Strands of DNA with four or more contiguous runs of 2'-deoxycytidine (dC) nucleotides have the potential to adopt i-motif folds, generally under mildly acidic conditions. Analysis of dC homo-oligonucleotide strands ranging in length from 10 to 30 nucleotides by five different pH-dependent methods identified a pattern in strand length vs stability. Beginning with dC, which does not fold, the transition pH (pH) increased with chain length with the addition of up to four nucleotides, after which the stability dramatically decreased, and the trend repeated this cycle up to dC. The analysis found dC strands of length 15, 19, 23, and 27 nucleotides (i.e., 4n-1) to have pH values >7.2 and thermal stabilities >37 °C at pH 7.0. Model studies using thymidine nucleotides to lock in i-motif loop lengths support the conclusion that the most stable dC i-motifs possess one nucleotide in each of the three loops and a core built of an even number of base pairs. The pattern identified from the model studies occurs with a frequency of four nucleotides at lengths of 15, 19, 23, and 27 in accordance with the results obtained for the dC strands. This observation led us to interrogate the human genome for dC runs. Inspection of the human genome indicates that dC runs are enriched in critical regions of the genome (promoters, UTRs, and introns), while being depleted in coding and intergenic regions, and these findings may have biological implications. Lastly, the ability to tune i-motif stabilities by the length of the strand might be harnessed for stimulus-responsive applications in DNA scaffolds, sensors, nanotechnology, and other chemical applications.

Epstein-Barr virus (EBV) SM protein is an RNA-binding protein that has multiple posttranscriptional gene regulatory functions essential for EBV lytic replication. In this study, we identified an interaction between SM and DHX9, a DExH-box helicase family member, by mass spectrometry and coimmunoprecipitation. DHX9 participates in many cellular pathways involving RNA, including transcription, processing, transport, and translation. DHX9 enhances virus production or infectivity of a wide variety of DNA and RNA viruses. Surprisingly, an increase in EBV late gene expression and virion production occurred upon knockdown of DHX9. To further characterize the SM-DHX9 interaction, we performed immunofluorescence microscopy of EBV-infected cells and found that DHX9 partially colocalized with SM in nuclear foci during EBV lytic replication. However, the positive effect of DHX9 depletion on EBV lytic gene expression was not confined to SM-dependent genes, indicating that the antiviral effect of DHX9 was not mediated through its effects on SM. DHX9 enhanced activation of innate antiviral pathways comprised of several interferon-stimulated genes that are active against EBV. SM inhibited the transcription-activating function of DHX9, which acts through cAMP response elements (CREs), suggesting that SM may also act to counteract DHX9’s antiviral functions during lytic replication. IMPORTANCE This study identifies an interaction between Epstein-Barr virus (EBV) SM protein and cellular helicase DHX9, exploring the roles that this interaction plays in viral infection and host defenses. Whereas most previous studies established DHX9 as a proviral factor, we demonstrate that DHX9 may act as an inhibitor of EBV virion production. DHX9 enhanced innate antiviral pathways active against EBV and was needed for maximal expression of several interferon-induced genes. We show that SM binds to and colocalizes DHX9 and may counteract the antiviral function of DHX9. These data indicate that DHX9 possesses antiviral activity and that SM may suppress the antiviral functions of DHX9 through this association. Our study presents a novel host-pathogen interaction between EBV and the host cell.