Flu has NA problem infecting guinea pigs: the not-so-random packaging of viral RNA

In the last article, I wrote about a study which looked at the ability for the flu virus to package all 8 segments of its RNA genome. In short: they often don’t package all 8. And if the process of stuffing flu particles with RNA isn’t that picky (hey, important pieces are supposedly missing here), you could think that any one RNA segment is just as likely as another to be left out.

But work published in 2014 provided a neat example of how this process can be far from random, and can be directly influenced by the virus itself.

It all focuses on a mutant of the flu NP (or NucleoProtein). NP smothers flu RNA, keeping it organised for transcription and copying the viral genome, as well as allowing it to be packaged into the virus. Following previous experimental infections with the mouse-adapted influenza A strain ‘PR8’, a single mutation in NP (F346S) was found to increase virus replication in guinea pig noses. Whatever this change in NP was doing, as far as making new viruses goes, it was doing a good job. But when the same lab group looked at infection in cell culture, they found something wrong with the virus: it didn’t make very much NA protein.

NA (or NeurAminidase) is an enzyme that sits on the surface of the flu particle and allows the virus to cut itself free from infected cells (and may also have a role in cleaving through mucus on the way into the host1). By all measures, flu needs NA for infection. Yet, compared to the starting PR8 strain, the mutant virus produced less NA protein, less NA mRNA, and populations of purified virus particles contained fewer copies of the NA RNA segment. In other words, there appeared to be less of the protein because there was a shortage of instructions to make it – and all seemingly because of a single mutation in NP.

Furthermore, the authors show in this work that the mutant flu virus (F346S) produced a greater proportion of “semi-infectious” viruses during replication. Semi-infectious means a flu virus without all 8 RNA segments and incapable of completing an infection cycle all by itself. When equal amounts of fully infectious particles were added to cells, the authors saw that the mutant virus infected 8x as many cells.2 This demonstrated that the vast majority of mutant virus particles were semi-infectious. See the following diagram for clarity:


When infectivity is different, you get more virus than your bargained for
A virus stock is made up of a) fully infectious particles (green circles) and b) semi- or non-infectious viruses (red crosses). Here, both mutant and normal flu samples total 10 particles each, but the proportion of circles and crosses is different for each virus. If you wanted to infect cells with 9 green circles from each stock, you would end up adding 90 total particles from the mutant and just 10 from the normal virus stock. This explains how so many more cells are infected during the mutant infection.


So how can a virus lacking such an important protein replicate better during guinea pig infection? Especially given that so many particles aren’t fully infectious on their own?

Firstly, why isn’t it just worse at replicating? Here, the authors suggest that the virus replicates well in guinea pigs because a large number of cells get infected with multiple viruses. If one virus lacks all 8 segments, then another can help out if they infect the same cell. Here’s this image again from my last article:

The power of co-infection
Lefty here only has 7 of the 8 pieces of RNA he needs, so this infection is doomed to fail. Righty and friend also only have 7 pieces, but between them they have the full set – they’ve all they need to make more virus.

I think the authors do a cool job of showing how co-infection occurs over time in the guinea pigs. When they took cells washed from infected noses at 9 hours after infection and flow sorted them, they saw that only a small proportion (25–44%) of cells produce both proteins HA and NA. But when they did the same thing at 48 hours after infection, 80–90% of cells contained both. These data suggest that early in infection, cells are generally infected with individual viruses, both fully or semi-infectious. But once more rounds of flu are produced in the guinea pig the majority of cells get infected with multiple viruses. Who cares about carrying all 8 RNA segments when you have a load of mates to help you out?

So while co-infection minimises the detrimental effect of semi-infectious particles, how exactly does this mutant virus replicate better in guinea pigs?

The authors suggest a number of possibilites in the discussion of this work, notably that some unknown beneficial effect of the NP mutation may outweigh the lack of NA, or that in combination with the NP mutation, reduced NA activity could be an advantage. It’s certainly not clear. To finish, I’ll posit a version of the second possibility.

Perhaps, in the absence of NA, the flu particles are simply aggregating together. This idea is supported by work from the Barclay lab at Imperial College London, in a 2013 paper.3 Specifically checking out figure 5, flu virus particles possessing a shorter form of NA aggregated together far more than those with the normal longer length NA. Why does this happen? Because sialic acid (the cell molecule to which the flu virus binds in order to infect cells) gets stuck on to newly formed virus particles and must be cleaved off by the NA protein. NA usually cuts flu free from cells, but it can also sever the tethers between viruses. The short NA is worse at this than the longer form, and you know what, I bet viruses lacking NA altogether are terrible at separating themselves.

Flu aggregation in the absence of NA
On the topmost cell, influenza buds and escapes normally due to the presence of NA (long red lines) on the virus surface, which cleaves the sialic acid molecules on the cell (very long green lines). On the bottom cell, the flu particles are covered in far fewer copies of NA, impairing the break down of sialic acid. The flu HA protein (short black lines) binds sialic acid on both the cell and virus membranes, leading to aggregation of virus particles. If some NA is present, the aggregate could come away from the cell and infect as one mass.

And if you’re a flu particle looking for a friend to go co-infecting with, what better way than to hold hands as you both enter the cell at the same time?

Whilst this hypothesis isn’t complete, as I have no answer as to how NP affects NA or what else it may be doing, I figure less NA on viruses = better virus aggregation and thus more efficient co-infection. Greater co-infection could allow for greater reassortment of novel mutations, and could aid the virus in the battle against intracellular immunity by overwhelming the cell before immune signalling is fully established.

Big clumps of virus sounds ideal for infection, but viruses don’t just ‘worry’ about what to do inside a host. What about getting between hosts?

More next time.

Main reference: Brooke et al., 2014. Influenza A virus nucleoprotein selectively decreases neuraminidase gene-segment packaging while enhancing viral fitness and transmissibility. (My apologies, this paper is not Open Access).

  1. Blumenkrantz et al., 2013. The short stalk length of highly pathogenic avian influenza H5N1 virus neuraminidase limits transmission of pandemic H1N1 virus in ferrets. (Open Access)
  2. Note: a virus doesn’t have to be fully infectious to get into a cell. So long as it can enter a cell and produce some virus proteins, that’s enough. The differentiation between semi and fully infectious is just about whether the virus can get in, copy itself AND get out again to repeat it all.
  3. This reference again: Blumenkrantz et al., 2013. The short stalk length of highly pathogenic avian influenza H5N1 virus neuraminidase limits transmission of pandemic H1N1 virus in ferrets. (Open Access)

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