Emily Bruce
@brucelab.bsky.social
#NewPI in #RNA #Virology, electron microscopy aficionado, gardener, parent, Assistant Professor
@UVMLarnerMed. Prev @Gates_Cambridge scholar
@UVMLarnerMed. Prev @Gates_Cambridge scholar
FIRST NOTICE OF AWARD!
July 8, 2025 at 3:59 PM
FIRST NOTICE OF AWARD!
As Rab11A/B aren't transmembrane proteins, we don't think Rab11B is acting directly to facilitate H3N2 binding. Instead its likely transporting a protein (or proteins) to the cell surface, which is required for the binding of recent H3N2 (but not H1N1) isolates- suggesting subtype specificity! 9/n
April 25, 2025 at 1:57 PM
As Rab11A/B aren't transmembrane proteins, we don't think Rab11B is acting directly to facilitate H3N2 binding. Instead its likely transporting a protein (or proteins) to the cell surface, which is required for the binding of recent H3N2 (but not H1N1) isolates- suggesting subtype specificity! 9/n
Next we developed an RT-qPCR assay to measure virion binding. We could remove bound virions with exogenous NA (panel A, as expected), but Rab11B depleted cells bound significantly fewer H3N2 virions (panel B). Binding of H1N1 virions to Rab11B- cells was unaffected. 8/n
April 25, 2025 at 1:56 PM
Next we developed an RT-qPCR assay to measure virion binding. We could remove bound virions with exogenous NA (panel A, as expected), but Rab11B depleted cells bound significantly fewer H3N2 virions (panel B). Binding of H1N1 virions to Rab11B- cells was unaffected. 8/n
We used an NH4Cl time of addition experiment to prevent endosomal acidification (and thus viral fusion) to map the kinetics of viral entry. Rab11B- cells had significantly decreased rates of entry, but the kinetics of internalization was not delayed (suggesting the problem was further upstream). 7/n
April 25, 2025 at 1:55 PM
We used an NH4Cl time of addition experiment to prevent endosomal acidification (and thus viral fusion) to map the kinetics of viral entry. Rab11B- cells had significantly decreased rates of entry, but the kinetics of internalization was not delayed (suggesting the problem was further upstream). 7/n
Since western blots measure bulk protein expression, and transient knockdowns may not reach 100% of cells, we wanted to see what was happening at a single cell level. Flow cytometry showed that most Rab11B- cells failed to produce any viral proteins, rather than a global decrease in translation. 6/n
April 25, 2025 at 1:55 PM
Since western blots measure bulk protein expression, and transient knockdowns may not reach 100% of cells, we wanted to see what was happening at a single cell level. Flow cytometry showed that most Rab11B- cells failed to produce any viral proteins, rather than a global decrease in translation. 6/n
Cells infected with the H1N1 isolate made viral proteins just fine, suggesting the defect in infectious virion production happened late in infection (fitting with Rab11A's well characterized role in transporting the RNPs to the site of budding). 5/n
April 25, 2025 at 1:55 PM
Cells infected with the H1N1 isolate made viral proteins just fine, suggesting the defect in infectious virion production happened late in infection (fitting with Rab11A's well characterized role in transporting the RNPs to the site of budding). 5/n
To make sure this wasn't an artifact of our particular H3N2, we repeated this experiment with a second H3N2 isolate from a different state (thanks to @andrewpekosz.bsky.social)! The 2nd virus showed the same phenotype- both Rab11s required for virus production, Rab11B needed early in infection. 4/n
April 25, 2025 at 1:54 PM
To make sure this wasn't an artifact of our particular H3N2, we repeated this experiment with a second H3N2 isolate from a different state (thanks to @andrewpekosz.bsky.social)! The 2nd virus showed the same phenotype- both Rab11s required for virus production, Rab11B needed early in infection. 4/n
Rab11 is well known to transport the genome segments late in IAV infection, but has not been reported to play a role prior to protein production. Which is why we were very surprised to see that cells lacking Rab11B and infected with the H3N2 isolate failed to efficiently make viral proteins. 3/n
April 25, 2025 at 1:53 PM
Rab11 is well known to transport the genome segments late in IAV infection, but has not been reported to play a role prior to protein production. Which is why we were very surprised to see that cells lacking Rab11B and infected with the H3N2 isolate failed to efficiently make viral proteins. 3/n
We started by depleting two Rab11 isoforms (A and B), separately or together (panel A), in human lung cells then infecting with recent H1N1 (panel B) or H3N2 (panel C) subtypes we isolated from VT clinical samples. Both Rab11 isoforms were required for efficient production of infectious virus. 2/n
April 25, 2025 at 1:53 PM
We started by depleting two Rab11 isoforms (A and B), separately or together (panel A), in human lung cells then infecting with recent H1N1 (panel B) or H3N2 (panel C) subtypes we isolated from VT clinical samples. Both Rab11 isoforms were required for efficient production of infectious virus. 2/n
Great to see lots of folks out for Stand Up For Science!!!
March 7, 2025 at 7:40 PM
Great to see lots of folks out for Stand Up For Science!!!
Join us today, in Burlington VT and around the nation, as we remind our communities what science brings to the country--solutions to our toughest challenges, new cures for disease and good jobs that pay a living wage.
March 7, 2025 at 12:30 PM
Join us today, in Burlington VT and around the nation, as we remind our communities what science brings to the country--solutions to our toughest challenges, new cures for disease and good jobs that pay a living wage.
Uncertain times are upon us folks. Grateful for those who meet them with the fortitude to drive a pallet jack full of (pre tariff) consumables across several buildings and an exciting downhill-ramp luge situation.
January 23, 2025 at 4:53 PM
Uncertain times are upon us folks. Grateful for those who meet them with the fortitude to drive a pallet jack full of (pre tariff) consumables across several buildings and an exciting downhill-ramp luge situation.
Morning cross country ski, took a page from @doctorloveday.bsky.social ! Almost a foot of powder in the last 24hr❄️
January 10, 2025 at 1:10 PM
Morning cross country ski, took a page from @doctorloveday.bsky.social ! Almost a foot of powder in the last 24hr❄️
Fantastic to have Paul Duprex here at UVM giving MMG seminar this week! Great talk about all things RNA virus, as well as the importance of showing complete stories even when things don’t work.
November 22, 2024 at 5:27 PM
Fantastic to have Paul Duprex here at UVM giving MMG seminar this week! Great talk about all things RNA virus, as well as the importance of showing complete stories even when things don’t work.
I had to buy a book of FarSide cartoons for my lab:)
November 19, 2024 at 11:27 PM
I had to buy a book of FarSide cartoons for my lab:)
The virology community is the best! Thanks @strangviruslab.bsky.social for sending me your spare Avicel all the way from London!! (Photography kudos to fantastic rotation student @gramstain.bsky.social)
November 14, 2024 at 7:04 PM
The virology community is the best! Thanks @strangviruslab.bsky.social for sending me your spare Avicel all the way from London!! (Photography kudos to fantastic rotation student @gramstain.bsky.social)
Figuring out the way forward this week. Chicken soup outing with the lab (and the Martorelli Di Genova group) and moving over here from X as a start.
November 8, 2024 at 2:45 PM
Figuring out the way forward this week. Chicken soup outing with the lab (and the Martorelli Di Genova group) and moving over here from X as a start.
Congratulations to my first graduate student, Dr. Hannah Despres, on the successful defense of her PhD thesis entitled "Characterization of SARS-CoV-2 Variants of Concern at Multiple Scales"!
August 6, 2024 at 5:39 PM
Congratulations to my first graduate student, Dr. Hannah Despres, on the successful defense of her PhD thesis entitled "Characterization of SARS-CoV-2 Variants of Concern at Multiple Scales"!
15/n We think that the novel disulfide bond in Delta stablizes higher order oligomers by bonding together pairs of dimers to form the oligomerized N that is the basis of the RNP inside the virion. Normally, coronavirus linker-linker interactions are more transient...
March 12, 2024 at 2:01 PM
15/n We think that the novel disulfide bond in Delta stablizes higher order oligomers by bonding together pairs of dimers to form the oligomerized N that is the basis of the RNP inside the virion. Normally, coronavirus linker-linker interactions are more transient...
14/n While our bulk biochemical data suggested that more N was being packaged per virion, we wanted to look at the single virion level. Mark Ladinsky was able to image fully budded virions inside cellular exit compartments and show the G215C virus had elongated virions w/more N!
March 12, 2024 at 2:00 PM
14/n While our bulk biochemical data suggested that more N was being packaged per virion, we wanted to look at the single virion level. Mark Ladinsky was able to image fully budded virions inside cellular exit compartments and show the G215C virus had elongated virions w/more N!
13/n Given N's role in encapsidation of viral RNA, we decided to look in virions. Not only was the dimerized form of N packaged at high levels in virus, total levels of N were higher in virions produced by the G215C virus, compared to both M (another structural protein) and FFU.
March 12, 2024 at 2:00 PM
13/n Given N's role in encapsidation of viral RNA, we decided to look in virions. Not only was the dimerized form of N packaged at high levels in virus, total levels of N were higher in virions produced by the G215C virus, compared to both M (another structural protein) and FFU.
11/n Representative H&E images of lungs shown below. I'm not a pathologist, but in the words of an esteemed keynote speaker at ASV last year "white space good, no white space bad"...and the G215C virus appeared to have higher immune cell infiltration and less open airway spaces.
March 12, 2024 at 1:59 PM
11/n Representative H&E images of lungs shown below. I'm not a pathologist, but in the words of an esteemed keynote speaker at ASV last year "white space good, no white space bad"...and the G215C virus appeared to have higher immune cell infiltration and less open airway spaces.
9/n Given the magnitude of the difference in vitro, @themenacherylab.bsky.social /Bryan Johnson examined the growth and pathogenesis of this virus in the syrian golden hamster model.
March 12, 2024 at 1:58 PM
9/n Given the magnitude of the difference in vitro, @themenacherylab.bsky.social /Bryan Johnson examined the growth and pathogenesis of this virus in the syrian golden hamster model.
8/n However, since Vero cells are not a physiologically relevant target for SARS2, we infected primary human bronchial cells differentiated on an air-liquid interface. Here, the G215C virus grew substantially (~100-fold) better than the mNG WT.
March 12, 2024 at 1:57 PM
8/n However, since Vero cells are not a physiologically relevant target for SARS2, we infected primary human bronchial cells differentiated on an air-liquid interface. Here, the G215C virus grew substantially (~100-fold) better than the mNG WT.
7/n Next, @themenacherylab.bsky.social used their infectious clone system to introduce the G215C mutation into the attenuated neon green WA1 background. G215C did make the N-N dimer, but it didn't appear to have any effect on the growth of this virus in Vero cells.
March 12, 2024 at 1:57 PM
7/n Next, @themenacherylab.bsky.social used their infectious clone system to introduce the G215C mutation into the attenuated neon green WA1 background. G215C did make the N-N dimer, but it didn't appear to have any effect on the growth of this virus in Vero cells.