Late endosome-resident interferon-induced transmembrane protein 3 (IFITM3) inhibits fusion of diverse viruses, including Influenza A virus (IAV), by a poorly understood mechanism. Despite the broad antiviral activity of IFITM3, viruses like Lassa virus (LASV), are fully resistant to its inhibitory effects. It is currently unclear whether resistance arises from a highly efficient fusion machinery that is capable of overcoming IFITM3 restriction or the ability to enter from cellular sites devoid of this factor. Here, we constructed and validated a functional IFITM3 tagged with EGFP or other fluorescent proteins. This breakthrough allowed live cell imaging of virus co-trafficking and fusion with endosomal compartments in cells expressing fluorescent IFITM3. Three-color single virus and endosome tracking revealed that sensitive (IAV), but not resistant (LASV), viruses become trapped within IFITM3-positive endosomes where they underwent hemifusion but failed to release their content into the cytoplasm. IAV fusion with IFITM3-containing compartments could be rescued by amphotericin B treatment, which has been previously shown to antagonize the antiviral activity of this protein. By comparison, virtually all LASV particles trafficked and fused with endosomes lacking detectable levels of fluorescent IFITM3, implying that this virus escapes restriction by utilizing endocytic pathways that are distinct from the IAV entry pathways. The importance of virus uptake and transport pathways is further reinforced by the observation that LASV glycoprotein-mediated cell-cell fusion is inhibited by IFITM3 and other members of the IFITM family expressed in target cells. Together, our results strongly support a model according to which IFITM3 accumulation at the sites of virus fusion is a prerequisite for its antiviral activity and that this protein traps viral fusion at a hemifusion stage by preventing the formation of fusion pores. We conclude that the ability to utilize alternative endocytic pathways for entry confers IFITM3-resistance to otherwise sensitive viruses. Expression of interferon-induced transmembrane proteins (IFITMs) in target cells potently inhibits fusion of many unrelated enveloped viruses, including the Influenza A virus, whereas arenaviruses, such as the Lassa fever virus, are resistant to these factors. The mechanism by which IFITMs interfere with the viral fusion step and the mechanism of virus escape from these restriction factors are poorly understood. Here, we tagged the late endosome-resident IFITM3 with fluorescent proteins and visualized single virus entry and fusion with endosomes in living cells expressing these constructs. Single virus and endosome tracking experiments demonstrate that the sensitive Influenza A virus is trapped within acidic IFITM3-positive endosomes that are not permissive for viral fusion. In contrast, the resistant Lassa virus consistently enters and fuses with endosomes lacking IFITM3. Our results imply that accumulation of IFITM3 in virus-carrying endosomes is a prerequisite for blocking fusion of diverse enveloped viruses and that viruses insensitive to this protein escape restriction by entering through distinct endosomal trafficking pathways that do not converge with IFITM3-positive compartments.

RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA three-dimensional structure prediction. With agreement from structural biologists, RNA structures are predicted by modeling groups before publication of the experimental structures. We report a large-scale set of predictions by 18 groups for 23 RNA-Puzzles: 4 RNA elements, 2 Aptamers, 4 Viral elements, 5 Ribozymes and 8 Riboswitches. We describe automatic assessment protocols for comparisons between prediction and experiment. Our analyses reveal some critical steps to be overcome to achieve good accuracy in modeling RNA structures: identification of helix-forming pairs and of non-Watson–Crick modules, correct coaxial stacking between helices and avoidance of entanglements. Three of the top four modeling groups in this round also ranked among the top four in the CASP15 contest.

Riboswitches are structural elements in the 5′ untranslated regions of many bacterial messenger RNAs that regulate gene expression in response to changing metabolite concentrations by inhibition of either transcription or translation initiation. The preQ₁ (7-aminomethyl-7-deazaguanine) riboswitch family comprises some of the smallest metabolite sensing RNAs found in nature. Once ligand-bound, the transcriptional Bacillus subtilis and translational Thermoanaerobacter tengcongensis preQ₁ riboswitch aptamers are structurally similar RNA pseudoknots; yet, prior structural studies have characterized their ligand-free conformations as largely unfolded and folded, respectively. In contrast, through single molecule observation, we now show that, at near-physiological Mg²⁺ concentration and pH, both ligand-free aptamers adopt similar pre-folded state ensembles that differ in their ligand-mediated folding. Structure-based Gō-model simulations of the two aptamers suggest that the ligand binds late (Bacillus subtilis) and early (Thermoanaerobacter tengcongensis) relative to pseudoknot folding, leading to the proposal that the principal distinction between the two riboswitches lies in their relative tendencies to fold via mechanisms of conformational selection and induced fit, respectively. These mechanistic insights are put to the test by rationally designing a single nucleotide swap distal from the ligand binding pocket that we find to predictably control the aptamers′ pre-folded states and their ligand binding affinities.

By examining the structure of phospholipase C-β, an autoinhibitory helix that interacts with the catalytic core of this enzyme is now identified. Disrupting this interaction enhances basal activity and decreases stimulation by Gαq, supporting an allosteric mechanism for PLCβ activation through displacement of the autoinhibitory helix by Gαq. The enzyme phospholipase C-β (PLCβ) is a crucial regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to members of the Gq family of heterotrimeric G proteins. We have determined atomic structures of two invertebrate homologs of PLCβ (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLCβ3 considerably increase basal activity and diminish stimulation by Gαq. Gαq binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLCβ.

Significance: Riboswitches are RNA molecules found mostly in bacteria that control genes by sensing cellular levels of metabolites, such as the simple organic compound preQ ₁ . The diversity of riboswitches and their potential as novel antibiotic targets continue to elicit interest in these regulatory sequences. Here we present the crystal structure of a newly discovered bacterial preQ ₁ -III riboswitch that senses preQ ₁ using an unusual, two-part architecture. A complementary analysis of flexibility and dynamics showed that recognition of preQ ₁ induces riboswitch compaction, while concomitantly enhancing formation of a distant double-helix possessing a regulatory signal that zips and unzips rapidly, producing gene “off” and “on” states. These observations expand our knowledge of riboswitch construction and suggest a broader role for dynamics than previously recognized.