The herpesviral antagonist m152 reveals differential activation of STING‐dependent IRF and NF‐κB signaling and STING's dual role during MCMV infection

Cytomegaloviruses (CMVs) are master manipulators of the host immune response. Here, we reveal that the murine CMV (MCMV) protein m152 specifically targets the type I interferon (IFN) response by binding to stimulator of interferon genes (STING), thereby delaying its trafficking to the Golgi compartment from where STING initiates type I IFN signaling. Infection with an MCMV lacking m152 induced elevated type I IFN responses and this leads to reduced viral transcript levels both in vitro and in vivo This effect is ameliorated in the absence of STING Interestingly, while m152 inhibits STING-mediated IRF signaling, it did not affect STING-mediated NF-κB signaling. Analysis of how m152 targets STING translocation reveals that STING activates NF-κB signaling already from the ER prior to its trafficking to the Golgi. Strikingly, this response is important to promote early MCMV replication. Our results show that MCMV has evolved a mechanism to specifically antagonize the STING-mediated antiviral IFN response, while preserving its pro-viral NF-κB response, providing an advantage in the establishment of an infection

Ehrlichia chaffeensis TRP120 nucleomodulin binds DNA with disordered tandem repeat domain.

Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis, secretes several effector proteins that bind host DNA to modulate host gene expression. The tandem repeat protein 120 (TRP120), one of the largest effector proteins, has four nearly identical tandem repeat (TR) regions that each consists of 80 amino acids. In addition to playing a role in ehrlichial binding and internalization, TRP120 translocates to the host nucleus where it is thought to function as a transcription factor that modulates gene expression. However, sequence analysis of TRP120 does not identify the presence of DNA-binding or trans-activation domains typical of classical eukaryotic transcription factors. Thus, the mechanism by which TRP120 binds DNA and modulates gene expression remains elusive. Herein, we expressed the TR regions of the TRP120 protein, and characterized its solution structure and ability to bind DNA. TRP120, expressed as either a one or two TR repeat, is a monomer in solution, and is mostly disordered as determined by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Using NMR spectroscopy, we further show that the 1 TR construct selectively binds GC-rich DNA. Although low pH was required for TRP120 TR-DNA interaction, acidic pH alone does not induce any significant structural changes in the TR region. This suggests that TRP120 folds into an ordered structure upon forming a protein-DNA complex, and thus folding of TRP120 TR is coupled with DNA binding

Structure of the HOPS tethering complex, a lysosomal membrane fusion machinery

Lysosomes are essential for cellular recycling, nutrient signaling, autophagy, and patho-genic bacteria and viruses invasion. Lysosomal fusion is fundamental to cell survival and requiresHOPS, a conserved heterohexameric tethering complex. On the membranes to be fused, HOPSbinds small membrane- associated GTPases and assembles SNAREs for fusion, but how the complexfulfills its function remained speculative. Here, we used cryo-electron microscopy to reveal the struc-ture of HOPS. Unlike previously reported, significant flexibility of HOPS is confined to its extremities,where GTPase binding occurs. The SNARE-binding module is firmly attached to the core, therefore,ideally positioned between the membranes to catalyze fusion. Our data suggest a model for howHOPS fulfills its dual functionality of tethering and fusion and indicate why it is an essential part ofthe membrane fusion machinery

TRP120-1TR selectively binds to GC-rich DNA.

<p>(A-B) HSQC spectra of ¹⁵N-labeled TRP120-1TR with GC-rich DNA at pH 7.0 or pH 5.5. TRP120-1TR (68 μM) alone (black) and with a 10-fold molar equivalent of unlabeled DNA (red, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194891#pone.0194891.t001" target="_blank">Table 1</a>) in 20 mM Tris pH 7.0 containing 100 mM NaCl or in 10 mM phosphate pH 5.5 containing 100 mM NaCl are shown. The lines highlight Asn or Gln side chain NH₂ peaks that show distinctly different chemical shifts in the bound form. (C-D) HSQC spectra of ¹⁵N-labeled TRP120-1TR with AT-rich DNA probe at pH 7.0 or pH 5.5. TRP120-1TR (30 μM) alone (black) and with a 10-fold molar equivalent of unlabeled DNA (blue, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194891#pone.0194891.t001" target="_blank">Table 1</a>) in 20 mM Tris pH 7.0 containing 100 mM NaCl or in 10 mM phosphate pH 5.5 containing 100 mM NaCl are shown.</p

Far-UV CD spectra of TRP120-1TR and TRP120-2TR.

<p>The CD spectrum of TRP120-1TR was measured in Tris pH 7.0 containing 300 mM NaCl (blue). The CD spectra of TRP120-2TR were measured at three different pHs—phosphate pH 7.0 (green), phosphate pH 6.0 (red), and citrate pH 4.5 (gray) containing 100 mM NaCl. Secondary structure content of TRP120 constructs was estimated by BestSel analysis (bottom).</p

NMR spectra of TRP120-1TR at different pHs.

<p>HSQC spectra of ¹⁵N-labeled TRP120-1TR were collected at four different pHs—Tris pH 7.0 (black), phosphate pH 6.5 (red), phosphate pH 6.0 (green), and phosphate pH 5.5 (blue) containing 100 mM NaCl.</p

DNA probes used in NMR titrations.

<p>DNA probes used in NMR titrations.</p

Sedimentation velocity profiles of TRP120-1TR and TRP120-2TR proteins.

<p>The molecular weights of TRP120-1TR and -2TR proteins were determined by sedimentation velocity experiments. Sedimentation coefficient distribution <i>c(s)</i> profile for TRP120-1TR and TRP120-2TR show one major species, which corresponds to the molecular weight of 12.3 or 22.2 kDa, respectively. The observed molecular weights indicate that both TRP120 constructs are monomers in solution.</p

TR units of TRP120.

<p>(A) Domain organization of TRP120 and sequences of each TR unit. The primary sequences of TR units are shown with residues that differ between TR units highlighted in yellow (top). The sequence was analyzed by secondary structure (middle) and disorder (bottom) predictions. Predicted α-helical and β-strand regions are indicated by H and E, respectively. (B) SDS-PAGE of purified TRP120-1TR and TRP120-2TR proteins. Molecular weights for TRP120-1TR and -2TR proteins are 11.5 and 20.3 kDa, respectively, but the proteins migrate at twice their expected size.</p