Localization of Rotavirus Antigens in Infected Cells by Ultrastructural Immunocytochemistry

Abstract

SUMMARYVirus structural antigens were localized within a line of monkey kidney (MA104) cells infected with the simian rotavirus SA11 using electron microscopic immunoperoxidase techniques. When hyperimmune guinea-pig anti-SA11 serum was used, virus particles, membranes of virus-associated endoplasmic reticulum, and viroplasmic inclusions were most heavily labelled. A general cytoplasmic reaction (ribosomes, intracytoplasmic membranes, etc.) with anti-SA 11 serum was also observed, but nuclei were unstained. In addition, several other virus-induced structures were found to contain rotavirus proteins, including convoluted smooth membrane within the endoplasmic reticulum, aberrant virus-like particles, and 15 to 20 nm diam. cytoplasmic tubules. Monospecific antiserum to VP7 (outer capsid glycoprotein, mol. wt. 38 000) reacted strongly with virus particles and the virus-associated endoplasmic reticulum, but reacted poorly with viroplasmic inclusions. The nucleus and general cytoplasm were unstained with anti-VP7. In contrast, monospecific antisera to VP2 and VP6 (inner capsid proteins, tool. wt. 94000 and 41000 respectively) reacted very strongly with viroplasmic inclusions. Virus particles, endoplasmic reticulum and cytoplasmic ribosomes were also labelled with these sera. These results indicate that rotavirus inner capsid proteins are synthesized throughout the cytoplasm and become concentrated in viroplasmic inclusions, while the outer capsid glycoprotein is synthesized primarily on ribosomes of the rough endoplasmic reticulum. Thus, the outer capsid layer appears to be acquired during virus budding into cisternae of the endoplasmic reticulum.

Biochemical Evidence for the Oligomeric Arrangement of Bovine Rotavirus Nucleocapsid Protein and Its Possible Significance in the Immunogenicity of This Protein

“…Since the nucleocapsid protein constitutes approximately 80 ~ of the protein in single-shelled particles, the arrangement of this protein has been the focus of many studies (Chasey & Labram, 1983;Martin et al, 1975;Palmer et al, 1977;Palmer & Martin, 1982;Petrie et al, 1982;Sabara et aL, 1985). Electron microscopical examination of virus particles suggested the arrangement of inner capsid subunits into an open mesh exhibiting a regular pattern of holes (Martin et al, 1975).…”

Rotavirus NSP5 phosphorylation is up-regulated by interaction with NSP2.

“…NSP2 and NSP5, are localized (Petrie et al, 1982(Petrie et al, , 1984Welch et al, 1989). These discrete structures are found in the cytoplasm at 2-3 h post-infection (p.i.…”

Association of rotavirus viroplasms with microtubules through NSP2 and NSP5

“…Viroplasms are composed of viral RNA and the proteins VP1, VP2, VP3, VP6, NSP2, and NSP5 (Petrie et al 1982, Gallegos & Patton 1989, and while virion assembly occur in these structures (Petrie et al 1982, Gallegos & Patton 1989) the mechanism of viroplasms formation is unknown. Recently, it was reported that the number of rotavirus viroplasms decrease with post-infection time (Eichwald et al 2004), and while the diameter of single viroplasms increased with time, the total number of viroplasms per cell diminishes, suggesting that growth of these inclusion bodies occur by fusion and probably also by stepwise addition of viral components to the viroplasms surface (Eichwald et al 2004).…”