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Infectious entry pathway of influenza virus in a canine kidney cell line
Abstract:
The entry of fowl plague virus, and avian influenza A virus, into Madin-Darby canine kidney (MDCK) cells was examined both biochemically and morphologically. At low multiplicity and 0 degrees C, viruses bound to the cell surface but were not internalized. Binding was not greatly dependent on the pH of the medium and reached an equilibrium level in 60-90 min. Over 90% of the bound viruses were removed by neuraminidase but not by proteases. When cells with prebound virus were warmed to 37 degrees C, part of the virus became resistant to removal b neuraminidase, with a half-time of 10-15 min. After a brief lag period, degraded viral material was released into the medium. The neuraminidase-resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride, a lysosomotropic agent, blocked both the infection and the degradation of viral protein. When the entry process was observed by electron microscopy, viruses were seen bound primarily to microvilli on the cell surface at 0 degrees C and, after warming at 37 degrees C, were endocytosed in coated pits, coated vesicles, and large smooth-surfaced vacuoles. Viruses were also present in smooth-surfaced invaginations and small smooth-surfaced vesicles at both temperatures. At physiological pH, no fusion of the virus with the plasma membrane was observed. When prebound virus was incubated at a pH of 5.5 or below for 1 min at 37 degrees C, fusion was, however, detected by ferritin immunolabeling. t low multiplicity, 90% of the prebound virus became neuraminidase-resistant and was presumably fused after only 30 s at low pH. These experiments suggest that fowl plague virus enters MDCK cells by endocytosis in coated pits and coated vesicles and is transported to the lysosome where the low pH initiates a fusion reaction ultimately resulting in the transfer of the genome into the cytoplasm. The entry pathway of fowl plague virus thus resembles tht earlier described for Semliki Forest virus.
Insights
Avian influenza virus enters host cells via endocytosis, a process involving coated pits and vesicles. Low pH in lysosomes triggers fusion, releasing viral genetic material into the cytoplasm for infection.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Fowl plague virus, a type of avian influenza A virus, requires entry into host cells to initiate infection.
- Understanding viral entry mechanisms is crucial for developing antiviral strategies.
Purpose of the Study:
- To biochemically and morphologically investigate the entry process of fowl plague virus into Madin-Darby canine kidney (MDCK) cells.
- To elucidate the role of pH and cellular compartments in viral internalization and infection.
Main Methods:
- Binding assays at low temperature (0°C) and physiological temperature (37°C).
- Neuraminidase and protease treatment to assess viral surface protein integrity.
- Electron microscopy to visualize virus-cell interactions and internalization pathways.
- Treatment with ammonium chloride (a lysosomotropic agent) to probe the role of endosomes/lysosomes.
- Low pH treatment to investigate membrane fusion.
Main Results:
- At 0°C, viruses bind to the cell surface primarily on microvilli but are not internalized.
- Upon warming to 37°C, viruses are endocytosed via coated pits and vesicles, and also appear in smooth-surfaced vacuoles.
- A portion of the virus becomes neuraminidase-resistant and infects cells, with infection and viral protein degradation inhibited by ammonium chloride.
- Low pH (≤5.5) at 37°C induces fusion of the virus with the cell membrane, observed via ferritin immunolabeling.
- No fusion was observed at physiological pH.
Conclusions:
- Fowl plague virus enters MDCK cells primarily through endocytosis involving coated structures.
- The low pH environment within lysosomes triggers viral fusion with the endosomal membrane.
- This fusion event facilitates the release of the viral genome into the cytoplasm, leading to infection.
- The entry pathway shares similarities with that of Semliki Forest virus.