Temperature control to track viral entry during live-cell Imaging

Mechanisms of viral entry

Viral infection begins with the attachment of viral particles to the host cell membrane. This is a complex process during which viral capsid proteins bind to receptors at the cell membrane. These receptors can be proteins, carbohydrates or lipids. The viral particle is then internalized by endocytosis or by membrane fusion with the host cell. The mechanism of entry depends on the type of viruses namely enveloped or non enveloped viruses.

Viral entry of non-enveloped viruses

Non-enveloped viruses such as Influenza virus or vesicular stomatitis viruses enter the cell through endocytosis, using both clathrin dependent or clathrin independent pathways. Poliovirus, a non-enveloped virus, uses two alternative strategies to enter the cell. The virus can be endocytosed: the hydrophobic part of the viral protein VP1 will insert itself inside the cell membrane of the endocytosed vesicles and form a pore to allow the penetration of the viral RNA. In the second strategy, the virus attaches to the cell membrane and directly releases its RNA material inside the cell.

Viral entry of enveloped viruses

Enveloped viruses, such as HIV, MLV or Ebola virus, enter the cells via membrane fusion. Viruses first dock to the cells; the binding of specific viral proteins to cell membrane receptors ensure the bridging of virus and host membranes. Subsequently both outer layer membranes of virus and host will fuse and the capsid or viral genome will be released inside the host cytoplasm. Since fusion is the first step towards cell infection, finding anti-fusion agents is a major area of research in pharmaceutics. An exhaustive lists of fusion proteins and entry mechanisms can be found in the excellent principles of virology book (Flint et al., 2009).

Studying viral entry with live cell Imaging

Key questions arise from studying viral entry: which viral and host proteins participate in viral entry? What are the entry mechanisms? What are the subsequent cytoplamic transport mechanisms? How to inhibit virus entry?

Live-cell imaging allows to track single viral particle as it enters and traffics inside the host cell.  The use of fluorescent proteins, fluorescent dyes such as amino-reactive dyes or lipophilic dyes enables the visualization of viral particles (Ayala-Nunez et al., 2011), however labeling can affect viral infectivity and the correct amount of dye has to be determined for each type of viruses-host studies. Viral entry into cells can be followed by fluorescently labeling the viral membrane, the viral content or the viral core (Hulme and Hope, 2014). If the viral membrane is labeled, fusion events will consist in a disappearance of fluorescence, however if viral content was also labeled it will then be possible to follow the transport of viral particles inside the cell.

Temperature control of viral entry with ElveflowTemp

References

Ayala-Nuñez NV, Wilschut J, JM Smit, Monitoring virus entry into living cells using DiD-labeled dengue virus particles, Methods, 2011

Flint SJ , Enquist LW,  Racaniello VR, and Skalka AM, principles of virology, asm press, 2009

Hulme AE.  and Hope TJ., Live Cell Imaging of Retroviral Entry, Annu. Rev. Virol, 2014

Brandenburg B, Lee LY, Lakadamyali M, Rust MJ , Zhuang X ,  Hogle JM Imaging poliovirus entry in live cells, PLoS Biol, 2007

Webb S, Alive and in focus, The scientist, 2012