MERS-CoV Accessory ORFs Play Key Role for Infection and Pathogenesis

ABSTRACT While dispensable for viral replication, coronavirus (CoV) accessory open reading frame (ORF) proteins often play critical roles during infection and pathogenesis. Utilizing a previously generated mutant, we demonstrate that the absence of all four Middle East respiratory syndrome CoV (MERS-CoV) accessory ORFs (deletion of ORF3, -4a, -4b, and -5 [dORF3-5]) has major implications for viral replication and pathogenesis. Importantly, attenuation of the dORF3-5 mutant is primarily driven by dysregulated host responses, including disrupted cell processes, augmented interferon (IFN) pathway activation, and robust inflammation. In vitro replication attenuation also extends to in vivo models, allowing use of dORF3-5 as a live attenuated vaccine platform. Finally, examination of ORF5 implicates a partial role in modulation of NF-κB-mediated inflammation. Together, the results demonstrate the importance of MERS-CoV accessory ORFs for pathogenesis and highlight them as potential targets for surveillance and therapeutic treatments moving forward. IMPORTANCE The initial emergence and periodic outbreaks of MERS-CoV highlight a continuing threat posed by zoonotic pathogens to global public health. In these studies, mutant virus generation demonstrates the necessity of accessory ORFs in regard to MERS-CoV infection and pathogenesis. With this in mind, accessory ORF functions can be targeted for both therapeutic and vaccine treatments in response to MERS-CoV and related group 2C coronaviruses. In addition, disruption of accessory ORFs in parallel may offer a rapid response platform to attenuation of future emergent strains based on both SARS- and MERS-CoV accessory ORF mutants

DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC–DNA binding

Drosophila origin recognition complex (ORC) localizes to defined positions on chromosomes, and in follicle cells the chorion gene amplification loci are well-studied examples. However, the mechanism of specific localization is not known. We have studied the DNA binding of DmORC to investigate the cis-requirements for DmORC:DNA interaction. DmORC displays at best six-fold differences in the relative affinities to DNA from the third chorion locus and to random fragments in vitro, and chemical probing and DNase1 protection experiments did not identify a discrete binding site for DmORC on any of these fragments. The intrinsic DNA-binding specificity of DmORC is therefore insufficient to target DmORC to origins of replication in vivo. However, the topological state of the DNA significantly influences the affinity of DmORC to DNA. We found that the affinity of DmORC for negatively supercoiled DNA is about 30-fold higher than for either relaxed or linear DNA. These data provide biochemical evidence for the notion that origin specification in metazoa likely involves mechanisms other than simple replicator–initiator interactions and that in vivo other proteins must determine ORC's localization

Discovery of tMAC: a Drosophila testis-specific meiotic arrest complex paralogous to Myb–Muv B

The Drosophila Myb–Muv B (MMB)/dREAM complex regulates gene expression and DNA replication site-specifically, but its activities in vivo have not been thoroughly explored. In ovarian amplification-stage follicle cell nuclei, the largest subunit, Mip130, is a negative regulator of replication, whereas another subunit, Myb, is a positive regulator. Here, we identified a mutation in mip40 and generated a mutation in mip120, two additional MMB subunits. Both mutants were viable, but mip120 mutants had many complex phenotypes including shortened longevity and severe eye defects. mip40 mutant females had severely reduced fertility, whereas mip120 mutant females were sterile, substantiating ovarian regulatory role(s) for MMB. Myb accumulation and binding to polytene chromosomes was dependent on the core factors of the MMB complex. In contrast to the documented mip130 mutant phenotypes, both mip40 and mip120 mutant males were sterile. We purified Mip40-containing complexes from testis nuclear extracts and identified tMAC, a new testis-specific meiotic arrest complex that contained Mip40, Caf1/p55, the Mip130 family member, Always early (Aly), and a Mip120 family member, Tombola (Tomb). Together, these data demonstrate that MMB serves diverse roles in different developmental pathways, and members of MMB can be found in alternative, noninteracting complexes in different cell types