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Study demonstrates SARS-CoV-2 ORF6 localization and interferon antagonism as independent events

Jacob Scott



The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antagonizes interferon (IFN)-mediated host defenses for infection establishment. SARS-CoV-2 nucleocapsid proteins further enhance its pathogenicity by suppressing IFN signaling to various degrees, with the open reading frame 6 (ORF6) protein being the strongest IFN antagonist.

Several previous research studies have demonstrated that ORF6 suppresses IFN signaling by localizing to multiple membranous compartments of host cells, including the nuclear pore complex (NPC). To this end, ORG6 directly interacts with nuclear-pore components Nup98-Rae1 through its C-terminal domain to impair docking of karyopherin/importin complex, thereby disrupting nuclear-translocation of activated STAT1/IRF3 transcription factors. However, these studies have not yet explored the direct cause-and-effect relationship between ORF6 localization and IFN antagonism experimentally.

Study: Decoupling SARS-CoV-2 ORF6 localization and interferon antagonism. Image Credit: creativeneko /

About the study

In a recent preprint study published on the bioRxiv* server, researchers deployed mutagenesis studies to investigate the relationship between ORF6 localization and IFN antagonism. Herein, they demonstrated that ORF6 uses two structural determinants for interacting with membranous compartments of host cells.

Further, the researchers demonstrate that ORF6 is a peripheral membrane protein, rather than a transmembrane protein as previously speculated. In addition, they demonstrated the potency of mislocalized ORF6 variants to induce cytoplasmic accumulation of importin-?, which inhibits nuclear trafficking.

Study findings

The researchers first identified protein regions of ORF6 that dictated its subcellular distribution. To this end, they generated a panel of ORF6-mCherry truncation mutants co-expressed in HeLa cells with markers for the endoplasmic reticulum (ER) and Golgi apparatus.

The C-terminal region of ORF6 interacts with Nup98-Rae1 NPC and induces the cytoplasmic accumulation of importin-?. It was observed during the analysis that the expression of amino acid residues 1-47 in HeLa cells resulted in localization to several membraneous organelles, which was similar to wild-type localization, while the residues 48-61 showed no discernable localization pattern.

Further analysis of the amino acid segments (1-47) showed that the first half (residues 1-23) partially co-localized with the Golgi marker, while the second half (residues 24-47) resulted in localization to an organelle distinct from the ER. Upon further truncating the 1-23 segment to residues 1-17, the researchers did not see a localization pattern, thus suggesting that segment 18-24 dictates Golgi retention.

To further validate these observations, they examined residues 18-47 and found that these did not mimic wild-type distribution. In lieu, this mutant localized to the ER-adjacent organelle, thereby suggesting that the putative helix contained within the 1-17 fragment also contributes to steady-state localization.

Taken together, these observations validated that ORF6 maintains steady-state localization through at least two distinct determinants. These include the first longer protein determinant, which resides within amino acid residues 1-47 that mediates steady-state membrane associations, and a second region from 18-24 that dictates Golgi retention.

Furthermore, the researchers examined a panel of localization disrupted mutants for their ability to block enhanced green fluorescent protein-karyopherin ?-2 (eGFP-KPNA2) trafficking. They stably introduced eGFP-KPNA2 in type II alveolar lung epithelial cell line A549 to test ORF6 activity. Remarkably, all the tested mutants induced cytoplasmic accumulation of eGFP-KPNA2 at efficiencies comparable to the wild-type strain of SARS-CoV-2.

The researchers then treated A549 cells with type I IFN and assessed STAT1 localization in the presence or absence of ORF6 proteins. As expected, all mutants could inhibit the nuclear accumulation of STAT1 at efficiencies comparable to wild-type, thereby suggesting that mutant ORF6 is potent in blocking the nuclear accumulation of activated STAT1. These findings further validated that ORF6 localization was independent of IFN antagonism.

Moreover, researchers established SARS-CoV-2 ORF6 as a peripheral membrane protein. They tested single amino acid substitution mutants that exchanged the wild-type residue with the one having opposing biophysical properties on the respective helical surfaces. The polar and hydrophobic amino acid residues were replaced with tryptophan and glutamate, respectively, and charged residues were exchanged with opposing charges, then assessed for localization.

The substitutions made only to the hydrophobic membrane-interacting surface would have exhibited disrupted localization if ORF6 was a peripheral membrane protein. The results demonstrated that all ORF6 mutants with substitutions on the hydrophobic surface showed mislocalized subcellular distribution patterns, thus indicating that SARS-CoV-2 ORF6 is a peripheral membrane protein that maintains steady-state localization through the 18-24 amino acid residues region and the adjacent hydrophobic surface.


The current study outlines several observations about the SARS-CoV-2 ORF 6, which is a nucleocapsid protein whose primary function is to suppress antiviral innate immune signaling or IFN signaling of the host for establishing SARS-CoV-2 infection. The results revealed that ORF6 actively associates with several membranous compartments through two distinct structural determinants during infection and is most likely a peripheral-membrane protein.

More importantly, the results demonstrated that mislocalized ORF6 variants potently induce cytoplasmic accumulation of importin-?, thus inhibiting nuclear trafficking. Further, the study results showed that ORF6 localization was independent of IFN antagonism, raising the possibility of ORF6 serving additional functions within membrane networks.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Wong, H. T., Cheung, V., & Salamango, D. J. (2021). Decoupling SARS-CoV-2 ORF6 localization and interferon antagonism. bioRxiv. doi:10.1101/2021.12.06.47141.

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Genetic bottlenecks could impact Wyoming toads’ ability to respond to new pathogens

Jacob Scott



A new study from North Carolina State University examines immune system diversity in the critically endangered Wyoming toad and finds that genetic bottlenecks could impact a species’ ability to respond to new pathogens. The findings could inform captive breeding strategies for endangered animal populations.

The Wyoming toad, Anaxyrus baxteri, suffered a severe population decline throughout the latter part of the 20th century due to factors including habitat destruction and fungal infection. The toad was brought into a captive breeding program in the 1990s in order to save the species. Scientists estimate a current wild population of only 400 to 1,500 animals, meaning that the toad is considered critically endangered.

Population reduction in this species created a genetic bottleneck to begin with, meaning the level of genetic diversity is already very small. This is the first study to look specifically at genetic diversity in the immune systems of these toads and how it could impact them as a population.”

Jeff Yoder, professor of comparative immunology at NC State and co-corresponding author of paper

Yoder, with co-corresponding author Alex Dornburg of the University of North Carolina at Charlotte, performed RNA sequencing on immune tissues from three healthy, retired Wyoming toad breeders. Study co-author Michael Stoskopf, who was on the Wyoming Toad Recovery Implementation Team established in 2008, obtained the samples.

“We were focused specifically on sequences encoding toll-like receptors – TLRs – and the proteins of the major histocompatibility complex, or MHC, expressed in these tissues,” says Kara Carlson, first author of the study and current Ph.D. candidate at NC State. “These sets of genes are major components of the immune system.”

TLRs are the first responders of the immune system, and are similar, or well-conserved, between species. The MHC, on the other hand, is a large and diverse group of genes that varies between species and individuals. It can determine why one group is more resistant to a particular pathogen than another.

“MHC genes are some of the most rapidly evolving sequences in the genome,” Carlson says. “So in a healthy population there’s a lot of variety that gets passed along to descendants, enabling the species at large to adapt to different pathogens. However, if disease survivors do so because of their MHC, then that group would have a similar MHC.

“The Wyoming toads that were brought into captivity to save the species were all able to resist the fungus that had decimated the population, but that could mean that their immune diversity is reduced.”

The researchers compared the TLR and MHC of the three Wyoming toads to each other, as well as to samples from a common toad and a cane toad. Both the common toad and the cane toad showed more MHC diversity than the Wyoming toad, even though the cane toad underwent a similar genetic bottleneck.

“The small sample size in this study – which was unavoidable due to the endangered status of the toad – nevertheless lays an important framework for conservation,” Carlson says.

“Amphibians in general don’t have as many genomic resources as other organisms,” Yoder says. “And captive breeding from a small population further decreases genetic diversity. But while these toads may be better protected against the fungal infection that nearly wiped them out, they may not be equipped to deal with new pathogens down the road.”

“While we weren’t necessarily surprised by the lack of immunogenic diversity in the Wyoming toad, it does spark an important question,” Dornburg says. “How equipped are other species of conservation concern for a battle with an emergent pathogen?”

“By understanding the genetic diversity of the immune system we can inform captive breeding to increase the chance of a species to resist disease in the wild,” Yoder adds. “Studies like this one are invaluable for captive breeding practices going forward.”

Journal reference:

Carlson, K.B., et al. (2022) Transcriptome annotation reveals minimal immunogenetic diversity among Wyoming toads, Anaxyrus baxteri. Conservation Genetics.

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Video conferencing hinders creativity

Jacob Scott



In-person teamwork has now transformed into virtual collaboration due to the COVID-19 pandemic. But does this affect innovation and creativity?

An interesting study on virtual communication led by Dr. Melanie S. Brucks from Columbia University and Prof. Jonathan Levav from Stanford University is published in the journal Nature. The study examines whether videoconferencing affects creative idea generation.

Study: Virtual communication curbs creative idea generation. Image Credit: Andrey_Popov / Shutterstock

Communication and collaboration

Collaboration is essential for several workplace tasks. It leads to innovation and idea generation. Historically, these collaborations have been in-person and required sharing of the same physical space. The use of traditional communication technologies like letters, emails, and phone calls hinder the smooth exchange of information and limits collaboration.

Now, due to advances in audio-visual technology, face-to-face interaction is possible via videoconferencing, allowing virtual collaboration. Videoconferencing has replaced in-person interactions due to pandemic-driven social distancing. Video interaction and in-person interaction allow communication of the same information.

While videoconferencing replacing in-person interaction has been beneficial in the social scenario, does it come with a cost in the workplace scenario? For example, does it affect collaborative idea generation?


The investigators performed a Laboratory experiment and a field experiment to test the difference between in-person interaction and videoconferencing in collaborative idea generation. First, they recruited participants and divided them into pairs: half of the pairs were assigned to an in-person setting, and the rest were assigned to a virtual setting.

Laboratory experiment

A total of 602 participants were recruited for the laboratory experiment and divided into pairs. The participants were in separate rooms in the virtual setting and communicated through videoconferencing. The pairs were allotted five minutes to generate creative uses for a frisbee (150 pairs) or bubble wrap (151 pairs) and then one minute to select their most innovative idea.

The pairs were evaluated by counting the number of creative ideas and ideas they generated. The virtual pairs generated significantly fewer total and creative ideas compared to in-person pairs.

So, virtual collaboration hampered creative ideas. This could be because the virtual space narrows the visual scope, which in turn narrows the cognitive scope. To assess the visual focus, two methods were used. Firstly, the participants had to recollect the individual props in the room and point them on a worksheet. Secondly, the participants’ eye gaze was recorded during the experiment.

The virtual pairs narrowed their focus to the screen. Compared to in-person pairs, they spent significantly more time looking directly at their partner and less time looking at the surrounding room, and remembered significantly fewer props in the surrounding room.

As a consequence, the virtual medium narrowed the visual focus and inhibited the generation of ideas.

However, these results are in the context of a controlled laboratory setting.

Field experiment

The experiment was repeated in ‘the field’ under actual work conditions within a large multinational telecommunications company to see if these results could be extrapolated to the real world. The field experiment was conducted in five country sites – in Europe, the Middle East, and South Asia. In this setting, the participants knew their partners and used video conferencing regularly for work. Moreover, it included domain experts highly invested in the outcome of the collaboration.

A total of 1,490 engineers were recruited to participate in an ideation workshop and randomly divided into pairs. The pairs were allotted an hour to generate product ideas and submit one idea as future product innovation for the company.

The engineer pairs who worked on the task virtually generated fewer total ideas and creative ideas than in-person pairs at all five sites.

However, the decision quality was not affected by virtual collaboration. The in-person pairs generated a significantly higher top-scoring idea, but the selected idea did not significantly differ in quality between the virtual pairs and in-person pairs.

Other reasons for reduced creativity

The could be other reasons why virtual collaboration negatively affected idea generation. Therefore, the investigators explored the alternative explanations.

More ideas

Since the in-person collaborators generated more total and creative ideas than the virtual collaborators, they could generate additional ideas similar to each other. However, upon semantic analysis, it was observed that they generated diverse and disconnected ideas.

Feelings of connection and trust

Studies have shown that feelings of connection and trust can foster team creativity. The virtual pairs may have reduced feelings of connection and trust toward their partner.

However, when assessed for subjective feelings of closeness, verbal and non-verbal behaviors, and mimicry, the virtual pairs were similar to in-person pairs in the laboratory experiment.

Thus, virtual and in-person interactions are very similar in terms of social connection or social behavior.

Communication coordination

Usually, there is a lack of coordination in a conversation in virtual interactions due to the absence of eye contact. However, it could not wholly explain the effect of virtual interaction on idea generation.

Interpersonal processes

This study also assessed the effect of interpersonal processes on idea generation. Interpersonal processes fear of evaluation, dominance, social facilitation, social loafing, social sensitivity, perceptions of performance, and production blocking were affected in virtual collaborations and these, in turn, affected idea generation.

Implications of the study

This study supports previous research suggesting that pairs perform better than large groups, both in-person and online. Therefore, this study recommends ideation in pairs and in person. Also, this study suggests that larger videoconferencing screens would not impact idea generation.

In-person collaborations offer a cognitive advantage. Now several workplaces are moving towards a hybrid setup. This study indicates that the creative idea generation should be reserved for in-person meetings.

Journal reference:
Brucks, M.S., Levav, J. (2022) Virtual communication curbs creative idea generation. Nature.,

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Gum Health Day 2022 calls for prevention, early detection, and effective treatment of gum diseases

Jacob Scott



“Treat your gums” is the slogan for Gum Health Day 2022, a worldwide awareness campaign organized by the European Federation of Periodontology (EFP). The event aims to inform the public of the detrimental effects of gum diseases – gingivitis, periodontitis, peri-implant mucositis, and peri-implantitis – on both oral and overall health. The campaign calls for the prevention, early detection, and – where necessary – effective treatment of gum diseases.

Although still poorly acknowledged by the public, gum diseases are chronic inflammatory conditions affecting a high proportion of adults worldwide, causing tooth loss and other problems in the mouth. Crucially, gum diseases are also linked to major systemic health issues including diabetes mellitus, cardiovascular diseases, chronic kidney disease, adverse pregnancy outcomes, rheumatoid arthritis, Alzheimer’s disease, erectile dysfunction, certain forms of cancer, and more severe Covid-19 outcomes. This means that gum diseases and their prevention and treatment are of major importance not only for oral health, but also for the whole body.

This year’s campaign focuses heavily on the treatment part – we know that millions of people suffer from gum diseases that can be treated effectively. ‘Treat your gums’ calls for this treatment – with all the documented positive effects for the mouth and whole body – to actually happen.”

Moritz Kebschull, coordinator of Gum Health Day 2022

That is why the hashtag for the campaign is #TreatYourGums, and why the recent EFP-produced clinical practice guidelines on the treatment of periodontitis are a major part of the Gum Health Day 2022 initiative.

“The new EFP-produced clinical practice guidelines on the treatment of all four stages of periodontitis are a crucial development, as they are the first high-quality international guidelines to outline a structured and easily implemented pathway for the efficient and effective treatment of gum disease,” Prof. Kebschull says. “In a nutshell, gum disease treatment that works!”

He adds: “It is important to underline that gum disease is one of the most widespread chronic diseases in the worldwide adult population, and that it is usually painless, so its early detection and successful treatment depends heavily on how fast the patient takes action.”

A major innovation of Gum Health Day 2022 is an EFP-designed “customized content generator”, a feature that allows the federation’s 37 affiliated national societies of periodontology, their individual members – as well as practices, hospitals, and members of the public – to customise their own Gum Health Day 2022 materials, based on a series of graphic templates and catchphrases.

In the framework of Gum Health Day 2022, the EFP encourages periodontists, dentists, researchers, and other health-related professionals to sign and disseminate the EFP Manifesto: Perio and General Health, an international call to action for the prevention, early detection, and treatment of gum disease. Individuals and organisations are invited to endorse it and join the 1,200+ professionals, dental practices, companies, and universities that have so far supported it.

The European Federation of Periodontology (EFP, is a non-profit organization dedicated to promoting awareness of periodontal science and the importance of gum health. Its guiding vision is “periodontal health for a better life.”

Founded in 1991, the EFP is a federation of 37 national periodontal societies that represents more than 16,000 periodontists, dentists, researchers, and oral-health professionals from Europe and around the world. It supports evidence-based science in periodontal and oral health, and it promotes events and campaigns aimed at both professionals and the public.

The EFP organizes EuroPerio, the world’s leading congress in periodontology and implant dentistry, as well as other important professional and expert events such as Perio Master Clinic and Perio Workshop. The annual Gum Health Day on May 12, organized by the EFP and its member societies, brings key messages on gum health to millions of people across the world.

The EFP also organizes workshops and outreach campaigns with its partners: projects to date have covered the relationship between periodontal disease and diabetes, cardiovascular disease, and caries, as well as women’s oral health during pregnancy.

The EFP’s Journal of Clinical Periodontology is the most authoritative scientific publication in this field. The federation also publishes JCP Digest, a monthly digest of research, and the Perio Insight magazine, which features experts’ views and debates.

The EFP’s work in education is also highly significant, notably its accreditation programme for postgraduate education in periodontology and implant dentistry.

The EFP has no professional or commercial agenda.

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