Watch this video to see why this paper is important: .https://www.youtube.com/watch?v=lIE_UElOk3c

I picked this paper: Host Cell Entry of Respiratory Syncytial Virus Involves Macropinocytosis Followed by Proteolytic Activation of the F Protein by Krzyzaniak et al, in PLoS Pathogens last week because of the depth and detail that they went to in order to determine one of the most basic and important processes in virus infection: cell entry.

journal.ppat.1003309.g002

Questions:

How important is it to find out how a virus enters a cell?

Could purification/concentration of the virus particles change how the virus enters?

Did they use the correct cells (not primary respiratory cells) and virus (not a clinical isolate) for their experiments?

Would you target this pathway as an antiviral strategy?

What would you do next?

Background:

Viruses are obligate intracellular parasites. Specifically, their extracellular particles lack many of the components (like ribosomes and metabolic enzymes) that they require for replication. That means that in order to propagate themselves they must get inside cells and hijack these host molecules. However, this poses a major problem for these parasites: how do you traverse your hosts phospholipid bilayered membrane (for animal viruses) / cell wall (for plant viruses and prokaryotic viruses)?

Viruses of bacteria, archaea and eukaryotes have evolved myriad entry strategies suited to their hosts biology. For the enveloped viruses (those enclosed in their own membrane), they must join their lipid membrane to that of their host cell. These viruses have adapted proteins on their surface that can latch onto host cells and induce these structural changes causing the virus to enter inside the cell.

This usually requires a physical association between virus particle and the cell (binding to receptors/co-receptors via viral proteins for example) followed by a change in the virus protein structure or an initial change in host cell structure (endocytosis for example) that subsequently leads to the activation of those viral entry proteins.

Being the initial events in the virus life cycle, this process is incredibly important. Regulation of cell entry can control what cells that virus infects, how it causes disease (or not) and how it is transmitted between infected hosts. Being so, it thus offers a therapeutic option of targeting this mechanism as an antiviral strategy.

Why did they do it?

But to do this we must first piece together the mechanism of each viruses entry pathway. This paper does just that, for one important virus: Respiratory Syncytial Virus, or RSV. This virus is dangerous for newborns and elderly people, where it can cause bronchiolitis and death. There is no vaccine for it yet and antiviral options are limited.

What did they do?

To investigate how RSV gets inside cells, they used pure, concentrated virus particles to infect cell lines (Figure 1) and tracked these events using confocal microscopy (fig 2). To determine specific host molecule/pathways involved they added a large number of protein inhibitors during infection. The paper describes how they determine that RSV can enter using a particular kind of endocytosis (Fig 2 – 5) : macropinocytosis (Fig 6 and 7) and intracellular movement inside membrane vesicles in the presence of specific host proteases induces the activation of RSV entry proteins allowing virus membrane/host membrane fusion (Fig 8 and 9).

This paper represents a great deal of good science backed up by data from a multitude of experiments using a variety of techniques

 

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