Packing it all

Last week I have started my final-year undergraduate research project and, not surprisingly, I made an effort to get a project on viruses. I was lucky enough to get a project with prof. Wendy Barclay who has a lab that works on various molecular aspects of Influenza virus infection. My project in particular, focuses on the packaging of viral genome into new budding viruses. At first glance this might not seem to be anything too exciting, however, although obviously during the short few months I’m probably not going to make big discoveries, actually, the current research on how Influenza packages its genome has given some really interesting but not clear-cut results. Therefore, this work allows me to try to synthesise work that has already been done and hopefully, if some interesting results will come out, contribute at least in a small part to what is already known.

Influenza Infected Cells. Cell's nucleus is in blue, cell's actin filaments in red and  viral NP and HA proteins are green and pink, respectively (these reflect positions of synthesised proteins and NP also RNA-NP protein complexes, HA also new budding viral particles).  From Vijayakrishnan, Swetha, et al.  PLoS pathogens 9.6 (2013): e1003413.
Influenza Infected Cells. Cell’s nucleus is in blue, cell’s actin filaments in red and viral NP and HA proteins are green and pink, respectively (these reflect positions of synthesised proteins and NP also RNA-NP protein complexes, HA also new budding viral particles).
From Vijayakrishnan, Swetha, et al. PLoS pathogens 9.6 (2013): e1003413.

Okay, so let me show you some pretty images and tell you why understanding the mechanism of Influenza’s genome packaging is still so little understood.

To do that I have to start from the very beginning: what is the genome of Influenza?
Influenza is an RNA virus, meaning that its genome is based on RNA and not DNA. In addition Influenza’s genome is a segmented one, namely, it has 8 RNA segments that code for different proteins (think of it as something like different chromosomes, although chromosomes are much more complex structures and have many more genes). 8 RNA segments are found in A and B type Influenza and 7 are present in C type, however, here I will only discuss Influenza A virus (IAV) as most of the research so far has been focused on it.

Influenza virus particle
Influenza virus particle

Considering the fact that IAV codes for only 12 proteins, it’s not surprising that they are all essential for successful viral replication and therefore, IAV requires to carry all of its 8 RNA segments through generation. That is 8 segments have to be in the viral particle not only when it infects the host cell but also when new viruses are made. Consequently, a mechanism has to be present to package all RNA segments. It might seem like a trivial thing but when you think of it it’s not as clear-cut as one might like it to be. The 8 different RNAs are not bound together during the viral life cycle in the cell’s nucleus, when they are replicated (to make more RNA that could be packaged into the new virions) each segment is coated by a special viral protein, called NP, and has to be exported out of the nucleus to the sight where the virus will bud and not just any segments but all 8 different ones need to be present at that site. Clearly, there has to be some mechanism that mediates successful RNA incorporation and traffics the segments to the budding site. Sure, it is possible that just by chance the correct segments are incorporated in some virions and so in the population of newly produced viruses only a few of them are properly infectious in the next round. However, even though indeed only 5 to 10% ratio of infectious to noninfectious particles have been estimated in the new IAV population, most of the recent research does not support this argument. Importantly, apart from filamentous-type viral particles (the function of which is still not known) all IAV particles always contain 8 different RNA segments. Moreover, the segments in electron microscopy studies always take a distinct 7+1 configuration, in which there is one central segment that is surrounded by a circle of other 7 segments. You can see this distinct arrangement in figure below.

How is this 7+1 complex formed is still not known, but it seems that both RNA and some RNA-associated viral proteins are required. Some research suggests that structurally, the complex consists of viral RNAs that are coated in NP protein hanging from a kind of platform made from a matrix protein (matrix protein makes up a protein coat that is located under a host-acquired plasma membrane after budding). This platform, called transition zone, is depicted in the movie below, where you can see viral RNA strands hanging down from it.

RNA and protein complex- movie 1

In addition, it also has been proposed that different segments interact with each other, which allows to select the specific 8 segments. A number of possible interactions have already been detected between some segments. Experiments are usually done by introducing so-called synonymous mutations in viral RNA sequence and then looking for changes in RNA content in the viral progeny. Synonymous mutations basically change the sequence of the RNA but not the amino acid sequence that it codes for, so you do not interfere with IAV protein production but you prevent any possible RNA-RNA interactions between segments because the mutated sequences can no longer pair up. Figure below illustrates the so-called kissing loop interaction that is proposed to take place between segment 8 and 2 of the H5N2 IAV virus. Kissing loops are also known to occur between the two RNAs on HIV virus.

Segments 2 and 8 interaction through a kissing loop. From  Gavazzi, Cyrille, et al. Proceedings of the National Academy of Sciences 110.41 (2013): 16604-16609.
Segments 2 and 8 interaction through a kissing loop.
From
Gavazzi, Cyrille, et al. Proceedings of the National Academy of Sciences 110.41 (2013): 16604-16609.

It might be all nice and simple like that; at one end all RNAs are held by the transition zone and RNA-RNA interactions between different RNAs take place to select for eight different segments that need to be packaged. But nature rarely makes things easy, not surprising though, as 4 billion years of evolution is likely to be enough time for complexity to develop ☺

The thing is that the current picture seems to differed for different virus strains, i.e. different RNA interactions form in say H1N1 compared to H5N2 viruses. Also, it is still far from clear how 8-RNA complexes are assembled. Once RNA is replicated and coated in NP it presumably exits the nucleus and associates with other RNAs but whether this association sequential or random is not known. Recently, scientists from NIH finally managed to couple 4 different segments to of IAV to different color fluorophores so that they could be visualized within the cell at the same time. Same group also coupled viral polymerase to green fluorescence protein so that they could track the movement of RNA inside the cell (one viral polymerase always associates with NP coated viral RNA so its movement reflects the movement of an RNA segment). Just look at the figure below- I always said that science is a form of art!!!

By coupling RNAs to fluorophores they can measure the distance they travel and which different fluorophores come in close proximity, which should indicate different RNAs interacting. Weirdly, they did not find any particular preferred interaction between different segments, i.e. they interact but do not seem to prefer to interact with one or other partner. They also measured the distance from nucleus that different number of segments containing complexes are found at. One might expect that closer to nucleus there would be more complexes with fewer RNAs and then as these complexes travel towards the plasma membrane more and more RNAs associate with complexes. Well, that’s not the case! You have as many four segments containing complexes close to nucleus as you have close to plasma membrane. The RNAs are definitely interacting as the movie 2 shows where you can see a cell in which white spots are RNAs and when spots get brighter that indicates the fusion of complexes. Some of these fusion complexes seem to be stable some brake apart and go interact with others.

RNA fusion- movie 2

So all in all, we have many bits and pieces about how RNA packaging in Influenza works but we are far from defining the exact mechanism. If you are still not sure why should anyone care about such thing, well then think about therapeutic potential. We have seasonal influenza vaccines which work but we need to change them every season to accommodate the antigenic changes acquired by the virus. Drugs against influenza also are present but viruses often become resistant to them and, to be honest, by the time you start using them you already probably had infection for a while, so effectiveness is not as good and drug only reduces the illness time by day or two. Finally, the pandemic potential of emerging Influenza strains (H1N5, H7N9 etc.) is always a bit of a threat to the global public health. Seeing that packaging correct viral RNAs into the virion so important for the virus maybe if we could understand the exact mechanism we might be able to develop some new and better drugs to inhibit the process. If there are some universal steps in the process among the different IAVs we could also hope for universal and not strain specific antivirals.

To finish, I’ll leave you with a beautiful video of cells getting infected with Influenza. The virus was placed on top of the cells and left overnight for imaging. Virus is marked by GFP so you can see more and more cells becoming green as the virus replicates and spreads.

Influenza infected Cells- movie 3

Video references:
Movie 1: Fournier, Emilie, et al. “A supramolecular assembly formed by influenza A virus genomic RNA segments.” Nucleic acids research 40.5 (2012): 2197-2209.
Movies 2 and 3: Lakdawala, Seema S., et al. “Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm.” PLoS pathogens 10.3 (2014): e1003971.

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