Citations

Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes.

Genome diversity is key to viral fitness and adaptability, especially for RNA viruses, which generate it through both mutation and recombination. Recombination occurs frequently in positive strand RNA virus infections, where the RNA‐dependent RNA polymerase can jump from one genomic template to another. On the other hand, template exchange events have been rarely detected in infections of negative strand RNA viruses even when sensitive functional assays were used. However, while characterizing defective interfering RNAs (DI RNA) generated by an innate immunity‐stimulating measles virus (MeV) mutant, we documented an unexpectedly high number of recombination events ( J. Virol. 89, 7735‐47, 2015 ). We asked here how frequently these events occur in the 15,894 bases genome of a MeV strain with intact innate immunity control functions. Towards this we purified MeV ribonucleocapsids and performed unbiased deep sequencing analyses. We detected more than 3000 recombination events while covering 32,000 genomes, or about one event every ten genomes. Mapping of breakpoints and re‐initiation sites indicated that recombination occurred along the entire genome, rather than being restricted to either end, where most DI RNAs were previously mapped. Recombination was phased neither by encapsidation (6‐base periodicity) nor by ribonucleocapsid helical turns (approximately 75‐base periodicity). Rather, most recombination events joined sequences located more than 1000 bases apart. Jump‐back events were prominent, occurring about 1.5 times more frequently than deletion or copy‐back events. Most events were unique, but a few short jump‐back events retaining 6‐base periodicity were detected up to 40 times, suggesting that the corresponding genomes were amplified. At new junctions, deletions or insertions were rare (<5%). Analyses of 10 nucleotides on each side of the new junctions suggested that base pairing guided about one third of the re‐initiation events. In addition to the recombination events, we detected clusters of adenosine deaminase acting on RNA 1 (ADAR1)‐like hypermutations in about 4% of the genomes. ADAR1 de‐stabilizes base pairing on double‐strand RNA, catalyzing C‐6 deamination of adenosine to produce inosine until double‐strand regions are unwound. Indeed, we document that in an infection with a MeV mutant known to generate many DI RNAs, the frequency of ADAR1‐like hypermutations was even higher than in standard infections. In summary, we show here that significant levels of RNA recombination occur even in standard lytic infections of a prototypic negative strand RNA virus. Even if some recombinant genomes may form double‐strand RNA, infections remain productive, probably due to the unwinding/modification activity of ADAR1. We discuss our data in the context of published observations indicating that recombination events, as well as ADAR1‐like hypermutations, contribute to the adaptation of other medically relevant negative strand RNA viruses to new hosts and environments. Support or Funding Information R21 AI 128037; Mayo Clinic Graduate School of Biomedical Sciences This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Abstract Viruses spread between hosts through particles, but within hosts, viral genomes can spread from cell to cell through fusion, evading antiviral defenses and obviating costly infectious virion production1-3. Billions of electromechanically coupled cardiomyocytes (CMs) make myocardium inherently vulnerable to pathological electromechanical short circuits caused by intercellular viral spread 4-6. Beyond respiratory illness, COVID-19 affects the heart7 and cardiac injury and arrhythmias are serious public health concerns8-12. By studying myocardium of a young woman who died suddenly, diagnosed postmortem with COVID-19, we discovered highly focal myocardial SARS-CoV-2 infection spreading from one CM to another through intercellular junctions identified by highly concentrated sarcolemmal t-tubule viral spike glycoprotein. SARS-CoV-2 permissively infected beating human induced pluripotent stem cell (hiPSC)-CMs building multinucleated cardiomyotubes (CMTs) through cell type-specific fusion driven by proteolytically-activated spike glycoprotein. Recombinant spike glycoprotein, co-localizing to sarcolemma and sarcoplasmic reticulum, produced multinucleated CMTs with pathological structure, electrophysiology and Ca2+ excitation-contraction coupling. Blocking cleavage, a peptide-based protease inhibitor neutralized SARS-CoV-2 spike glycoprotein pathogenicity. We conclude that SARS-CoV-2 spike glycoprotein, efficiently primed, activated and strategically poised during biosynthesis, can exploit the CM’s inherent membranous connectivities to drive heart damage directly, uncoupling clinically common myocardial injury from lymphocytic myocarditis, often suspected but rarely confirmed in COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic is associated with significant morbidity and mortality throughout the world, predominantly due to lung and cardiovascular injury. The virus responsible for COVID-19—severe acute respiratory syndrome coronavirus 2—gains entry into host cells via ACE2 (angiotensin-converting enzyme 2). ACE2 is a primary enzyme within the key counter-regulatory pathway of the renin-angiotensin system (RAS), which acts to oppose the actions of Ang (angiotensin) II by generating Ang-(1–7) to reduce inflammation and fibrosis and mitigate end organ damage. As COVID-19 spans multiple organ systems linked to the cardiovascular system, it is imperative to understand clearly how severe acute respiratory syndrome coronavirus 2 may affect the multifaceted RAS. In addition, recognition of the role of ACE2 and the RAS in COVID-19 has renewed interest in its role in the pathophysiology of cardiovascular disease in general. We provide researchers with a framework of best practices in basic and clinical research to interrogate the RAS using appropriate methodology, especially those who are relatively new to the field. This is crucial, as there are many limitations inherent in investigating the RAS in experimental models and in humans. We discuss sound methodological approaches to quantifying enzyme content and activity (ACE, ACE2), peptides (Ang II, Ang-[1–7]), and receptors (types 1 and 2 Ang II receptors, Mas receptor). Our goal is to ensure appropriate research methodology for investigations of the RAS in patients with severe acute respiratory syndrome coronavirus 2 and COVID-19 to ensure optimal rigor and reproducibility and appropriate interpretation of results from these investigations.

Abstract Background: Viral vectors based on the Edmonston strain of measles virus (MV-Edm) selectively destroy all tumor cell lines tested in vitro. The oncolytic activity of the virus is enhanced by expression of the thyroidal sodium iodide symporter (MV-NIS) that allows selective 131I uptake by infected tumor cells and eliminates myeloma tumor xenografts that are resistant to the parent virus. MV-NIS is being considered for therapy of patients with relapsed or refractory multiple myeloma. Advanced myeloma is associated with significant immunosuppression with the potential risk of uncontrolled virus proliferation. The number of agents with activity against MV is limited. Low energy (Auger) electrons have a short path length and selectively damage cells in which the isotope decays. Thus, we hypothesized that the Auger electron emitting isotope 125I, selectively taken up by cells expressing NIS, can be used to control viral proliferation. Methods: A replication competent MV that expressed both a soluble form of carcinoembryonic antigen (CEA) and NIS (MV-NICE) was rescued and characterized. Cells were infected with MV-NICE or control vectors and exposed to 125I with appropriate controls. CEA expression and viral titers were determined at different time points. The role of free radical generation on virus replication was explored. In vivo control of MV-NICE replication with 125I was attempted. Results: MV-NICE replication in vitro is inhibited by the selective uptake of 125I by cells expressing NIS. Extracellular decay of the isotope has no effect on virus proliferation. Auger electron damage is in part mediated by free radicals and abrogated by glutathione. In myeloma xenografts, control of MV-NICE with 125I was not possible under the conditions of the experiment. Conclusion: MV-NICE does not replicate faster in the presence of radiation under our experimental conditions. Auger electron emitting isotopes effectively stop propagation of MV vectors expressing NIS in vitro. Additional work is necessary to translate these observations in vivo.

The ability of viral particles to directly spread cell to cell within the airways without particle release is considered to be highly advantageous to many respiratory viruses. Our previous studies in well-differentiated, primary human airway epithelial cells suggest that measles virus (MeV) spreads cell to cell by eliciting the formation of intercellular membrane pores. Based on a newly generated ribonucleoprotein complex (RNP) “tracker” virus, we document by live-cell microscopy that MeV RNPs move along F-actin rings before entering a new cell. Thus, rather than diffusing through the cytoplasm of a newly infected columnar cell, RNPs take advantage of the cytoskeletal infrastructure to rapidly spread laterally across the human airway epithelium. This results in rapid horizontal spread through the epithelium that does not require particle release.

Replication of negative-strand RNA viruses relies on two components: a helical ribonucleocapsid and an RNA-dependent RNA polymerase composed of a catalytic subunit, the L protein, and a cofactor, the P protein. We show that the measles virus (MeV) C protein is an additional component of the replication complex. We provide evidence that the C protein is recruited to the ribonucleocapsid by the P protein and map the interacting segments of both C and P proteins. We conclude that the primary function of MeV C is to improve polymerase processivity and accuracy, rather than uniquely to antagonize the type I interferon response. Since most viruses of the Paramyxoviridae family express C proteins, their primary function may be conserved.

Cedar virus (CedV) is a bat-borne henipavirus related to Nipah virus (NiV) and Hendra virus (HeV), zoonotic agents of fatal human disease. CedV receptor-binding protein (G) shares only ∼30% sequence identity with those of NiV and HeV, although they can all use ephrin-B2 as an entry receptor. We demonstrate that CedV also enters cells through additional B- and A-class ephrins (ephrin-B1, ephrin-A2, and ephrin-A5) and report the crystal structure of the CedV G ectodomain alone and in complex with ephrin-B1 or ephrin-B2. The CedV G receptor-binding site is structurally distinct from other henipaviruses, underlying its capability to accommodate additional ephrin receptors. We also show that CedV can enter cells through mouse ephrin-A1 but not human ephrin-A1, which differ by 1 residue in the key contact region. This is evidence of species specific ephrin receptor usage by a henipavirus, and implicates additional ephrin receptors in potential zoonotic transmission.

ABSTRACT Here, we show that cells expressing the adherens junction protein nectin-1 capture nectin-4-containing membranes from the surface of adjacent cells in a trans-endocytosis process. We find that internalized nectin-1–nectin-4 complexes follow the endocytic pathway. The nectin-1 cytoplasmic tail controls transfer: its deletion prevents trans-endocytosis, while its exchange with the nectin-4 tail reverses transfer direction. Nectin-1-expressing cells acquire dye-labeled cytoplasmic proteins synchronously with nectin-4, a process most active during cell adhesion. Some cytoplasmic cargo remains functional after transfer, as demonstrated with encapsidated genomes of measles virus (MeV). This virus uses nectin-4, but not nectin-1, as a receptor. Epithelial cells expressing nectin-4, but not those expressing another MeV receptor in its place, can transfer infection to nectin-1-expressing primary neurons. Thus, this newly discovered process can move cytoplasmic cargo, including infectious material, from epithelial cells to neurons. We name the process nectin-elicited cytoplasm transfer (NECT). NECT-related trans-endocytosis processes may be exploited by pathogens to extend tropism. This article has an associated First Person interview with the first author of the paper.

Morbilliviruses such as measles virus, rinderpest virus, and canine distemper virus (CDV) are highly contagious. Despite extensive knowledge of how morbilliviruses interact with their receptors, little is known about how those interactions influence viral transmission to naive hosts. In a ferret model of CDV contact transmission, we showed that sequential use of the signaling lymphocytic activation molecule (SLAM) and nectin-4 receptors is essential for transmission. In one animal infected with a SLAM-blind CDV, we documented mild viremia due to the acquisition of three compensatory mutations within or near the SLAM-binding surface. The interaction, however, was not sufficient to cause disease or sustain transmission to naive contacts. This work confirms the sequential roles of SLAM and nectin-4 in morbillivirus transmission and highlights the selective pressure directed toward productive interactions with SLAM.