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Abstract
Influenza viruses are known to cause seasonal epidemics that claim nearly half a million lives each year. In addition, influenza viruses can also cause occasional pandemics which, in the past, have resulted in a significant increase in the number of influenza-related deaths worldwide. Pandemics arise when influenza viruses with novel hemagglutinin (HA) and neuraminidase (NA) surface proteins are introduced into the human population to which there is no prior immunity. In addition to causing disease, emerging viruses with pandemic potential must also be capable of sustaining efficient human-to-human transmission. Transmission of human influenza viruses occurs mainly through contact of the mucosal membranes with respiratory droplets containing infectious viral particles. Previous studies have shown that both environmental and molecular factors can influence the efficiency of influenza virus transmission such as air temperature and humidity, and viral protein stability. Another factor which is likely to affect the efficiency of influenza virus transmission is virion morphology. Influenza virions are known to have multiple forms, ranging from 100 nm spheres to filamentous particles with lengths of up to 20 µm. Furthermore, there is a known correlation between the size of respiratory droplets and the distance in which they can travel. The smallest droplets measuring between 1 and 5 µm are carried the greatest distances and are therefore thought to be responsible for long range influenza virus transmission. For this reason, influenza viruses that produce an abundance of small spherical virions may exhibit higher transmission efficiencies than viruses with large filamentous phenotypes.
In April of 2009, a novel influenza virus (pH1N1) with an extremely high rate of transmission efficiency emerged in the human population. This virus was identified as an H1N1 reassortant between two influenza viruses that had been circulating throughout pig populations for many years. Most of the pH1N1 gene segments were originated from the triple reassortant (TR) swine lineage, while the M and NA segments are from viruses of the Eurasian swine lineage. Despite being genetically similar, TR viruses rarely infected humans and were incapable of sustaining efficient human-to-human transmission. We therefore tested whether pH1N1 and TR viruses which differ in their ability to transmit between humans also differ in virion morphology. We discovered using immunofluorescence and electron microscopy that the highly transmissible pH1N1 strain produced predominantly spherical virions, whereas the poorly transmissible TR isolates induced a filamentous phenotype. We also showed that the difference in morphology between these viruses was due to specific amino acid residues with the influenza matrix (M1) protein at positions 30, 207, and 209. Substitution of these pH1N1 M1 residues with the corresponding TR residue by site-directed mutagenesis resulted in recombinant pH1N1 viruses with filamentous morphology, reduced viral growth kinetics, and small plaque phenotypes. Altogether, our findings strongly suggest a direct correlation between spherical influenza morphology and efficient viral spread in vitro.
Although M1 has previously been shown to be the major determinant of influenza virion morphology, the release of viruses as either spherical or filamentous particles is likely to involve interactions between multiple viral proteins. Previous studies have shown that M1 interaction with the cytoplasmic tail domains of the three transmembrane proteins HA, NA and M2 affect virion morphology. In addition, M1 interaction with the nucleoprotein (NP) has been suggested to affect virion morphology. To gain further insight into the mechanism that determines influenza morphology, we tested the effect of M1 interaction with each of these viral proteins individually. Using a recombinant virus system, we discovered that while A/WSN/33 (WSN) and A/Aichi/2/68 (Aichi) wild-type viruses are predominantly spherical strains, a recombinant WSN virus expressing the Aichi M1 protein (WSN-AichiM1), exhibited filamentous virion morphology. By expressing addition Aichi proteins in a WSN-AichiM1 background, we found that coexpression of Aichi M1 with NP, but not the three transmembrane proteins, inhibit filament formation. Moreover, we showed that WSN-AichiM1 viruses expressing only residues 214 and 217 from the Aichi NP sequence could produce virions with spherical morphology. The results of our study revealed specific amino acids in the NP sequence that are required for the production of spherical virions, a characteristic of highly transmissible viruses. This specific interaction site can potentially serve as a target for the development of novel therapeutics against emerging pandemic viruses.
