ABSTRACT
Protein screening/detection is an essential tool in many laboratories. Owing to the relatively large time investments that are required by standard protocols, the development of methods with higher throughput while maintaining an at least comparable signal-to-noise ratio would be highly beneficial to many researchers. This chapter describes how cold microwave technology can be used to enhance the rate of molecular interactions and provides protocols for dot blots, western blots, and ELISA procedures permitting a completion of all incubation steps (blocking and antibody steps) within 45 min.
Subject(s)
Blotting, Western/methods , Microwaves , Proteins/analysis , Staining and Labeling/methods , Animals , Cells, Cultured , Cold Temperature , Electrophoresis, Polyacrylamide Gel/methodsABSTRACT
Coronaviruses cause a variety of respiratory and enteric diseases in animals and humans including severe acute respiratory syndrome. In these enveloped viruses, the filamentous nucleocapsid is formed by the association of nucleocapsid (N) protein with single-stranded viral RNA. The N protein is a highly immunogenic phosphoprotein also implicated in viral genome replication and in modulating cell signaling pathways. We describe the structure of the two proteolytically resistant domains of the N protein from infectious bronchitis virus (IBV), a prototype coronavirus. These domains are located at its N- and C-terminal ends (NTD and CTD, respectively). The NTD of the IBV Gray strain at 1.3-A resolution exhibits a U-shaped structure, with two arms rich in basic residues, providing a module for specific interaction with RNA. The CTD forms a tightly intertwined dimer with an intermolecular four-stranded central beta-sheet platform flanked by alpha helices, indicating that the basic building block for coronavirus nucleocapsid formation is a dimeric assembly of N protein. The variety of quaternary arrangements of the NTD and CTD revealed by the analysis of the different crystal forms delineates possible interfaces that could be used for the formation of a flexible filamentous ribonucleocapsid. The striking similarity between the dimeric structure of CTD and the nucleocapsid-forming domain of a distantly related arterivirus indicates a conserved mechanism of nucleocapsid formation for these two viral families.
Subject(s)
Coronavirus/chemistry , Nucleocapsid Proteins/chemistry , Nucleocapsid/chemistry , Virus Assembly , Animals , Coronavirus/metabolism , Coronavirus Infections/metabolism , Coronavirus Nucleocapsid Proteins , Crystallography, X-Ray , Dimerization , Humans , Nucleocapsid/metabolism , Nucleocapsid Proteins/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Viral/chemistry , RNA, Viral/metabolism , Structure-Activity RelationshipABSTRACT
Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid protein (N) may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. 32P-orthophosphate labeling and Western blot analyses confirmed that N was the only viral protein that was phosphorylated. Pulse labeling with 32P-orthophosphate indicated that the IBV N protein was phosphorylated in the virion, as well as at all times during infection in either chicken embryo kidney cells or Vero cells. Pulse-chase analyses followed by immunoprecipitation of IBV N proteins using rabbit anti-IBV N polyclonal antibody demonstrated that the phosphate on the N protein was stable for at least 1 h. Simultaneous labeling with 32P-orthophosphate and 3H-leucine identified a 3.5-fold increase in the 32P:3H counts per minute (cpm) ratio of N in the virion as compared to the 32P:3H cpm ratio of N in the cell lysates from chicken embryo kidney cells, whereas in Vero cells the 32P:3H cpm ratio of N from the virion was 10.5-fold greater than the 32P:3H cpm ratio of N from the cell lysates. These studies are consistent with the phosphorylation of the IBV N playing a role in assembly or maturation of the viral particle.
