Adeno-associated virus (AAV) vectors are preeminent in emerging clinical gene therapies. Generalizing beyond the most tractable genetic diseases will require modulation of cell specificity and immune neutralization. Interactions of AAV with its cellular receptor, AAVR, are key to understanding cell-entry and trafficking with the rigor needed to engineer tissue-specific vectors. Cryo-electron tomography shows ordered binding of part of the flexible receptor to the viral surface, with distal domains in multiple conformations. Regions of the virus and receptor in close physical proximity can be identified by cross-linking / mass spectrometry. Cryo-electron microscopy with a two-domain receptor fragment reveals the interactions at 2.4 Å resolution. AAVR binds between AAV's spikes on a plateau that is conserved, except in one clade whose structure is AAVR-incompatible. AAVR's footprint overlaps the epitopes of several neutralizing antibodies, prompting a re-evaluation of neutralization mechanisms. The structure provides a roadmap for experimental probing and manipulation of viral-receptor interactions.
Electron microscopy maps and atomic coordinates will be available from the electron microscopy and protein data banks (https://www.ebi.ac.uk/pdbe/emdb/ & https://www.rcsb.org/). For the high resolution PKD1-2/AAV2 complex, the accession numbers are EMD-0553, PDB ID 6NZ0, respectively. Reconstructions for the 4 tomographic classes have accession numbers of EMD-0621, EMD-0622, EMD-0623 and EMD-0624.
Cryo-EM structure of AAV-2 in complex with AAVR PKD domains 1 and 2Protein Data Bank, 6NZ0.
Cryo-EM structure of AAV-2 in complex with AAVR PKD domains 1 and 2Unified Data Resource for 3DEM, EMD-0553.
Structure of the AAV2 with its Cell Receptor AAVRUnified Data Resource for 3DEM, EMD-0621.
Structure of the AAV2 with its Cell Receptor AAVRUnified Data Resource for 3DEM, EMD-0622.
Structure of the AAV2 with its Cell Receptor AAVRUnified Data Resource for 3DEM, EMD-0623.
Structure of the AAV2 with its Cell Receptor AAVRUnified Data Resource for 3DEM, EMD-0624.
- Andrew Trzynka
- Michael Stewart Chapman
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Sriram Subramaniam, University of British Columbia, Canada
© 2019, Meyer et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Microsporidia are eukaryotic, obligate intracellular parasites that infect a wide range of hosts, leading to health and economic burdens worldwide. Microsporidia use an unusual invasion organelle called the polar tube (PT), which is ejected from a dormant spore at ultra-fast speeds, to infect host cells. The mechanics of PT ejection are impressive. Anncaliia algerae microsporidia spores (3–4 μm in size) shoot out a 100-nm-wide PT at a speed of 300 μm/s, creating a shear rate of 3000 s-1. The infectious cargo, which contains two nuclei, is shot through this narrow tube for a distance of ∼60–140 μm (Jaroenlak et al, 2020) and into the host cell. Considering the large hydraulic resistance in an extremely thin tube and the low-Reynolds-number nature of the process, it is not known how microsporidia can achieve this ultrafast event. In this study, we use Serial Block-Face Scanning Electron Microscopy to capture 3-dimensional snapshots of A. algerae spores in different states of the PT ejection process. Grounded in these data, we propose a theoretical framework starting with a systematic exploration of possible topological connectivity amongst organelles, and assess the energy requirements of the resulting models. We perform PT firing experiments in media of varying viscosity, and use the results to rank our proposed hypotheses based on their predicted energy requirement. We also present a possible mechanism for cargo translocation, and quantitatively compare our predictions to experimental observations. Our study provides a comprehensive biophysical analysis of the energy dissipation of microsporidian infection process and demonstrates the extreme limits of cellular hydraulics.
Angiotensin-converting enzyme 2 (ACE2) is a major cell entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The induction of ACE2 expression may serve as a strategy by SARS-CoV-2 to facilitate its propagation. However, the regulatory mechanisms of ACE2 expression after viral infection remain largely unknown. Using 45 different luciferase reporters, the transcription factors SP1 and HNF4α were found to positively and negatively regulate ACE2 expression, respectively, at the transcriptional level in human lung epithelial cells (HPAEpiCs). SARS-CoV-2 infection increased the transcriptional activity of SP1 while inhibiting that of HNF4α. The PI3K/AKT signaling pathway, activated by SARS-CoV-2 infection, served as a crucial regulatory node, inducing ACE2 expression by enhancing SP1 phosphorylation—a marker of its activity—and reducing the nuclear localization of HNF4α. However, colchicine treatment inhibited the PI3K/AKT signaling pathway, thereby suppressing ACE2 expression. In Syrian hamsters (Mesocricetus auratus) infected with SARS-CoV-2, inhibition of SP1 by either mithramycin A or colchicine resulted in reduced viral replication and tissue injury. In summary, our study uncovers a novel function of SP1 in the regulation of ACE2 expression and identifies SP1 as a potential target to reduce SARS-CoV-2 infection.