Role of viral genome dinucleotide content on replication, gene expression, and interferon evasion

Abstract

Significant suppression of CpG dinucleotides has been observed in some DNA and RNA viruses. Based on this observation, it has long been suggested that dinucleotide frequency may play a role in host defenses by allowing the host cell to distinguish between self and non-self RNA. Recently, the interferon-stimulated Zinc-finger Antiviral Protein (ZAP) has been shown to target viral RNA containing high numbers of CpG dinucleotides for degradation.

Previous work from the Grey lab determined that ZAP is a human cytomegalovirus (HCMV) host restriction factor. HCMV displays a striking pattern of CpG dinucleotides, with suppression limited to the immediate-early (IE) gene, IE1. Previously, the lab demonstrated that suppressed CpG levels in IE1 enabled evasion of ZAP in the context of a co-plasmid expression system. To test whether CpG dinucleotides are important in the context of virus infection, we created a high CpG IE1 mutant HCMV.

Based on growth kinetics following reconstitution of the recombinant virus, increased CpG levels in IE1 attenuated the virus. While ZAP depletion increased replication of the high CpG IE1 virus, similar results were observed for the control virus.

Subsequent sequence analysis showed that virus stocks contained the parental virus in addition to the high CpG recombinant, suggesting small amounts of parental virus outcompeted the high CpG virus due to the associated attenuation.

In addition to investigating ZAP evasion, we investigated the role of CpG dinucleotide content in viral gene expression. Nucleotide bias has previously been shown to influence gene expression in a splicing-dependent manner. To determine the effect of CpG levels on unspliced herpesviruses genes, we created reduced CpG constructs of an early gene, pp52, by way of synonymous mutations. We showed that reducing CpG levels in pp52 drastically reduced gene expression efficiency, but also resulted in a decrease in RNA levels. Further studies are needed to investigate how transcription may be affected, but together these experiments suggest that the high CpG frequencies of these genes may be maintained as a means to increase viral fitness.

Finally, herpesviruses express a family of RNA binding proteins that increase the export of unspliced transcripts. The best-studied of these are ICP27 of the alphaherpesvirus Herpes Simplex Virus-1 (HSV-1), and ORF57 of the gammaherpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV). Using GFP constructs that contain synonymous mutations, we investigated whether these RNA binding proteins recognise transcripts with the same dinucleotide composition as their respective subfamilies. While preliminary, our data suggest that both ICP27 and ORF57 increased expression of a GFP construct with high CpG frequencies, while only ORF57 increased the expression of a low CpG construct. These studies suggest that dinucleotide content plays a fundamental role in herpesviruses biology, including immune evasion and viral gene expression.