limit the replication of recoded viruses. Several elements present in viral RNA may be recognized and eliminated by various mechanisms. CpG-rich RNA is detected by ZAP, whose interaction with TRIM25 may facilitate the coalescence of other cellular proteins that determine its fate. Viral RNA that is recognized by ZAP can be degraded by the endonuclease KHNYN or relocalized to stress granules, where it may become a substrate of the RNA exosome. The presence of dsRNA, a frequent product of virus replication, is detected by OAS3, leading to the production of 2′-5′-oligoadenylate (2-5A) from ATP. The ankyrin repeats of RNase L interact with 2-5A, promoting the formation of a dimeric, active state of this protein. RNase L interacts with Dom34/Pelota and cleaves mRNA 3′ to UpA dinucleotides. This reaction is inhibited by ABCE1. The presence of rare codons or codon pairs may lead to slow ribosome translocation, causing ribosome collisions. Stalled ribosomes are sensed by Dom34/Pelota, recruiting Cue2/N4BP2 that cleaves the translating mRNA. Ribosome dissociation is promoted by ABCE1, while Xrn1 and other exonucleases degrade mRNA containing inhibitory codon pairs.