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Flavivirus infection enhancement in macrophages: an electron microscopic study of viral cellular entry
Abstract:
The mode of entry of West Nile virus (WNV) into the macrophage-like cell line P388D1 was investigated at the electron microscopical level using synchronized infections. The presence of the antiviral monoclonal antibody F6/16A at a concentration that enhanced viral attachment to P388D1 cells ninefold made no difference to the entry pathway of WNV. In both the absence and presence of F6/16A the initial uptake of single viral particles was mediated by coated pits, and started within 30 s of warming the cells to 37 degrees C. Viral particles later appeared in fully or partially coated vesicles and later in uncoated prelysosomal endocytic vacuoles before degradation in lysosomes. However, aggregates of viral particles (five or more virus particles in cross-section), appeared to be phagocytosed whole by cells in a process which involved aggregates being engulfed by extensions of the plasma membrane. This process exhibited a slower time course than the uptake of single viral particles, becoming prominent 15 to 30 min after warming the cells to 37 degrees C. The involvement of a prelysosomal vacuolar compartment in the entry process was shown by a failure to stain for acid phosphatase. This compartment could be specifically loaded with viral particles when viral internalization occurred at 20 degrees C in the presence of 50 mM-ammonium chloride.
Insights
West Nile virus (WNV) enters macrophages via coated pits for single particles and phagocytosis for aggregates. An antiviral antibody did not alter these distinct WNV entry pathways into P388D1 cells.
Area of Science:
- Virology
- Cell Biology
- Immunology
Background:
- West Nile virus (WNV) is a significant public health concern.
- Understanding WNV entry mechanisms into host cells is crucial for developing antiviral strategies.
- Macrophage-like cell lines are valuable models for studying viral infections.
Purpose of the Study:
- To elucidate the distinct pathways of West Nile virus entry into P388D1 macrophage-like cells.
- To investigate the role of an antiviral monoclonal antibody in WNV entry.
- To characterize the cellular compartments involved in WNV internalization.
Main Methods:
- Synchronized infections of P388D1 cells with WNV.
- Electron microscopy to visualize viral entry.
- Use of an antiviral monoclonal antibody (F6/16A) to assess its impact on entry.
- Temperature and chemical treatments (ammonium chloride) to probe entry mechanisms.
Main Results:
- Single WNV particles entered cells via coated pits and coated vesicles, progressing to prelysosomal vacuoles.
- Viral aggregates were internalized through phagocytosis, a distinct and slower process.
- The antiviral antibody F6/16A enhanced viral attachment but did not alter the entry pathway.
- A prelysosomal compartment, negative for acid phosphatase, was identified and could be loaded with WNV.
Conclusions:
- WNV utilizes distinct entry pathways depending on particle aggregation: coated pit-mediated endocytosis for single virions and phagocytosis for aggregates.
- Antiviral antibodies may enhance attachment but do not fundamentally change the cellular entry route.
- The identified prelysosomal compartment plays a role in WNV internalization before lysosomal degradation.
