SARS-CoV-2 Entry: The Potential EV Hitchhiker

You can find the original article here, and the Ya-Wen Chen Lab (Icahn Medical School at Mount Sinai) here.

Introduction

The COVID-19 pandemic, caused by SARS-CoV-2, is an event whose modern-day impact precludes introduction. In December 2019, an outbreak of SARS-CoV-2 emerged from a cluster of patients in Wuhan, situated in China’s Hubei province (1). By November 2025, upwards of 7.1 million deaths worldwide had been attributed to the pandemic, especially among adults above the age of 65 (2). SARS-CoV-2 infection is usually characterized, within the first 0-7 days post-infection, by its constitutional symptoms (the generic, body-wide symptoms associated with “normal” disease progression). In the vast majority of infections, this is followed by a gradual return to health following bodily elimination of the virus. For certain groups of individuals (especially older adults, immunocompromised individuals, and those with underlying pulmonary or respiratory illness), however, internal dysregulation or severe COVID-19 disease may manifest instead. This is usually due to the emergence of a “cytokine storm” (essentially an overproduction of molecules that cause inflammation), and SARS-CoV-2 infection of cell types outside of its normal hosts. There is also “Long COVID”, a poorly understood condition wherein patients experience internal dysregulation months to years after virus elimination (2). Overall, SARS-CoV-2 creates a very diverse set of illnesses and conditions, and the reasons for this remain poorly understood. Given these discrepancies, there has been a major focus in the scientific enterprise over the last several years to fully understand and characterize SARS-CoV-2 infection mechanisms, with the hope of producing more effective and specific therapies for affected individuals, especially given the large number of coronaviruses circulating in mammal populations. This paper, published from the Ya-Wen Chen Lab at the Icahn Medical School at Mount Sinai, explores a poorly understood but intriguing aspect of SARS-CoV-2 infection: how it can infect cells that do not possess its attachment receptors (molecules on cells that the virus uses to gain entry).

An Upheaval in SARS-CoV-2 Entry

Viruses build their outer proteins into super-specific shapes, and in the world of proteins, shape determines what you can do and what you can interact with. By doing so, viruses, under evolutionary pressure, design their outer proteins to specifically and strongly interact with host cells' outer proteins. This allows them to gain entry to these cells and determines which cells they can interact with. In the case of SARS-CoV-2, established models affirmed that the virus interacts with Angiotensin Converting Enzyme 2, or ACE2, on pulmonary epithelial cells (cells that comprise the surfaces of much of the lungs. This places the virus in an endosome (an internal cellular compartment) that then showers the virus with acids that allow it to release its genetic material. An alternate entry mechanism, however, involves TMPRSS2, a protein that changes SARS-CoV-2’s outer protein shapes, which in turn allows the virus’s outer membrane to fuse with the membrane of the host cell. 

The latter explanation generally assumes that these proteins are on the surface of cells, since the virus needs to access them first in order to gain entry. However, imaging of the cells reveals that TMPRSS2 is almost exclusively found on the inside of cells, near but not breaching the surface:

Fig. 1A, C: These results show that TMPRSS2 exists inside the cells. To the left, Figure 1A shows images of cells with dyes that stain TMPRSS2 and EPCAM, a surface protein. As can be seen, TMPRSS2 exists in an inner band within the EPCAM signals, indicating that it exists inside the cells, rather than on the surface. On the right, we can see the results of flow cytometry, where the further along an axis a signal is, the greater the amount of protein is in that sample. In the surface samples, we can see high levels of EPCAM-APC but low levels of TMPRSS2. In the inner samples, we observe high levels of both signals, indicating that TMPRSS2 is exclusively intracellular.

Fig. 2A and Fig. S5A: These results show that TMPRSS2 exists in extracellular vesicles or EVs (which are small compartments that carry stuff out of cells). To the left, there are more images showing TMPRSS2 in green and common “markers” of EVs in red. As can be seen, there is significant overlap between these signals. To the right, we can see that a great proportion of these EV markers are in the same places as TMPRSS2 (and vice versa), indicating that TMPRSS2 may exist inside of EVs.

Fig 3A-B: These results show that TMPRSS2 is expressed on the surface of pulmonary epithelial cells in diseased lungs. In A, we see that healthy lung cells have low levels of TMPRSS2 compared to EPCAM, indicating little to no TMPRSS2 is on their surfaces. In B, we see that amongst patients with idiopathic pulmonary fibrosis (IPF), fibrosis, or interstitial lung disease (ILD), there are small but nonzero levels of TMPRSS2 on cell surfaces

From the above results, the authors begin to challenge the latter SARS-CoV-2 entry mechanism. Only in diseased lungs is TMPRSS2 on the surface, since in healthy lung cells, TMPRSS2 remains in extracellular vesicles. EVs are notable in that they tend to be absorbed by other cells, especially phagocytic cells (cells that engulf and consume given materials). Based on these findings, then, the authors began to look for cells that contain TMPRSS2 but aren’t capable of producing the molecule, as they may be absorbing it through these EVs.

A Striking Discovery: SARS-CoV-2 can enter Cell Types that do not Produce TMPRSS2

In their search, the authors find that alveolar macrophages, or AMs, and endothelial cells (ECs) expressed TMPRSS2 on their surfaces despite not creating the genetic material needed to produce TMPRSS2. Alveolar macrophages are phagocytic cells that patrol the alveoli for incoming disease threats. Endothelial cells are cells that comprise the lining of blood vessels, and do not normally express TMPRSS2 at all. However, the authors find that some of these cells nevertheless have TMPRSS2 on their surfaces:

Fig 5A-B: These results show that, in some diseased lungs, endothelial cells and alveolar macrophages possess TMPRSS2 on their surfaces, despite these cell types being incapable of doing so. We can see in A and in B that TMPRSS2 levels are high in both cell populations. 

Fig 6C, E: These results show that, within purified alveolar macrophages and endothelial cells, adding the aforementioned EVs caused the sudden appearance of TMPRSS2 on their surfaces. In C and E, we can see that adding EVs caused a massive increase in the TMPRSS2 signal. This perhaps explains why, in certain cases, cells have TMPRSS2 on their surfaces even if they are incapable of producing it, and a potential way in which they then acquire TMPRSS2. 

Fig 7B-C: These results show that adding EVs to stem cells increases their susceptibility to SARS-CoV-2 infection. We can see in both panels that viral titers (concentrations) are massively increased when EVs are added. Stem cells are cells that haven’t developed into any given cell type, so they shouldn’t normally produce TMPRSS2 since they haven’t developed in pulmonary epithelial cells. However, by adding EVs, SARS-CoV-2 was able to infect them nonetheless, suggesting that the EVs were installing TMPRSS2 onto their surfaces, and thus allowing the virus to gain entry.

The aggregate of these experiments reveals a striking new mechanism through which SARS-CoV-2 gains access to cells. Extracellular vesicles can carry TMPRSS2 and install it onto the surface of phagocytic cells, expanding the range of cells SARS-CoV-2 can infect. This newfound host diversity may begin to explain some of the greater discrepancies regarding COVID-19 disease, especially within severe COVID-19. As aforementioned, this installation of TMPRSS2 onto cell surfaces occurs more frequently in diseased lungs, providing potential directions regarding COVID-19’s abnormal pathologies (the manifestation of disease) in such cases. One major question that remains is whether this release of EVs is a deliberate mechanism of SARS-CoV-2, meant to prolong or intensify infection, or if this release is a hijacked or deliberate mechanism of the body. The authors posit that these EVs may serve as “decoys”, meant to absorb viruses so that they cannot enter actual cells. It’s also possible, as the authors also consider, that SARS-CoV-2 deliberately “hitchhikes” within the EVs and waits for them to be absorbed by cells, which would allow the viruses to enter undetected. More experiments in these regards will be needed to meaningfully explore these avenues, but this paper nonetheless explores a fascinating aspect of SARS-CoV-2 infection. In exploring such questions, it may be possible to create better treatments against coronaviruses, especially for vulnerable individuals, and to better understand SARS-CoV-2 infection. Coronaviruses are diverse and circulate in animal populations, so there is an omnipresent threat that a new virus may emerge in humans, as has occurred a few times this century (such as SARS-CoV in 2006, MERS-CoV in 2011, and of course, SARS-CoV-2 in 2019). By studying SARS-CoV-2 in its entirety, we can better safeguard ourselves against such new diseases, improving outcomes for untold numbers of people as we continue in our ever-advancing day.

References

N.A., (2024, July 4).  CDC Museum COVID-19 Timeline. Center for Disease Control,

    https://www.cdc.gov/museum/timeline/covid19.html#print.

Zhu, Y., Sharma, L., & Chang, D. (2023). Pathophysiology and clinical management of 

coronavirus disease (COVID-19): a mini-review.Frontiers in immunology, 14, https://doi.org/10.3389/fimmu.2023.1116131. 

Rea-Moreno, M., Tian, L., Tavakol, T.N. et al. (2026). Unveiling alternate pathways for 

SARS-CoV-2 infection via extracellular vesicle-mediated transfer of ACE2 and TMPRSS2. Nature Communications, https://doi.org/10.1038/s41467-026-71680-w. 

Inapparent maternal ZIKV infection impacts fetal brain development and postnatal behavior

You can find the original article here, and the Daniels Lab can be found here. 

Zika virus became a household name in 2015, when an epidemic began in Brazil and spread throughout the Americas (1). Zika is spread through mosquito bites, and in adults it causes mild illness. The primary concern with Zika is not its impact on adults, but its effects on developing fetuses. When a pregnant woman is infected with Zika virus, the virus can cross the placenta and infect the fetus in a process known as vertical transmission. This can cause severe birth defects known collectively as congenital Zika syndrome, or CZS. CZS is as dramatic as it is rare: an estimate from the CDC in 2017 estimated that one in twenty children exposed to Zika during their mother’s pregnancy would develop birth defects (2). What happens to the other nineteen children? This paper, out of Brian Daniels’ lab at Rutgers University, looks into how milder Zika virus infections reshape the developing brain and can potentially have effects that reach far into adulthood.

           

A Mouse Model for Mild Zika

            As with many other diseases, we study Zika virus through observing the course of infection in mice. Zika virus presents a unique challenge, however. Adult mice are not naturally susceptible to Zika virus infection due to differences between their immune system and ours. Traditionally, the solution to this problem was to use immunodeficient mice. While this allowed Zika virus to take hold, the resulting disease progression looks very different to what we see in most humans. The authors of this paper take a different approach. They use a specialized lineage of mice genetically altered to have a crucial immune response regulator, Stat2, replaced with the human analogue. These mice are susceptible to Zika virus because of their humanized immune systems without having to be immunocompromised, allowing for more nuanced studies like this one to go forward.

            The authors show their refinement of this mouse model in Figure 1. Their goal here is “inapparent maternal Zika infection”: infection with Zika virus that is successful but does not produce severe symptoms in the mother or the pups. This is achieved through exposing the mothers to Zika virus during the middle of their pregnancy, rather than in earlier stages. The resulting procedure has the researchers infecting the mothers at E12.5 (roughly equivalent to the late first trimester in humans) and collecting samples for analysis at E18.5 (late second to early third trimester).

            The most important elements of Figure 1 are the experiments showing that the pups of infected mothers are not showing severe symptoms of Zika infection. This figure shows that few fetuses are resorbed regardless of Zika virus exposure; resorption occurs in mice when a fetus is not viable, similar to miscarriage in humans. Additionally, the researchers show that there is no difference between the brain weights of fetuses with and without Zika virus exposure. This shows a lack of microcephaly, a classic sign of fetal Zika virus exposure that involves a newborn’s head being much smaller than normal. Taken together, these confirm that the infant mice are “clinically normal” and allow the researchers to look at the less-obvious effects of Zika virus exposure. Having confirmed that their mouse model works the way they want it to, the researchers move forward.

Figure 1I-J: This data shows that the fetuses of mice infected with Zika virus following this paper’s protocol are viable at the same rate as uninfected mice. The authors show data from two timepoints, E15.5 and E18.5. Black bars represent fetuses resorbed by the mother, a process that occurs in mice when a fetus is not viable similar to miscarriage in humans. There is no significant difference between resorption in Zika virus-infected mothers and mothers not infected.

Figure 1K: Microcephaly (small heads) can occur as a result of Zika virus in a fetus, so the authors checked brain weights to confirm that their mice were not experiencing this symptom. The bars show the average brain weights of mice exposed to Zika virus as fetuses and those not exposed, and show that there is no significant difference between the two. This demonstrates that microcephaly is not a problem in the Zika virus-exposed fetuses.

 

The transcriptome: a snapshot of activity

            Much of this paper revolves around analysis of the cellular transcriptome. While the genome is a collection of all the genes in a cell, the transcriptome provides a record of all the genes that are being expressed; that is, what is turned on and affecting the cell. This paper is looking at the transcriptome of mouse pup brains and how it is different if the mice have been exposed to Zika virus through their mothers. They find that there is a shift in activity briefly around E18.5, the timepoint used here as an analogue to late-second and early-third trimester pregnancies. Figure 2 shows this divergence: the transcriptomes of Zika virus-exposed mice and healthy mice overlap at E15.5 (1^st trimester) and at P21 (early infancy), but diverge at E18.5.

Figure 2B: These three charts visualize the transcriptomes of Zika virus-exposed and unexposed fetal mice at three timepoints (E15.5, E18.5, and P21). The exposed and unexposed samples overlap except at E18.5, suggesting that this is when the viral infection is affecting which genes are active in the cell.

            The next step after identifying if and when the transcriptomes are different is finding out how they differ. This paper does this using gene ontology enrichment analysis, which analyzes a transcriptome sample and identifies the functions of the genes found to be active. The results from this analysis show that there is a difference in the regulation of genes associated with synapse development between Zika virus-exposed and healthy mice. Synapses are connections between neurons that allow for communication between individual cells, and proper synapse formation is essential for healthy brain function. Abnormal expression of genes related to synapse function in fetal mice exposed to Zika virus suggests that these mice may have issues with their brain function, particularly since these differences are seen right as the brain is beginning to come together in earnest. It’s also important to remember that these differences are in mice who appear “clinically normal” – this is the first sign in the paper that Zika-exposed children might have underlying health issues that aren’t immediately obvious.

 

Figure 2D: This table shows the results of an initial gene ontology analysis, which looks at the functions of the genes that have different activation levels between healthy and Zika virus-exposed mice. The lines highlighted in blue show genes associated with synapse function and development, suggesting that the brain cells exposed to Zika virus will form connections with other cells differently.

 

            The next four figures build on this finding by narrowing the focus to two specific types of cells that have the greatest change in activity and studying them in depth.

 

Beyond the transcriptome: problems in later life

            Although finding these changes to cellular activity is interesting, it may be just a temporary shift as the cells deal with a brief visit from a virus. Do the cells return to normal after their encounter with Zika virus, or are there effects that last beyond the brief window of observed transcriptomic change? Figure 7 provides compelling evidence for the latter.

            The authors study the brains of P30 (early infancy) mice and observe differences in the number of excitatory synapses in the hippocampus when the mouse was exposed to Zika virus. Excitatory synapses increase the likelihood that a neuron they are connected to fires, or transmits an electrical signal. They’re necessary for normal brain function, but too many can cause a condition called excitotoxicity. Excitotoxicity happens when a neuron is damaged by too many excitatory signals, and it is a factor in neurodegenerative diseases and stroke. More excitatory synapses in the brains of Zika-exposed mice suggests that these mice are more prone to these kinds of brain damage.

Figure 7B: Here, the authors show a significant increase in the amount of excitatory synapses in the hippocampi of mice exposed to Zika virus as fetuses. This is done through measuring levels of Homer1, a fluorescent tag that marks excitatory synapses.

            The authors show how an increase in excitatory neurons might result in brain damage by testing how susceptible to stroke their Zika-exposed mice are. This is done by inducing strokes in the mice and recording how severe the stroke is. Compared with nonexposed mice, Zika-exposed mice have much more severe strokes, including a death rate twice as high and no “mild” strokes.

Figure 7H and 7I: The authors show a scale for grading seizure severity in 7H and apply it to the seizures observed in their mice in 7I. Mice exposed to Zika virus as fetuses have a greater risk of death from induced seizure, and they did not have any observed mild seizures.

            Finally, the authors test how the behavior of Zika-exposed pups differs from their unexposed counterparts as adults. These differences seem to be divided between sexes: female Zika-exposed mice show differences in movement distance, movement speed, and behaviors exhibited that aren’t present in the male mice. Although mapping mouse behavior onto human diseases is difficult, the fact that there are differences after the mice are fully grown is a concerning finding that shows how long Zika virus exposure can have an effect.

            The authors began this study asking if there are health outcomes of Zika virus exposure during development that aren’t immediately obvious at birth, and they come away from it with strong evidence that there are. Although mice are far from humans (no matter how tailored the model may be), these results are concerning and suggest that some children may have slipped through the cracks of the medical system without awareness of their underlying risk for neurological conditions. The authors suggest keeping a closer watch on children exposed to Zika virus before they are born and following up with them as they grow. Do these children have more strokes than is typical as they grow? Are there higher rates of learning disabilities or memory issues among them? While there is no guarantee that there will be, it might be better to be overcautious to ensure that these children grow up to be as healthy as possible.  

Figure 8B and 8C: 8B shows differences in distance traveled, while 8C shows differences in velocity (speed) between female mice exposed to Zika virus as fetuses (red line) and unexposed mice. The authors do not see a difference in exposed and unexposed males.

Figure 8E: This graph shows behaviors that have different likelihoods in female mice exposed to Zika virus as fetuses and unexposed females. Gray dots show behaviors that are equally likely in either mouse, red dots above the trendline show behaviors more likely in unexposed mice, and blue dots below the trendline show behaviors more likely in Zika virus-exposed mice.

 

Citations

Chou, T. et al. (2026). Inapparent maternal ZIKV infection impacts fetal brain development and         postnatal behavior. PLOS Pathogens 22(1): e1013850.             https://doi.org/10.1371/journal.ppat.1013850

1.     Hennessey, M., Fischer, M., & Staples, J. E. (2016, January 29). Zika virus spreads to new areas – Region of the Americas, May 2015-January 2016. CDC Morbidity and Mortality Weekly Report. http://dx.doi.org/10.15585/mmwr.mm6503e1.

2.     Shapiro-Mendoza, C. K. et al. (2017, June 16). Pregnancy outcomes after maternal Zika virus infection during pregnancy – U.S. Territories, January 1, 2016 – April 25, 2017. CDC Morbidity and Mortality Weekly Report. http://dx.doi.org/10.15585/mmwr.mm6623e1

The Ability of Hantaan Virus to Interfere with Host Immune Responses via the Manipulation of Autophagy

Paper: Wang, K., Ma, H., Liu, H., Ye, W., Li, Z., Cheng, L., ... & Zhang, F. (2019). The Glycoprotein and Nucleocapsid Protein of Hantaviruses Manipulate Autophagy Flux to Restrain Host Innate Immune Responses. Cell reports, 27(7), 2075-2091. [1]

Hantavirus is an infective agent relevant to public health concerns worldwide. As human-to-human transmission is rare, infection is primarily passed from rodent reservoir hosts into humans through aerosolization of mouse or rat bodily secretions [2]. Ultimately, the impacts of this infection pose great epidemiological threats through the syndromes they are linked to induce. In the Americas, for instance, “New World” hantaviruses primarily cause hantavirus pulmonary syndrome (HPS), which in turn leads to severe respiratory stress and high mortality rates. (Click here to view the CDC interactive map of hantavirus incidents within the United States). Additionally, in Europe and Asia, “Old World” hantaviruses primarily manifest as hemorrhagic fever with renal syndrome (HFRS) upon infection. The major causative agent of this syndrome is Hantaan virus (HTNV), which is able to disrupt the permeability of kidney endothelial cells, ultimately resulting in acute kidney failure and mortality rates as high as 15% [2]. Although there are established vaccines and antiviral drugs to combat HTNV-induced syndromes in China and Korea, further public health interventions are being developed for hantavirus. Specifically, many clinical trials are currently being conducted for vaccines against causative agents of HPS, as therapeutic measures against these hantaviruses have yet to be successfully achieved in the United States and greater western hemisphere [3]. 

Therefore, many researchers continue to look into the pathogenic mechanisms of hantavirus infection with the hopes of uncovering new steps and proteins present in the viral pathway to target clinically. In this particular study, researchers were investigating the ability of HTNV to interfere with autophagy flux pathways facilitated by the host’s antiviral response to infection (see diagram A below and click link to cartoon) [4]. Specifically, this study aimed to investigate how HTNV is able to manipulate the autophagy pathway and which viral proteins are involved. HTNV is an enveloped virus containing a segmented genome composed of a single minus-sense strand of RNA. Therefore, viral proteins of particular interest to this study were the HTNV surface glycoprotein (Gn), as well as the nucleoprotein functioning to coat its genome within the viral particle (NP). These structures are depicted in diagram B below. 

Diagram A: Host Cell Autophagy. At the start of the pathway, a double-membraned particle (phagophore) grows to surround specific cytosol contents to target for degradation. This structure matures into an autophagosome that ultimately fuses with a lysosome (forming an "autolysosome") to complete the degradation of contained substrates. [1]
Image source: https://www.med.uio.no/ncmm/english/news-and-events/profiles/images/cropped-illustration.jpg

Diagram B: Structure of Hantavirus. The virus contains three segments of (-)-sense ssRNA that each encode key viral proteins: small (S) encodes the nucleocapsid to coat the genome, medium (M) encodes the surface glycoproteins, and large (L) encodes the viral polymerase needed for replication. [1]
Image source: https://jasn.asnjournals.org/content/jnephrol/16/12/3669/F1.large.jpg

To first verify that HTNV infection acts to both induce and manipulate autophagy flux, researchers analyzed various known indicators of the pathway. Specifically, human umbilical vein endothelial cells (HUVECs) were infected with HTNV at various multiplicities of infection (amount of viral particles per cell) and immunoblots were performed 6 and 24 hours after infection. As shown below, two key indicators analyzed for autophagy were p62 and LC3B. The p62 protein is a receptor for substrates that are being sent for degradation, while LC3B is a microtubule-associated protein that acts within the selection process of this cargo [5; 6]. It is important to look at these two proteins together, as they have been found in previous studies to bind to one another and become degraded as a result of autophagy flux completion [7]. Therefore, the decrease of both p62 and LC3B at increased MOIs indicates that at 6 hours post infection (hpi), HTNV was indeed inducing complete autophagy flux. This is further supported by the increase across rising MOIs within Beclin1 levels, as this protein is an established indicator of autophagy initiation. However, at 24 hpi, these indicators did not demonstrate the same response to increased levels of HTNV infection, implying a disruption of host autophagy.

Figure 1B: HTNV induces host autophagy at early stages of infection. Levels of proteins p62, LC3B, and Beclin1 at differential MOIs of HTNV were detected in HUVECs at 6hpi. Reduced p62 and LC3B, as well as increased Beclin1 at high MOIs indicate complete autophagy at this time point post infection.

Figure 1C: HTNV blocks host autophagy at late stages of infection. Levels of proteins p62, LC3B, and Beclin1 at differential MOIs of HTNV were detected via immunoblot in HUVECs at 24hpi. Lack of changes in expression at high MOIs indicate incomplete autophagy at this time point post infection.

After visualizing these cells via electron microscopy, these findings were further supported by quantifying the number of autophagosomes as compared to autolysosomes at both time points. At 6 hpi, cell cultures exhibited more autolysosomes (the last step of autophagy flux in which substrates are degraded). Whereas at 24 hpi, cultures showed an accumulation of autophagosomes (an intermediate step demonstrating incomplete fusion with lysosomes). This prompted researchers to conclude that despite HTNV’s induction of autophagy at early stages, the virus is somehow able to manipulate the pathway to be incomplete at later stages of its infection.

In hopes of uncovering the viral mechanism beneath this manipulation, HEK293T cells were transfected with various proteins of HTNV and levels were detected via immunoblot. Of particular interest to researchers was the resulting levels of the viral surface glycoprotein (Gn) and nucleoprotein (NP). As shown below, with Gn expression alone, decreased p62 and LC3B levels indicated complete autophagy, just as they had in the prior experiment. An additional immunoblot also demonstrated that with decreased levels of Gn, there was a loss of this p62/LC3B response, indicating that Gn may be able to drive autophagy flux upon infection. However, with the addition of NP expression to that of Gn, there was a noticeable loss in the robust nature of this Gn-induced autophagy. This suggested that NP was somehow interfering with Gn’s ability to complete autophagy— a hypothesis supported further by subsequent detection of autophagy progress via fluorescence microscopy. Using a reporter construct that is able to induce a color change from yellow to red when autophagosome to lysosome fusion occurs, increased yellow fluorescence was detected in Gn-NP expressing cells. This supported the conclusion that NP was blocking Gn at the particular autophagy step of fusion.

Figure 3A: Gn drives autophagy flux, unless inhibited by NP. Levels of autophagy indicators (p62 and LC3B) were detected via immunoblot in HEK293T cells transfected with HTNV viral proteins. Reductions in LC3B and p62 with Gn expression alone suggest the occurrence of complete autophagy, which is less robust in cells expressing NP+Gn.   

To build upon their findings that HTNV infection and viral Gn expression are able to induce autophagy flux within the host, researchers then aimed to determine the type of autophagy occurring as well as the mechanism of action. Given previous findings of the impact of viral infection on mitochondria within host cells, they investigated the possibility of the observed autophagy being the preferential degradation of mitochondria, termed “mitophagy” [8]. This hypothesis was supported via electron microscopy and flow cytometry of HUVECs that were either infected with HTNV or transfected to express Gn. Specifically, electron microscopy allowed researchers to visualize mitochondrial swelling within these cells, while the ability of flow cytometry to measure mitochondrial membrane potentials further demonstrated disturbed mitochondrial function under these conditions. 

Drawing again upon previous findings, researchers hypothesized that this mitophagy may be facilitated via the interaction of Gn with LC3B, the protein from their first experiments that functions in autophagy substrate selection. After identifying a particular sequence within the C-terminus of Gn that commonly binds with LC3B in other protein-protein interactions, mutants of the HTNV Gn were created by the researchers. Two mutants lacking the LC3B-binding motif (YRTL) were unable to cause the same level of mitochondrial damage as compared to full-length Gn and truncated mutants still able to interact with LC3B, as shown in the figure below. In addition, further investigations into Gn protein interactions revealed that the viral protein may also facilitate mitophagy through binding to a translation elongation factor associated with mitochondria (TUFM). Specifically, immunofluorescence imaging showcased colocalization of the proteins, and immunoblot revealed that TUFM knockdown cell lines exhibited significantly attenuated expression of mitophagy indicators. From these findings, a conclusion was made that Gn interacts with both the autophagy-associated protein (LC3B) and the elongation factor (TUFM) to trigger the degradation of mitochondria in infected cells.

Figure 4H: Loss of mitochondrial damage with disrupted Gn-LC3B interaction. HUVECs transfected with HA-tagged Gn mutants were imaged using a Mitotracker dye and cells with fragmented mitochondria were counted. Mutants lacking an LC3B binding motif (GnΔ35 and GnΔ42) showed decreased mitochondrial damage. 

Given this potential role in mitochondrial disruption, the question was then asked if HTNV Gn is also capable of interfering with the Mitochondrial Antiviral-Signaling Protein (MAVS) and its effects further downstream on the host innate immune system. Upon transfection of HUVECs to overexpress Gn, an immunoblot showing decreased MAVS levels led the researchers to claim that the viral protein does act to disrupt this antiviral signaling protein. Interestingly, looking at this result within cell lines depleted of the TUFM protein actually showed a restoration of MAVS levels, despite overexpression of Gn.The researchers in turn concluded that TUFM is a factor required for the degradation of mitochondria by HTNV Gn. Next, to look at subsequent impacts of HTNV infection on innate immunity, researchers then tracked the activity of reporter genes being expressed via promoters of either type 1 interferon (IFN) or the IFN-stimulated response element (ISRE). Interferons are a key indicator for the innate immune response to infection, as they are proteins released upon viral invasion and activate many genes via interactions with sequences such as the ISRE. Bringing the Gn mutants from prior experiments back into use, those unable to bind to LC3B (due to deletions or mutations of their YRTL motif) showcased significantly decreased IFN and ISRE reporter activity. At this point in their study, researchers concluded that at early infection HTNV Gn is able to facilitate complete mitophagy via its interactions with LC3B and TUFM, a process that in turn disrupts the host’s innate immune response to produce interferon. 

However, it is important to note that an ultimate consequence of this complete autophagy is, ironically, the eventual degradation of HTNV Gn. Not only is this an issue for progeny viruses being produced within the host cell, which need this glycoprotein to go off and infect other cells, but also this limits the suppression of MAVS and interferon antiviral responses. Therefore, the need for Gn to be maintained brings us back to two of the researchers' earlier findings: that HTNV infection leads to incomplete autophagy at late stages of infection and that HTNV NP has a role in blocking Gn autophagy (refer back to Figures 1c and 3a above). After recognizing via immunofluorescence that NP colocalizes with LC3B at later stages of infection, researchers hypothesized that NP is disrupting autophagy flux via preventing LC3B binding to Gn. Co-immunoprecipitation assays that utilized antibodies against a tag on the NP protein to detect interactions with LC3B, verified that this physical interaction was indeed occurring. Furthermore, additional co-immunoprecipitation assays revealed another key interaction between NP and a SNARE protein, SNAP29. Like other SNARE proteins, SNAP29 typically interacts with a syntaxin (Stx17) to drive the fusion of two membranes, which in this case are those of the autophagosome and lysosome. However, as shown below, researchers argue that NP’s binding to SNAP29 resulted in a lessened interaction of the protein with Stx17, thus indicating incomplete fusion, and in turn, the incomplete autophagy seen at late stages of HTNV infection. 

Figure 6G: HTNV NP disrupts SNAP29-Stx17 interaction. HEK293T cells were transfected with tagged Gn and/or NP HTNV proteins. Levels of coimmunopreciptation between SNAP29 and Stx17 were reduced in cells expressing both Flag-Gn and Myc-NP, as compared to cells expressing Flag-Gn alone.

Graphical Conclusion of Findings

To conclude their study, Wang et al. hoped to apply these findings to in vitro and in vivo models of HTNV infection via the application of autophagy inhibitors. Their reasoning behind this experiment was to interfere with autophagy early in infection, thereby limiting Gn’s ability to drive HTNV replication within hosts. Interestingly, infected mice showed increased levels of interferon, decreased organ damage, and reduced viral loads as a result of this treatment. Despite the benefits of this potential HTNV treatment, however, it is hard to imagine an autophagy inhibitor without a plethora of side effects. For instance, one concern of autophagy inhibitor-based treatments in the past has been potential neurodegeneration [9]. Thus, next steps to determine the viability of this treatment would be to track the long-term impact of their application, especially since the antiviral effects of the inhibitors in this study were only seen for 4 days post infection. In addition, a number of the conclusions within this paper could use further support. For instance, in Figure 6G above, the conclusion that NP significantly prevents SNAP29 binding to Stx17 is questionable, as there is still a clear interaction detected in the presence of NP. Perhaps further experiments, such as immunofluorescence and pull-down assays, are necessary to further support this claim. Additionally, it is still unclear how the virus is able to differentiate between Gn-induced autophagy and NP-induced incomplete autophagy. As mentioned by the researchers, the fact that Gn (as a surface glycoprotein) is a first contact to the host cell could be facilitating this early autophagy flux, before NP has an impact. Thus, a future study could possibly look into how applying HTNV infection to cells already transiently expressing viral NP would impact the completion of autophagy at early stages of infection. With these future considerations in mind, it is exciting to see such comprehensive research being done on a virus still in need of effective treatments worldwide. 

References

[1] Wang, K., Ma, H., Liu, H., Ye, W., Li, Z., Cheng, L., ... & Zhang, F. (2019). The Glycoprotein and Nucleocapsid Protein of Hantaviruses Manipulate Autophagy Flux to Restrain Host Innate Immune Responses. Cell reports, 27(7), 2075-2091.

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Understanding Astrocyte Apoptosis: New Developments in Venezuelan Equine Encephalitis Virus Research

In Response to Dahal, B., Lin, S. C., Carey, B. D., Jacobs, J. L., Dinman, J. D., van Hoek, M. L., ... & Kehn-Hall, K. (2020). EGR1 upregulation following Venezuelan equine encephalitis virus infection is regulated by ERK and PERK pathways contributing to cell death. Virology, 539, 121-128.

The Venezuelan Equine Encephalitis Virus (VEEV) is a rare, but debilitating virus that can cause severe neurological symptoms in both humans and horses. VEEV is a positive-sense single-stranded RNA Alphavirus from the Togaviridae virus family. There are fourteen different strains of VEEV of which four, IA/B, IC, ID, and IE, can infect humans (Aguliar et al. 2011). The virus is spread to humans primarily through mosquito bites or contact with infected horses (Aguliar et al. 2011). VEEV infections occur in two stages. During the first stage, the virus infects the periphery organs, in particular, glands such as the lymph nodes and the spleen. During this stage, patients typically experience symptoms such as fever, vomiting, and diarrhea. The second, and more serious, stage of the infection begins when the virus progresses to the central nervous system (CNS). Infection in the CNS causes encephalitis, inflammation of the brain. If the patient develops encephalitis, they may experience convulsions or feel disorientated. Sometimes encephalitis can lead to death which occurs in around 1% of people infected with VEEV (Zacks and Paessler, 2010). Twelve countries in the Americas have reported outbreaks of this debilitating virus with the first documented outbreak occurring in Columbia in the 1950s (Aguliar et al. 2011).

VEEV is of concern to public health officials because of its potential use as a biochemical weapon of terror. During the Cold War, both the Soviet Union and the United States weaponized the virus by developing aerosol forms to use as a biochemical agent against enemy combatants (Davis, 1999). When the virus is in aerosol form, VEEV causes a more serious infection because it can directly infect the CNS through the olfactory system (Vogel et al., 1996). The potential for this virus to be used as a weapon of terror has prompted scientists to study the viral mechanism of the VEEV to develop drugs that prevent the debilitating neuropathology from occurring once the disease progresses to the second stage of the infection.

To gain a better understanding of how to address and prevent VEEV infection, scientists have begun investigating the changes in the brain that occur once VEEV has infected neurons. Astrocytes are one type of brain cells heavily targeted in the second stage of VEEV infection (Peng et al., 2013). Astrocytes are glial cells found in the brain that have many functions ranging from promoting CNS homeostasis to reducing brain inflammation following infection (Guttenplan and Liddelow, 2018). After VEEV infection, there is an increase in astrocyte apoptosis, cell-initiated cell death (Aronson et al., 2000). Dr. Kehn-Hall’s laboratory group at George Mason University recently released a paper in Virology which seeks to uncover the mechanism of VEEV infection that results in high levels of astrocyte apoptosis. The lab group sought to build off their previous research which showed that VEEV infection caused an increase in the level of early growth response 1 (EGR1) mRNA and proteins in astrocytes in a cancer cell line (Baer et al., 2016). EGR1 is an immediate-early gene associated with cell growth, survival, and apoptosis (Pagel and Deindl, 2011). In their 2019 paper, these researchers sought to replicate their findings in primary human astrocytes, because the primary astrocyte culture would be more reflective of what occurs in a human brain during VEEV infection. Additionally, the researchers were interested in uncovering which cellular pathways are responsible for the upregulation of ERG1 expression and, consequently, greater levels of cellular apoptosis.

Researchers in the Kehn-Hall lab first determined whether or not ERG1 expression is elevated in primary human astrocyte cultures after VEEV infection. They tested their hypothesis by infecting primary human astrocyte cell cultures with the TC-83 strain of VEEV. TC-83 is a live attenuated strain of the VEEV virus. Live attenuated strains are forms of viruses that can infect cells, but which have been modified so they are less virulent than unmodified strains of the viruses (Lauring et al., 2010). These strains of viruses are safer for laboratory use. Following infection, the scientists ran real-time quantitative polymerase chain reactions (RT-qPCR) to measure the respective amounts of ERG1 mRNA transcript levels in the primary human astrocytes after VEEV infection. RT-qPCR measures increases in levels of mRNAs of interest compared levels of control mRNAs, which are held at reliable levels in the cell. For this experiment, ribosomal RNA was used as a control for comparing levels of ERG1 mRNA level. The Kehn-Hall laboratory found a significant upregulation of EGR1 gene expression nine hours post-infection as displayed by the increase in fold change of EGR1 mRNA transcript (Figure 1). Thus, their RT-PCR analysis of VEEV infected primary human astrocyte cultures demonstrated that VEEV infected astrocytes have increased expression of the EGR1 mRNA.

Figure 1. Quantification of EGR1 Gene Expression Following VEEV Infection in Primary Human Astrocytes. EGR1 RNA levels in primary human astrocyte cell cultures incubated with either VEEV or a control MOCK injection were quantified at 3, 6, 8, and 18 hours post-infection. Analysis was completed with RT-qPCR. Significant upregulation of ERG1 transcripts was found in VEEV infected cells 9 and 18 post-infection.

After demonstrating that EGR1 levels were elevated in astrocytes following VEEV infection, the research group wanted to assess which internal cellular pathways were responsible for the upregulation in EGR1 expression. The researchers examined four pathways previously implicated in EGR1 upregulation: the extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK), phosphoinositide 3-kinase (PI3K) and the protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway. The researchers infected cells with VEEV and small kinase inhibitor molecules previously shown to inhibit either the ERK, p38 MAPK, PI3K, or PERK pathway. As before, the researchers quantified the levels of EGR1 expression with RT-qPCR. Their results are displayed in figure 2. The researchers found that inhibition of the ERK and PERK pathways caused a significant decrease in EGR1 gene expression in primary astrocyte cultures infected with VEEV when compared to control cell cultures, those treated with dimethyl sulfoxide (DMSO). Inhibition of p38 MAPK did not yield any significant changes in EGR1 expression when compared to control cells. Interestingly enough, treatment with PI3K inhibitors significantly increased the expression of EGR1 gene transcripts in astrocytes treated with VEEV. The researchers posited no explanation for why PI3K inhibitors triggered a rise in EGR1 transcripts, but in the future, the researchers should investigate this finding to determine why their results were inconsistent with previous papers that found that inhibition of PI3K triggered a decrease in EGR1 gene expression (Pagel and Deindl, 2011).

Figure 2. The Impact of Pathway Inhibition on EGR1 Transcript Expression. Researchers utilized RT-qPCR to assess the difference in EGR1 transcript levels following inhibition of the ERK, p38 MAPK, PI3K, and PERK pathways. Expression of EGR1 in cells treated with inhibitors was compared to the expression of EGR1 in cells treated with DMSO, a nonreactive solution. VEEV infected cells expressions were compared to MOCK control cells expressions. Researchers found a significant decrease in ERG1 expression following inhibition of the ERK and PERK pathways.

After identifying which cellular pathways were responsible for the upregulation of EGR1 after VEEV infection, the researchers examined whether inhibition of the PERK or ERK pathway would reduce astrocyte apoptosis and, consequently, increase cell viability. To test this hypothesis, the researchers used small interference RNAs (siRNAs) to target the ERK and PERK pathways in primary astrocytes before infecting the cells with VEEV. siRNAs target mature RNA sequences in cells by binding to commentary RNA base-pair sequences found in the targeted RNA sequence. siRNAs are designed in laboratories to fuse with specific mRNA and thus can reliably and consistently inhibit the expression of target molecules. Following binding, the siRNA directs the cleavage of the targeted RNA, effectively removing the RNA sequence from the cell (Lopez-Sierra and Esteller, 2012). Researchers transfected cells with siRNAs that targeted the ERK pathway, the PERK pathway and a negative siRNA which was used as a control. Cell viability assays measured ATP production in cells as a proxy measurement for cell viability; increased ATP production was used to demonstrated increased cell viability. The researchers also performed caspase 3/7 assays to assess the levels of apoptosis occurring in the primary human astrocyte cultures. Caspase 3/7 is a molecule released in late-stage apoptosis; an upregulation of this molecule in cell cultures indicates that a greater level of apoptosis is occurring in the cell. (Carrasco et al., 2003). Through these two assays, the researchers found that inhibition of both the ERK and PERK pathways increased astrocyte cell viability and decreased caspase 3/7 activity following VEEV infection (Figure 3). These results suggest that these two cellular pathways are significantly involved in triggering astrocyte apoptosis following VEEV infection.

Figure 3. Cell Viability and Caspase 3/7 Assays in VEEV infected Cells following ERK and PERK Inhibition. Levels of astrocyte viability and apoptosis following VEEV infection were analyzed through cell viability and caspase 3/7 assays. In cell cultures where ERK and PERK pathways were inhibited, there was a significant increase in cell viability and a decrease in caspase 3/7 activity. These results suggest that ERK and PERK pathways induce apoptosis in human astrocytes following VEEV infection.

Based on their experimental data, the researchers in the Kehn-Hall laboratory developed a model for the upregulation in EGR1 expression and resulting in apoptosis increase in astrocytes following VEEV infection (Figure 4). The researchers proposed that when VEEV infects the cell, the ERK pathway is directly triggered and begins to stimulate the expression of ERG1. While ERK is directly upregulated by VEEV, the virus’s proteins accumulate in the host cell’s endoplasmic reticulum which the cell recognizes as foreign proteins. The presence of foreign proteins causes the cell to activate the unfolded protein response (UPR) which stimulates the PERK pathway. The PERK pathway interacts with the ERK pathway and also increases the amount of EGR1 expression. Increased EGR1 expression ultimately leads to an increase in apoptosis in astrocytes.

Figure 4. Proposed Model for Human Astrocyte Apoptosis Upregulation Following VEEV Infection. Researchers at the Kehn-Hall lab proposed that VEEV infection directly triggers the ERK pathway and indirectly triggers the PERK pathway through activation of the cell’s unfolded protein response (UPR). Both of these pathways stimulate greater expression of the EGR1 gene which triggers apoptosis in human astrocytes.

Looking towards the future, the Kehn-Hall lab is interested in understanding why ERG1 expression causes an increase in cellular apoptosis. There are no experimentally verified mechanisms that explain why an upregulation of EGR1 causes an increase in apoptosis. The experimenters have theorized that ERG1 stimulates other genes in the cell which directly cause apoptosis, but they need to perform more research to support their theory on ERG1’s role in apoptosis. Beyond uncovering the role of ERG1 in apoptosis, the researchers need to develop an improved way to test the influence of ERK on ERG1 transcript expression and caspase 3/7 activity. In their RT-qPCR analysis of ERG1 transcripts and caspase 3/7 assays following ERK inhibition, the reduction in ERG1 expression and caspase 3/7 activity in the VEEV infected cells was remarkably similar to the reduction in ERG1 expression and caspase activity in the control cells. Thus, it is imperative for the researchers to perform additional tests on the influence of ERK inhibition on ERG1 expression to verify that the pathway does stimulate the gene expression following VEEV infection and not just generally cause a greater expression of all genes in astrocytes. On a broader level, the VEEV community should investigate how the rates of the disease could potentially increase with the dawn of climate change. VEEV is spread through mosquito vectors, and currently, climate change has caused a shift global temperature that facilitates the breeding of mosquitos (Aguliar et al. 2011). A global rise in the mosquito population could potentially trigger a massive surge in mosquito-transmitted illnesses such as VEEV. Scientists must continue to perform research on diseases like VEEV to prepare the global community for an exponential increase in mosquito-spread diseases.

References
Aguilar, P. V., Estrada-Franco, J. G., Navarro-Lopez, R., Ferro, C., Haddow, A. D., & Weaver, S. C. (2011). Endemic Venezuelan equine encephalitis in the Americas: hidden under the dengue umbrella. Future virology, 6(6), 721-740.

Aronson, J. F., Grieder, F. B., Davis, N. L., Charles, P. C., Knott, T., Brown, K., & Johnston, R. E. (2000). A single-site mutant and revertants arising in vivo define early steps in the pathogenesis of Venezuelan equine encephalitis virus. Virology, 270(1), 111-123.

Baer, A., Lundberg, L., Swales, D., Waybright, N., Pinkham, C., Dinman, J. D., ... & Kehn-Hall, K. (2016). Venezuelan equine encephalitis virus induces apoptosis through the unfolded protein response activation of EGR1. Journal of virology, 90(7), 3558-3572.

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Peng, B. H., Borisevich, V., Popov, V. L., Zacks, M. A., Estes, D. M., Campbell, G. A., & Paessler, S. (2013). Production of IL-8, IL-17, IFN-gamma and IP-10 in human astrocytes correlates with alphavirus attenuation. Veterinary microbiology, 163(3-4), 223-234.

Vogel, P., Abplanalp, D., Kell, W., Ibrahim, M. S., Downs, M. B., Pratt, W. D., & Davis, K. J. (1996). Venezuelan equine encephalitis in BALB/c mice: kinetic analysis of central nervous system infection following aerosol or subcutaneous inoculation. Archives of pathology & laboratory medicine, 120(2), 164-172.

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