Feeling under the weather? Are you congested, maybe having a hard time breathing, especially at night? Don’t worry – this is your body’s natural response to an infection. Mucus production is generally regarded as antimicrobial – it traps particles like virions before they can infect nearby host cells. But in some cases, microbes can turn the tables and use our defenses to their advantage.
New research in the Journal of Virology investigates the role of mucus during viral infection. The study began by comparing alveolar epithelial cells infected with influenza A virus to those uninfected, when lead scientist Dr. Keizo Tomonaga and first author Shoko Nakamura identified two miRNAs (miR17-3p and miR-221) that regulate human UDP-GalNAC transferase (also called GALNT3). Influenza-infected cells had lower expression of both these miRNAs, and GALNT3 expression was concurrently upregulated. What does GALNT3 do?
Mucus is a mixture of glycoproteins, antibodies, antimicrobial factors, and water. A major component of mucus is the mucin proteins produced on epithelial cell surfaces. Mucins undergo O-linked glycosylation (a sugar group is covalently bonded through an oxygen atom) before being either attached to the epithelial cell surface or secreted into the extracellular space of the respiratory tract. GALNT3 is one of the enzymes necessary for glycosylation, and therefore full function, of mucins. But if mucins (and mucus) are generally antimicrobial, why would influenza infection indirectly upregulate GALNT3 expression?
When the research team looked at GALNT3 expression in a time-course assay, they found that induction of GALNT3 in infected cells was highly correlated with release of viral genomic RNA, suggesting that GALNT3 expression aids influenza replication (see figure, right). A further experiment suggested that GALNT3 influences the viral polymerase activity, which could explain the increased viral production. While GALNT3 plays a role in mucus production, the virus also benefits from its activity, which may be why influenza virus regulates miRNA that influence GALNT3 expression.
If GALNT3 increases viral production, logic suggests that cells that lack GALNT3 should have lower viral replication. The scientific team tested this and confirmed that an infection in Galnt3-/- cells produced less viral RNA and fewer virions compared to normal cells. So if influenza virus increases GALNT3 expression to promote viral replication, and cells that don’t have GALNT3 make less virus, it sounds like GALNT3 should be a viable drug target – if scientists can block GALNT3 expression or activity in influenza patients, it should shorten the duration of their illness. Right?
All of the experiments to this point were performed in cell culture, which is a great way to test and understand molecular interactions. However, the more complex environment of an animal host is a better model of the variables encountered during infection, including multiple cell types, tissue architecture, vasculature that brings nutrients and immune responders, and other microbial constituents of the host microbiome with which to interact. The scientists tested infection of wild-type mice compared to the Galnt3-/- mice, expecting the knockout mice to survive better, since they should support less viral replication.
However, what they observed was the opposite – the Galnt3-/- animals succumbed to their infection much more quickly than the normal animals. Although the decreased virus production, observed in cell lines, initially held up in the animal model, after three days, the tides turned and the Galnt3-/- animals had higher virus titers. Replication during the onset of infection may still have been affected, but the virus was able to overcome its initial deficit.
These results make it seem likely that GALNT3 plays a dual role during infection: at first, it helps the virus by promoting viral replication, but at later stages of disease, it helps the host to control the infection, perhaps by promoting mucus production to trap and clear the virus particles. This duality illustrates the complexity of molecular interactions during infection, as an all-or-nothing result is rare in the context of disease (or biology in general, in which redundant systems are common). And since modulating enzyme activity level is much more difficult than inactivating with an inhibitor, this last experiment reveals GALNT3 itself is not a strong antiviral drug candidate.
What the scientists did take away is the importance of understanding how influenza regulates GALNT3 expression. Infected cells changed expression of two key miRNAs, but how the infected cells changed their miRNA levels is still a mystery. Understanding this may provide a viral (rather than host) target that could manipulate viral replication and facilitate control earlier in the course of infection.
-- Julie Wolf