Use of a small DNA virus model to investigate mechanisms of CpG dinucleotide-induced attenuation of virus replication.

Abstract

Suppression of the CpG dinucleotide is widespread in RNA viruses infecting vertebrates and plants, and in the genomes of retroviruses and small mammalian DNA viruses. The functional basis for CpG suppression in the latter was investigated through the construction of mutants of the parvovirus, minute virus of mice (MVM) with increased CpG or TpA dinucleotides in the VP gene. CpG-high mutants displayed extraordinary attenuation in A9 cells compared to wild-type MVM (>six logs), while TpA elevation showed no replication effect. Attenuation was independent of Toll-like receptor 9 and STING-mediated DNA recognition pathways and unrelated to effects on translation efficiency. While translation from codon-optimized VP RNA was enhanced in a cell-free assay, MVM containing this sequence was highly attenuated. Further mutational analysis indicated that this arose through its increased numbers of CpG dinucleotides (7→70) and separately from its increased G+C content (42.3→57.4 %), which independently attenuated replication. CpG-high viruses showed impaired NS mRNA expression by qPCR and reduced NS and particularly VP protein expression detected by immunofluorescence and replication in A549 cells, effects reversed in zinc antiviral protein (ZAP) knockout cells, even though nuclear relocalization of VP remained defective. The demonstrated functional basis for CpG suppression in MVM and potentially other small DNA viruses and the observed intolerance of CpGs in coding sequences, even after codon optimization, has implications for the use of small DNA virus vectors in gene therapy and immunization.

• Authors: Lisa Loew, Niluka Goonawardane, Jeremy Ratcliff, Dung Nguyen, Peter Simmonds
• Journal: The Journal of general virology (J Gen Virol) Vol. 101 Issue 11 Pg. 1202-1218 (11 2020) ISSN: 1465-2099 [Electronic] England
• PMID: 32783803 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Codon
• Dinucleoside Phosphates
• RNA, Viral
• RNA-Binding Proteins
• Viral Nonstructural Proteins
• Viral Proteins
• Viral Structural Proteins
• ZC3HAV1 protein, human
• cytidylyl-3'-5'-guanosine

Topics

• A549 Cells
• Base Composition
• Codon
• Dinucleoside Phosphates (genetics, metabolism)
• Humans
• Minute Virus of Mice (genetics, physiology)
• Mutation
• RNA, Viral (genetics, metabolism)
• RNA-Binding Proteins (metabolism)
• Viral Nonstructural Proteins (genetics, metabolism)
• Viral Proteins (genetics, metabolism)
• Viral Structural Proteins (genetics, metabolism)
• Virus Replication