The coronavirus nucleocapsid (N) protein packages viral genomic RNA into a ribonucleoprotein complex. Interactions between N proteins and RNA are thus crucial for the assembly of infectious virus particles. The 45 kDa recombinant nucleocapsid N protein of coronavirus infectious bronchitis virus (IBV) is highly sensitive to proteolysis. We obtained a stable fragment of 14.7 kDa spanning its N-terminal residues 29-160 (IBV-N29-160). Like the N-terminal RNA binding domain (SARS-N45-181) of the severe acute respiratory syndrome virus (SARS-CoV) N protein, the crystal structure of the IBV-N29-160 fragment at 1.85 A resolution reveals a protein core composed of a five-stranded antiparallel beta sheet with a positively charged beta hairpin extension and a hydrophobic platform that are probably involved in RNA binding. Crosslinking studies demonstrate the formation of dimers, tetramers, and higher multimers of IBV-N. A model for coronavirus shell formation is proposed in which dimerization of the C-terminal domain of IBV-N leads to oligomerization of the IBV-nucleocapsid protein and viral RNA condensation.

The N-terminal domain of the coronavirus nucleocapsid (N) protein adopts a fold resembling a right hand with a flexible, positively charged β-hairpin and a hydrophobic palm. This domain was shown to interact with the genomic RNA for coronavirus infectious bronchitis virus (IBV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Based on its 3D structure, we used site-directed mutagenesis to identify residues essential for the RNA-binding activity of the IBV N protein and viral infectivity. Alanine substitution of either Arg-76 or Tyr-94 in the N-terminal domain of IBV N protein led to a significant decrease in its RNA-binding activity and a total loss of the infectivity of the viral RNA to Vero cells. In contrast, mutation of amino acid Gln-74 to an alanine, which does not affect the binding activity of the N-terminal domain, showed minimal, if any, detrimental effect on the infectivity of IBV. This study thus identifies residues critical for RNA binding on the nucleocapsid surface, and presents biochemical and genetic evidence that directly links the RNA binding capacity of the coronavirus N protein to the viral infectivity in cultured cells. This information would be useful in development of preventive and treatment approaches against coronavirus infection.

Lipid raft is an important element for the cellular entry of some viruses, including coronavirus infectious bronchitis virus (IBV). However, the exact role of lipid rafts in the cellular membrane during the entry of IBV into host cells is still unknown. In this study, we biochemically fractionated IBV-infected cells via sucrose density gradient centrifugation after depleting plasma membrane cholesterol with methyl-β-cyclodextrin or Mevastatin. Our results demonstrated that unlike IBV non-structural proteins, IBV structural proteins co-localized with lipid raft marker caveolin-1. Infectivity assay results of Vero cells illustrated that the drug-induced disruption of lipid rafts significantly suppressed IBV infection. Further studies revealed that lipid rafts were not required for IBV genome replication or virion release at later stages. However, the drug-mediated depletion of lipid rafts in Vero cells before IBV attachment significantly reduced the expression of viral structural proteins, suggesting that drug treatment impaired the attachment of IBV to the cell surface. Our results indicated that lipid rafts serve as attachment factors during the early stages of IBV infection, especially during the attachment stage.

Abstract. When doing an interactive proof about a piece of software, it is important that the underlying programming language's semantics does not make the proof unnecessarily difficult or unwieldy. Both smallstep and big-step semantics are commonly used, and the latter is typically given by an inductively defined relation. In this paper, we consider an alternative: using a recursive function akin to an interpreter for the language. The advantages include a better induction theorem, less duplication, accessibility to ordinary functional programmers, and the ease of doing symbolic simulation in proofs via rewriting. We believe that this style of semantics is well suited for compiler verification, including proofs of divergence preservation. We do not claim the invention of this style of semantics: our contribution here is to clarify its value, and to explain how it supports several language features that might appear to require a relational or small-step approach. We illustrate the technique on a simple imperative language with C-like for-loops and a break statement, and compare it to a variety of other approaches. We also provide ML and lambda-calculus based examples to illustrate its generality.

This article proves the completeness of an axiomatization for differential equation invariants described by Noetherian functions. First, the differential equation axioms of differential dynamic logic are shown to be complete for reasoning about analytic invariants. Completeness crucially exploits differential ghosts, which introduce additional variables that can be chosen to evolve freely along new differential equations. Cleverly chosen differential ghosts are the proof-theoretical counterpart of dark matter. They create a new hypothetical state, whose relationship to the original state variables satisfies invariants that did not exist before. The reflection of these new invariants in the original system then enables its analysis. An extended axiomatization with existence and uniqueness axioms is complete for all local progress properties, and, with a real induction axiom, is complete for all semianalytic invariants. This parsimonious axiomatization serves as the logical foundation for reasoning about invariants of differential equations. Indeed, it is precisely this logical treatment that enables the generalization of completeness to the Noetherian case.

Animal coronaviruses, such as infectious bronchitis virus (IBV), and arteriviruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), are able to manifest highly contagious infections in their specific native hosts, thereby arising in critical economic damage to animal industries. This review discusses recent progress in studies of virus-host interactions during animal and human coronavirus and arterivirus infections, with emphasis on IBV-host cell interactions. These interactions may be directly involved in viral replication or lead to the alteration of certain signaling pathways, such as cell stress response and innate immunity, to facilitate viral replication and pathogenesis.

COVID-19, the disease caused by SARS-CoV-2 (1), was declared a pandemic by the World Health Organization (WHO) in March 2020 (2). While awaiting a vaccine, several antivirals are being used to manage the disease with limited success (3, 4). To expand this arsenal, we screened 4 compound libraries: a United States Food and Drug Administration (FDA) approved drug library, an angiotensin converting enzyme-2 (ACE2) targeted compound library, a flavonoid compound library as well as a natural product library. Of the 121 compounds identified with activity against SARS-CoV-2, 7 were shortlisted for validation. We show for the first time that the active form of Vitamin D, calcitriol, exhibits significant potent activity against SARS-CoV-2. This finding paves the way for consideration of host-directed therapies for ring prophylaxis of contacts of SARS-CoV-2 patients.

Coronaviruses RNA synthesis occurs in the cytoplasm and is regulated by host cell proteins. In a screen based on a yeast three-hybrid system using the 5′-untranslated region (5′-UTR) of SARS coronavirus (SARS-CoV) RNA as bait against a human cDNA library derived from HeLa cells, we found a positive candidate cellular protein, zinc finger CCHC-type and RNA-binding motif 1 (MADP1), to be able to interact with this region of the SARS-CoV genome. This interaction was subsequently confirmed in coronavirus infectious bronchitis virus (IBV). The specificity of the interaction between MADP1 and the 5′-UTR of IBV was investigated and confirmed by using an RNA pull-down assay. The RNA-binding domain was mapped to the N-terminal region of MADP1 and the protein binding sequence to stem–loop I of IBV 5′-UTR. MADP1 was found to be translocated to the cytoplasm and partially co-localized with the viral replicase/transcriptase complexes (RTCs) in IBV-infected cells, deviating from its usual nuclear localization in a normal cell using indirect immunofluorescence. Using small interfering RNA (siRNA) against MADP1, defective viral RNA synthesis was observed in the knockdown cells, therefore indicating the importance of the protein in coronaviral RNA synthesis.