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Localization of rotavirus antigens in infected cells by ultrastructural immunocytochemistry
Abstract:
Virus 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-SA11 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. 38000) 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, mol. 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.
Insights
Simian rotavirus SA11 infection reveals distinct cellular locations for viral proteins. Inner capsid proteins concentrate in inclusions, while outer capsid glycoprotein is synthesized on the endoplasmic reticulum.
Area of Science:
- Virology
- Cell Biology
- Immunology
Background:
- Simian rotavirus SA11 is a model for studying rotavirus replication.
- Understanding the cellular localization of viral proteins is crucial for elucidating replication mechanisms.
Purpose of the Study:
- To localize simian rotavirus SA11 structural antigens within infected monkey kidney cells.
- To differentiate the synthesis and assembly pathways of rotavirus inner and outer capsid proteins.
Main Methods:
- Electron microscopic immunoperoxidase techniques were employed.
- Infected MA104 cells were probed with hyperimmune guinea-pig anti-SA11 serum and monospecific antisera against VP7, VP2, and VP6.
Main Results:
- Virus particles, endoplasmic reticulum membranes, and viroplasmic inclusions showed heavy labeling with anti-SA11 serum.
- Outer capsid glycoprotein (VP7) localized to virus particles and ER, while inner capsid proteins (VP2, VP6) strongly labeled viroplasmic inclusions.
- Aberrant virus-like particles and cytoplasmic tubules also contained rotavirus proteins.
Conclusions:
- Rotavirus inner capsid proteins are synthesized in the cytoplasm and accumulate in inclusions.
- The outer capsid glycoprotein is synthesized on the rough endoplasmic reticulum.
- Outer capsid acquisition occurs during virus budding into the ER, suggesting a specific assembly pathway.