Dengue Induced Autophagy Through ER Stress: A Mechanism for Viral Replication

The article reviewed in this post is titled: "Dengue-induced autophagy, virus replication and protection from cell death require ER stress (PERK) pathway activation" by Datan et al 2016. The article was released in March of 2016. 

Threatening 40% of the world’s population, dengue fever infection has doubled in the past 20 years, (Datan et. al. 2016). A viral disease transmitted to humans by the mosquito genus Aedes, dengue fever involves sudden fever and acute pain in the muscles and joints, (Morens, 2009). Severe dengue fever can turn into dengue hemorrhagic fever and dengue shock syndrome, which can be fatal, (Rajapakse et. al. 2012). As a vector-borne illness, dengue infection varies with seasonal climate changes, but as global warming begins to alter region’s climates, the tropics are no longer the only locations with high risk of dengue. Public health issues associated with the spread of dengue fever make research involving viral mechanisms pertinent to understand how the virus replicates for potential treatments and preventions. In a recent article, investigation of dengue-2 virus reveals that infected cells require ER stress, or PERK, pathway activation for dengue-induced autophagy, viral replication, and protection from other cell stressors, (Datan et. al. 2016). New insight into mechanisms of how dengue replicates can leads to possible treatments and vaccinations as dengue infection advances on the world’s population.

In order to ensure the virus’s own survival, dengue induces autophagy enabling the viral replication, (Datan et. al. 2016). Previously, dengue-2 was found to kill mice macrophages, yet protect epithelial cells and fibroblasts creating an optimal environment for viral survival. Induced autophagy increased viral replication and viral gene NS4A is upregulated, (McLean et. al. 2011). Inhibition of the NS4A gene limited viral replication indicating the NS4A is sufficient and necessary to trigger PI3K-dependent autophagy, (McLean et. al. 2011).

Building off this concept, researchers investigated the mechanism of dengue-induced autophagy. Autophagy involved the transport of proteins, lipids, and organelles to autophageosomes, and then they are targeted for lysosomes for degradation as part of a cell’s normal homeostatic response, (Datan et. al. 2016). Typically, the mediators of this process include sensors of cell energy, such as AMPK, however, research has shown dengue virus mediates autophagy through activation of ER, or PERK, pathway activation. Through tracking of calreticulin, a marker of global ER stress, and markers of PERK pathway, ATF-4 (cyclic-AMP-dependent transcription factor 4), and GADD34 (growth arrest and DNA damage-inducible protein 34), ER stress is activated through signaling via the PERK pathway, (Datan et al 2016). Levels of calreticulin, ATF-4, and GADD34 all increased in host cells after infection with dengue virus indicating a strong correlation between viral infection and induced ER stress and PERK pathway signaling, (Figure 1)(Datan et al 2016). Salubrinal, an inhibitor of PERK pathways, eliminates dengue virus induced increases of calreticulin in host cells also strengthening the correlation between ER stress and PERK pathway activation for mechanism of autophagy in dengue-infected cells (Figure 1). The article also described the importance of PERK pathway in viral replication and transcription by treating cells with salubrinal and using specific primers for qPCR. Presence of the PERK pathway inhibitor drastically reduced the transcription of NS4A, the gene previously found to be responsible for autophagy and viral replication in dengue infected cells (Figure 1). Since it is known dengue replication relies on autophagy, the PERK pathway is necessary for viral replication. This information was confirmed with testing in MDCK cells, (Datan et al 2016).

The study also reviewed how an activated PERK pathway increases authophagy turnover in MEF cells. Using PERK knock-outs and wild-type cells, they measured green puncta of p62, a lysosomal protein and marker for autophagy turnover, via antibodies and observed decreased p62 in PERK wild-types infected with dengue compared to the control, yet not difference in p62 concentrations in PERK knock-outs, (Datan et al, 2016). Since p62 degrades as a result of elevated rates of digestion of autophagosome content in lysosomes, the final step of autophagy, the results from this study further confirm PERK has a necessary role in dengue induced autophagy and thus viral replication.

The researchers extended the study to determine if ATM (ataxia telangiectasia mutated) signaling followed by the production of ROS (reactive oxygen species), but the results revealed little importance. While direct inhibition of ER stress causes decreases in autophagy turnover, curtails production of ROS and suppresses viral replication, ATM signaling inhibition does little to viral titers and ROS accumulation, (Datan et al 2016). However, they were able to determine that ROS regulates dengue-induced autophagy by implementing a ROS inhibitor linking autophagy to ROS accumulation and then observed high levels of ROS in dengue-infected cells, (Datan et al 2016). Cumulatively, the results demonstrate that dengue virus induces and replicates through autophagy via ER stress through ROS accumulation through a PERK dependent pathway.

Knowing this mechanism of dengue replication opens up the possibility to treat with pharmaceuticals that interfere with PERK pathway signaling during viral infection. It also poses the possibility that other virus may work this way, providing basis for investigation of other unknown viral mechanisms. For dengue, knowing the mechanisms and inner workings of the virus can help medical professionals and researchers attack the emerging and expanding outbreaks.

The threat of the reemergence of Dengue Fever also exists for Australia. A 2004 study revealed that intense El Niño Southern Oscillation (ENSO) events combined with surface temperature increases of 0.3-0.6°C correlate with higher incidence rates of mosquitoes-borne illnesses such as Malaria and Dengue Fever, (Zell 2004). Heavy precipitation events lead to floods and development of stagnant water, especially in urban environments, providing viable breeding grounds for mosquito vectors, (Khasnis & Nettleman 2005). Periods of severe rainfall most likely result from increased frequency and intensity of ENSO events due to climate change. Sea level rise creates more extensive saltmarsh habitats along coastlines, providing stagnant, moist environments for mosquitoes, (Russell, 1998). Urban area are also prone to developing more area of stagnant water as heavy rains pool water in tanks, trash bins, gutters and stray rubbish, co-inhabiting locations where mosquitoes reproduce. Increases in temperatures enable geographical shifts of species that carry Dengue Fever, such as Aedes albopictus and Aedes aegypti, further south in Australia, (Russell et. al. 2009). Temperature increases also contribute to quicker virus incubation and elevated rates of vector feeding, demonstrating the potential of rapid transmission to humans.

A majority of Australia’s population is concentrated in urban areas along the coast. High-density urban areas combined with intense ENSO and QBO events and sea level rise leave the country susceptible to Dengue Fever outbreaks. Present in Australia since the 1880’s, the disease was largely eliminated in the 1950’s, but reappeared in 1981, (Mackenzie et. al. 2006)(Russell, 2009).  Localized around the northern coastlines, dengue fever is expected to move further south and inland, corresponding to climate zone shifts, (Figure 8).  If average surface temperatures in Australia increase by 2.0-3.0°C Dengue Fever could become a common occurrence in Brisbane, (Russell, 2009). Temperature increases of 3.0-4.0°C could stabilize the virus transmission as far south as Sydney. In absence of mitigation strategies to address global warming, the spread of Dengue further south in Australia could occur by 2100 based on warming trends, (Head et. al., 2014).

Seeing the increased threat of dengue on a global scale, understanding the mechanisms of how the virus replicates initiates better intervention strategies and perhaps prevent the disease from emerging in area affected by climate change. 

1. Datan, E. Roy, S. G., Germain, G., Zali, N., McLean, J. E., Golshan, G., Harbajan, S. Lockshin, R. A. & Zaken, Z. (2016) Dengue-induced autophagy, virus replication and protection from cell death require ER stress (PERK) pathway activation. Cell Death and Disease. 7: e2127
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