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Emily Bruce @brucelab.bsky.social · 25/04/2025

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

graphical schematic showing Rab11B vesicles transporting a protein requied for H3N2 virions to bind, while a second (unknown) mechanism is required to transport a molecule required for H1N1 binding.

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Emily Bruce @brucelab.bsky.social · 25/04/2025

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

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Emily Bruce @brucelab.bsky.social · 25/04/2025

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

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Emily Bruce @brucelab.bsky.social · 25/04/2025

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

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Emily Bruce @brucelab.bsky.social · 25/04/2025

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

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Emily Bruce @brucelab.bsky.social · 25/04/2025

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

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Emily Bruce @brucelab.bsky.social · 25/04/2025

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

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Emily Bruce @brucelab.bsky.social · 25/04/2025

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

graphs showing knockdown of Rab11A, Rab11B, Rab8 and infectious virus production.

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Emily Bruce @brucelab.bsky.social · 07/03/2025

Great to see lots of folks out for Stand Up For Science!!!

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Emily Bruce @brucelab.bsky.social · 07/03/2025

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.

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Emily Bruce @brucelab.bsky.social · 23/01/2025

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.

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Emily Bruce @brucelab.bsky.social · 10/01/2025

Morning cross country ski, took a page from @doctorloveday.bsky.social ! Almost a foot of powder in the last 24hr❄️

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Emily Bruce @brucelab.bsky.social · 22/11/2024

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.

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Emily Bruce @brucelab.bsky.social · 19/11/2024

I had to buy a book of FarSide cartoons for my lab:)

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Emily Bruce @brucelab.bsky.social · 14/11/2024

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)

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Emily Bruce @brucelab.bsky.social · 08/11/2024

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.

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Emily Bruce @brucelab.bsky.social · 06/08/2024

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"!

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Emily Bruce @brucelab.bsky.social · 12/03/2024

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...

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Emily Bruce @brucelab.bsky.social · 12/03/2024

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!

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Emily Bruce @brucelab.bsky.social · 12/03/2024

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.

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Emily Bruce @brucelab.bsky.social · 12/03/2024

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.

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Emily Bruce @brucelab.bsky.social · 12/03/2024

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.

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Emily Bruce @brucelab.bsky.social · 12/03/2024

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.

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Emily Bruce @brucelab.bsky.social · 12/03/2024

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.

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Emily Bruce @brucelab.bsky.social · 12/03/2024

6/n To determine if the cysteines were sufficient for N-N dimer formation we introduced a cysteine in the WA1 N background at residue 185 (Beta), 215 (Delta) and 243 (Iota) individually. The G215C mutation recapitulated the N-N dimer we saw in Delta reproducibly.

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Emily Bruce @brucelab.bsky.social · 12/03/2024

5/n In order to determine if these residues were necessary for dimer formation, we cloned the N sequences & transfected them into 293T cells. In each case, the variant Ns were capable of forming the N-N dimer (in the absence of other viral proteins) in a cysteine-dependent manner

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Emily Bruce @brucelab.bsky.social · 12/03/2024

4/n We predicted that this cysteine could form a disulfide bond, leading to a stable N-N dimer. And sure enough, when we ran non-reducing SDS-PAGE gels that is exactly what we saw!

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Emily Bruce @brucelab.bsky.social · 12/03/2024

3/n While coronavirus N proteins are known to form dimers, this cysteine was in the linker region. This is not a place you expect to find a cysteine based on other coronas (none in SARS/MERS), but oddly we found 3 different cysteines in 3 of our variants- Beta, Delta and Iota).

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Emily Bruce @brucelab.bsky.social · 12/03/2024

2/n As Delta was emerging, we saw that most of the viral sequencing surveillance showed that a cysteine had been introduced into the nucleocapsid protein, at residue 215 (great work from the Schuck group showed this was advantageous in the absence of other mutations in N or S)

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Emily Bruce @brucelab.bsky.social · 08/09/2023

2/n We sampled 739 nasal or throat samples from VT wildlife throughout the 2021-2022 harvest season, including red/gray foxes, fishers, river otters, coyotes, bobcats, black bears and white-tailed deer. As this figure shows, wildlife were sampled from all over the state of VT.

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Emily Bruce @brucelab.bsky.social · 25/08/2023

Plaques!! Anyone have a lead on how to get a bulk supply (more than the initial “free sample”) of Avicel? Has anyone managed to actually buy it?

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Emily Bruce @brucelab.bsky.social · 25/08/2023

Good science day! First time on Twiv, (illicitly) painted the lab office area, and got a great new piece of data.

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