ABSTRACT
Early defibrillation is an essential step in the "chain of survival" for patients with in-hospital cardiac arrest. To increase the rate of early defibrillation by nurse first responders in noncritical care areas, our institution employed a quality resuscitation consultant, implemented nursing education programs, and standardized equipment and practices. Automated external defibrillator application by nurse first responders prior to advanced cardiac life support team arrival has improved from 15% in 2011 to 76% in 2013 (P < .001).
Subject(s)
Cardiopulmonary Resuscitation , Electric Countershock , Heart Arrest/therapy , Time-to-Treatment , Cardiopulmonary Resuscitation/methods , Education, Nursing , HumansABSTRACT
The coronavirus membrane (M) protein carboxy tail interacts with the nucleocapsid during virus assembly. Previous studies demonstrated that the two terminal residues are important, and the charged residue (R227) in the penultimate position in the mouse hepatitis coronavirus (MHV) A59 M protein was suggested to participate in intermolecular interactions with negative charges in the nucleocapsid (N) protein. To determine the significance of the positive charge at position 227, we substituted the arginine with lysine (K), aspartic acid (D), glutamic acid (E), or alanine (A) and studied these by reverse genetics in the context of a MHV full-length infectious clone. Viruses with wild-type phenotype were readily recovered with the K or A substitutions. In contrast, negative-charge substitutions were not tolerated as well. In all recovered R227D viruses the negative charge was replaced with heterologous residues resulting from apparent template switching during negative-strand synthesis of subgenomic RNA 7. An additional second-site compensatory V202I substitution was present in some viruses. Recovered R227E viruses had second-site changes within the M protein carboxy tail that were partially compensatory. Significantly, most of the second site changes in the R227E mutant viruses were previously shown to compensate for the removal of negative charges in the N protein. Our results strongly indicate that a positive charge is not absolutely required. It is clear that other regions within the tail must also be involved in helping mediate interactions between the M protein and the nucleocapsid.
Subject(s)
Murine hepatitis virus/physiology , Viral Matrix Proteins/physiology , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Line , Coronavirus M Proteins , Coronavirus Nucleocapsid Proteins , Cricetinae , Mice , Microscopy, Confocal , Microscopy, Fluorescence , Molecular Sequence Data , Murine hepatitis virus/genetics , Murine hepatitis virus/growth & development , Mutagenesis, Site-Directed , Nucleocapsid Proteins/metabolism , Protein Binding , Suppression, Genetic , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Viral Plaque AssaySubject(s)
Murine hepatitis virus/metabolism , Nucleocapsid Proteins/chemistry , Amino Acid Sequence , Animals , Cell Line , DNA, Complementary/metabolism , Glycine/chemistry , Mice , Molecular Sequence Data , Mutation , Phosphorylation , Plasmids/metabolism , Protein Biosynthesis , Protein Structure, TertiaryABSTRACT
The coronavirus nucleocapsid (N) protein is a multifunctional viral gene product that encapsidates the RNA genome and also plays some as yet not fully defined role in viral RNA replication and/or transcription. A number of conserved negatively charged amino acids are located within domain III in the carboxy end of all coronavirus N proteins. Previous studies suggested that the negatively charged residues are involved in virus assembly by mediating interaction between the membrane (M) protein carboxy tail and nucleocapsids. To determine the importance of these negatively charged residues, a series of alanine and other charged-residue substitutions were introduced in place of those in the N gene within a mouse hepatitis coronavirus A59 infectious clone. Aspartic acid residues 440 and 441 were identified as functionally important. Viruses could not be isolated when both residues were replaced by positively charged amino acids. When either amino acid was replaced by a positively charged residue or both were changed to alanine, viruses were recovered that contained second-site changes within N, but not in the M or envelope protein. The compensatory role of the new changes was confirmed by the construction of new viruses. A few viruses were recovered that retained the D441-to-arginine change and no compensatory changes. These viruses exhibited a small-plaque phenotype and produced significantly less virus. Overall, results from our analysis of a large panel of plaque-purified recovered viruses indicate that the negatively charged residues at positions 440 and 441 are key residues that appear to be involved in virus assembly.
