<TITLE: Virus Club
ACADEMIC DOMAIN: medicine
DISCIPLINE: virology
EVENT TYPE: post-graduate seminar discussion
FILE ID: USEMD310
NOTES: seminar includes presentations USEMP15A-B

RECORDING DURATION: 16 min 25 sec

RECORDING DATE: 10.5.2007

NUMBER OF PARTICIPANTS: 35

NUMBER OF SPEAKERS: 10

S1: NATIVE-SPEAKER STATUS: Romanian; ACADEMIC ROLE: research student; GENDER: female; AGE: 24-30

S2: NATIVE-SPEAKER STATUS: Finnish; ACADEMIC ROLE: senior staff; GENDER: male; AGE: 51-over

S3: NATIVE-SPEAKER STATUS: Russian; ACADEMIC ROLE: research student; GENDER: male; AGE: 31-50

S4: NATIVE-SPEAKER STATUS: Czech; ACADEMIC ROLE: senior staff; GENDER: male; AGE: 31-50

S5: NATIVE-SPEAKER STATUS: Lithuanian; ACADEMIC ROLE: research student; GENDER: female; AGE: 24-30

S6: NATIVE-SPEAKER STATUS: Finnish; ACADEMIC ROLE: junior staff; GENDER: female; AGE: 24-30

S7: NATIVE-SPEAKER STATUS: Finnish; ACADEMIC ROLE: junior staff; GENDER: female; AGE: 31-50

S8: NATIVE-SPEAKER STATUS: Lithuanian; ACADEMIC ROLE: research student; GENDER: male; AGE: 24-30

S9: NATIVE-SPEAKER STATUS: Finnish; ACADEMIC ROLE: research student; GENDER: female; AGE: 24-30

S10: NATIVE-SPEAKER STATUS: Finnish; ACADEMIC ROLE: unknown; GENDER: male; AGE: unknown

SS: several simultaneous speakers>


<S3> and today we have three presentations so far now (xx) and the first would be <NAME S1> who should tell us the tale about some spikes </S3>

<PRESENTATION USEMP15A by S1>

<S3> questions </S3>
<S4> a question it seems that still in in the reconstruction the N-terminus of P5 is still more or less disordered you modelled some sort of a piece which er looks like this collagen like motif <S1> yes </S1> (expanded but er er part of it) is not resolved and the C-terminal part or whatever is seen (the crystal) structure seems to be rather (unresolved) there is it er er s- are you certain that this this this er (xx) expands erm exactly this distance or is it possible that there is a lot more sticking up but it's just disordered like er we saw in the SAXS </S4>
<S1> well from the data that we had and the er atomic reconstruct- m- atomic models it seems that the length of P5 is 170 angstroms and er whatever was fitted into the SAXS model er fit i mean erm it was (added) exactly this length with the N-terminus folded not extended but er it can be very well [(xx)] </S1>
<S4> [but if] if you have this fold you should see in the capsid that that the most most of the <S1> [well because it it has] </S1> [(xx)] density there </S4>
<S1> but it has er homology with P31 er pentamer so it's very difficult to say which is which </S1>
<S4> well i mean if you if you would s- er s- well so you so you you your de- you don't see extra density there except (what is there) would be <S1> [yeah] </S1> [certain] (xx) and and from that you sort of then get this this heteropentamer <S1> yes </S1> , okay i got the idea </S4>
<P:07>
<S3> i have also a question so maybe just for for information maybe on the fourth slide or something there was fitting into the the the structure yeah into the density so erm for example at the neck so the the domains they fit the density <S1> [this one] </S1> [quite full] yeah and the necks they they like the @density@ it's it's a lot of erm i mean it's a lot of [grey] </S3>
<S1> [you mean] this grey surface <S3> [yeah] </S3> [which is] not well [SAXS] </S1>
<S3> [so what] what is yeah </S3>
<S1> the SAXS model er has quite low resolution <S3> [so] </S3> [so] if you i mean if you fit these at- atomic models there er the distances they span are quite similar to what we expect </S1>
<S3> okay so this was just basically the (after-effect) of the resolution <S1> [yeah] </S1> [because] you cannot see this precisely the (xx) there </S3>
<S2> so what is the the SAXS model resolution approximately </S2>
<S1> er i have to confess that i don't remember @exactly@ </S1>
<S2> but it's low it's very low </S2>
<S1> it's quite [low] </S1>
<S2> [(xx)] </S2>
<S4> and this is er </S4>
<S2> in the order of er </S2>
<S4> well 30 angstroms [or so] <S2> [yeah] 40 yeah </S2> 30 40 angstroms but but the main thing is i- it sees even disordered parts <S2> [yeah] </S2> [so] it's more like the EM where you have for example P2 <S1> [yeah] </S1> [possibilities] or the densities are more more more diffuse <S2> yeah </S2> but you still see so it's probably what happens <S2> yeah </S2> so it all co- kind of fits together well so and so </S4>
<S3> another question is er so this this symmetry mismatch problem <S1> [yes] </S1> [which] you have like trimer over pentamer so it it seems to be like so do viruses (align) are they always phaged they so if they need to fit the trimer over pentamer that always happen with some kind of trick for that for example like additional spike or something else or sometimes or do you think like sometimes it just goes let's say nicely without anything else so can trimer fit pentamer without any special tricks </S3>
<S1> er you mean in the viral structure or when you try to reconstruct the structure </S1>
<S3> no no no i mean in the in the viral structure so if the </S3>
<S1> well i mean there are plenty of viruses which have only [(xx)] </S1>
<S3> [yes and] and many of them have symmetry mismatches <S1> yeah </S1> and do they invent something for that or sometimes it works like this </S3>
<S1> well i don't know <S3> [so th-] </S3> [about] other viruses but for example er the erm avian aden- adenovirus it had been noticed that they have two spikes as well and they are binding the second one is binding to a secondary receptor in PRD1 however this has not been er discovered i mean it seems that P5 is not binding to a secondary er receptor but i mean this can be er pressure er evolutive pressure so it's not something that applies to all viruses it depends you know on the host and and and how the virus evolved so </S1>
<S3> mhm okay </S3>
<S4> i have a lot of questions i- it was shown by <NAME> by genetic means that the C-terminus of P5 is is important for P2 incorporation because er er when you truncate just the C-terminal trimerisation domain it still doesn't incorporate P2 <S1> yes well </S1> erm what's the explanation based on your model which interacts with the N-terminal part erm of the yes </S4>
<S1> well it seems that if you have erm a mut- so you you cannot have er just a a a P5 mutant because then the P2 is not binding so what we have er observed from this is that actually er the interaction between P5 and P2 has to be somewhere erm near the capsids so basically er the N-terminal of the P5 and erm the tail domain of P2 so i don't know what happens when you know how how the C-terminus of P5 is is affecting P2 <P:06> because from our model you could think that they are quite apart </S1>
<S4> yeah well they don't they don't seem to interact directly <S1> yes </S1> but somehow the C-terminus still s- somehow plays a role [some mysterious erm] </S4>
<S1> [yeah it's very possible] @@ </S1>
<S3> okay , thank you very much , now it's <NAME S5>'s talk </S3>
<SS> (xx) </SS>
<GETTING ORGANISED, P:39>

<PRESENTATION USEMP15B by S5>

<S3> questions please </S3>
<S6> wow i'm impressed why didn't i w- why wasn't i i able to do to do that when i was still working on on the project erm <SS> @@ </SS> any- anyway er the role of P8 er well it seems that it's essential but then again it's not a P98 interaction maybe so do you have some nice model for what role the P8 now is </S6>
<S5> well not @yet@ <S6> @@ </S6> these are quite quite fresh results and and we are thinking to test also some other phages with terminal proteins and so on so we we can't tell but from what we did it seems like it it's necessary for packaging , i i can't really say what's the role it's it's it's coming <S6> yes </S6> hopefully <S6> @sure@ </S6> , yes <NAME S3> </S5>
<S3> er <NAME S4> </S3>
<S4> er maybe just one question to clarify th- th- the bam-35 DNA came with bam-35 P8 protein and still was recognised by PRD1 that's [the case] <S5> [yes] yes </S5> <S3> yes </S3> that's quite remarkable [because the other way around it doesn't work] </S4>
<S5> [well it's it's not P8 we] <S3> yeah </S3> it it's it's a diff- different protein but [it's a terminal protein yes yes] </S5>
<S4> [yeah but w- will it be a terminal protein which is] (xx) </S4>
<S2> it wasn't expected </S2>
<S4> yeah well </S4>
<SS> @@ </SS>
<S3> and what what is the general structure for terminal protein is it polypeptide is it er some globular protein or is it just a [stretch or what it what it is] </S3>
<S5> [i i i i] i can't @really say i'm i'm sorry@ yes </S5>
<S6> er so if you put bam-35 DNA into a PRD1 capsid can you actually transfer those into cells say the normal bam-35 host by i don't know electroporation or something like that or i mean can you get the PRD1 capsid into gram-positive cells and see if you actually get bam-35 out </S6>
<S5> we haven't @tried@ </S5>
<SS> @@ </SS>
<S6> i mean of course you can't use the normal infection route because the <S5> [yes yes] </S5> [(xx)] system is very different but er is there any </S6>
<S5> yes but i mean wh- wh- which is the host then </S5>
<S7> yeah yeah </S7>
<S6> [i mean if you i] </S6>
<S5> [because for] the infection you would if you have the the outside of PRD1 you would need PRD1 host but then af- [after that what what do you] </S5>
<S6> [yeah tha- that's what i meant so you] is there any system how you could get the capsid inside a gram-positive cell so that you could actually get bam-35 out <S5> i i </S5> (therapulse) or something </S6>
<S2> i have a suggestion <SS> @@ </SS> now er of course you might not get er it's like the second cycle so if PRD1 would deliver this DNA into the cytosol the chances that you know that there's a small chance that it replicates <S5> yes </S5> and it but it makes bam-35 and <S5> [yes] </S5> [maybe] a gram-negative cell would be supporting that er assembly process er that you but the first thing of the system wouldn't the lysis system wouldn't work because it's a gram-positive but if you open up the cells and plate them on the host on on the bam-35 host you might get a plaque remember plaque is a single molecule er device system so y- you you might be able to see a a few plaques </S2>
<S5> we can @try@ <SS> @@ </SS> er <NAME S8> has has a question </S5>
<S8> have you tried packaging erm PRD1 particles with the er , bam-35's ATPase and bam-35's P8 proteins </S8>
<S5> no , no we haven't , yes </S5>
<S4> i have a question wh- when you were on the gradient you were just looking at the er protein position and and just looking at P3 did you try to look (xx) for P9 whether the [empty particles are (avoided) (xx) for example] </S4>
<S5> [not yet but but we're actually planning that] because it would be very nice to to have the er th- the plotted for like er coat protein and then P9 which should overlap at some point and and then [er yeah it's it's] </S5>
<S4> [oh to see w- why it is and maybe] if (xx) don't have P9 and then [then] </S4>
<S7> [they] are not having </S7>
<S4> they don't [have] </S4>
<S5> [empty] empties they are not having [(PRD1)] </S5>
<S4> [even] a- after the packaging reaction [i mean sometimes they don't have (xx)] </S4>
<S7> [i don't know but they] should </S7>
<S5> [they would (clash)] </S5>
<S4> [oh but basically] if you add (xx) they should acquire it and then er the question is whether it is the DNA bonding to it or whether it is the P9 bonding with capsid which is the (xx) factor which might then help you out with the er the efficiency (xx) </S4>
<S5> so that that's in our plans we we are planning to do it </S5>
<S2> one more question what do you think about the efficiency <SS> @@ </SS> why do you laugh it's a serious [this is a serious question] </S2>
<S5> [i got i got this question] previously </S5>
<SS> @@ </SS>
<S6> yeah well with the previous system i was trying to calculate something and it was not very efficient it was one one of the 100,000 particles which are added packaged but with this system if we see it's on the gradient it must be some per cents maybe ten if we see the shift in on on the gradient of of the particles so we will try to to somehow quantitate that but , didn't come up with a good method of quantification </S6>
<S3> thank you very much now it's the the next is <NAME S9>'s paper about the phages from hot springs </S3>

<PRESENTATION USEMP15C by S9, NOT TRANSCRIBED>

<S3> i i have a question if erm how do you how do you define so like you name these the phage of thermus thermophilus but er how th- this whole the whole range is defined in that sense can it infect anything else the thermus thermophilus or or </S3>
<S9> yes er in that er study which i mentioned in the in the beginning of this my seminar they were tried er something like six strains which they had ordered from PTCC and they just tried to isolate the phages by titring the host cells </S9>
<S3> so it's just the most efficient the [thermus thermophilus yeah] </S3>
<S9> [yeah yeah yeah] it can infect some other PTCC strains also </S9>
<S3> are they the same (few) like the others [the other strains the same (few)] </S3>
<S9> [yes yes they all are] thermus phages , yes </S9>
<S3> but E-coli for example it's not [possible for (xx)] </S3>
<S9> [no i i don't have tried] but i don't think that <S3> [yes yeah] </S3> [E-coli (in cold)] has (xx) </S9>
<S2> at least it doesn't grow that high up in temperature </S2>
<SS> @@ </SS>
<S9> yes <NAME S4> </S9>
<S4> well i wonder if er in y- in your in the cryo-EM (xx) whether you see the membrane or whether you (at 14 angstroms) you should be able to see the phages clearer <S9> yeah </S9> as well as the DNA is it similar to SH1 on er on the membrane level as well or is it </S4>
<S9> er they're they can see something but they don't wanna speak @about it@ <SS> @@ </SS> because it's so early but for sure [or how else (xx)] </S9>
<S2> [there were a few empty] particles in the (prep so far) </S2>
<S9> yeah yeah </S9>
<S10> but i can tell you that it kind of it looks like (xx) but we haven't done any exact measurements yet </S10>
<S4> (xx) or or you might be right </S4>
<S2> same benefits you see in the in the <S10> [yeah] </S10> [(xx)] so you (could) go back and er sort of sort of er do the cryo in that one </S2>
<S9> [or this one] </S9>
<S4> [yeah that one] </S4>
<S2> [this one] so there is the the empty particle [and look at the] </S2>
<S4> [yeah] yeah there was </S4>
<S2> yeah so it's a landmark yeah <P:06> okay now we have thermo-stable some things somewhere </S2>
<S9> thank you </S9>
<S2> okay any more questions are we happy everyone is happy </S2>
