No abstract

Cohesins, which mediate sister chromatin cohesion, and CTCF, which functions at chromatin boundaries, play key roles in the structural and functional organization of chromosomes. We examined the binding of these two factors on the Kaposi's sarcoma-associated herpesvirus (KSHV) episome during latent infection and found a striking colocalization within the control region of the major latency transcript responsible for expressing LANA (ORF73), vCyclin (ORF72), vFLIP (ORF71), and vmiRNAs. Deletion of the CTCF-binding site from the viral genome disrupted cohesin binding, and crippled colony formation in 293 cells. Clonal instability correlated with elevated expression of lytic cycle gene products, notably the neighbouring promoter for K14 and vGPCR (ORF74). siRNA depletion of RAD21 from latently infected cells caused an increase in K14 and ORF74, and lytic inducers caused a rapid dissociation of RAD21 from the viral genome. RAD21 and SMC1 also associate with the cellular CTCF sites at mammalian c-myc promoter and H19/Igf2 imprinting control region. We conclude that cohesin subunits associate with viral and cellular CTCF sites involved in complex gene regulation and chromatin organization.

The 59 untranslated region (UTR) of the mouse hepatitis virus (MHV) genome contains cis-acting sequences necessary for transcription and replication. A consensus secondary structural model of the 59 140 nucleotides of the 59 UTRs of nine coronaviruses (CoVs) derived from all three major CoV groups is presented and characterized by three major stem-loops, SL1, SL2, and SL4. NMR spectroscopy provides structural support for SL1 and SL2 in three group 2 CoVs, including MHV, BCoV, and HCoV-OC43. SL2 is conserved in all CoVs, typically containing a pentaloop (C47-U48-U49-G50-U51 in MHV) stacked on a 5 base-pair stem, with some sequences containing an additional U 39 to U51; SL2 therefore possesses sequence features consistent with a U-turn-like conformation. The imino protons of U48 in the wild-type RNA, and G48 in the U48G SL2 mutant RNA, are significantly protected from exchange with solvent, consistent with a hydrogen bonding interaction critical to the hairpin loop architecture. SL2 is required for MHV replication; MHV genomes containing point substitutions predicted to perturb the SL2 structure (U48C, U48A) were not viable, while those that maintain the structure (U48G and U49A) were viable. The U48C MHV mutant supports both positive-and negative-sense genome-sized RNA synthesis, but fails to direct the synthesis of positive-or negative-sense subgenomic RNAs. These data support the existence of the SL2 in our models, and further suggest a critical role in coronavirus replication.

SummaryThe leader RNA of the 5' untranslated region (UTR) of coronaviral genomes contains two stem-loop structures denoted SL1 and SL2. Herein, we show that SL1 is functionally and structurally bipartite. While the upper region of SL1 is required to be paired, we observe strong genetic selection against viruses that contain a deletion of A35, an extrahelical nucleotide that destabilizes SL1, in favor of genomes that contain a diverse panel of destabilizing second-site mutations, due to introduction of a non-canonical base pair near A35. Viruses containing destabilizing SL1-ΔA35 mutations also contain one of two specific mutations in the 3' UTR. Thermal denaturation and imino proton solvent exchange experiments reveal that the lower half of SL1 is unstable and that second-site SL1-ΔA35 substitutions are characterized by one or more features of the wild-type SL1. We propose a "dynamic SL1" model, in which the base of SL1 has an optimized lability required to mediate a physical interaction between the 5' and 3' UTRs that stimulates subgenomic RNA synthesis. Although not conserved at the nucleotide sequence level, these general structural characteristics of SL1 appear to be conserved in other coronaviral genomes.