<TITLE: Virus Club
ACADEMIC DOMAIN: medicine
DISCIPLINE: virology
EVENT TYPE: post-graduate seminar presentation
FILE ID: USEMP15B
NOTES: continuation of and continued in USEMD310, seminar also includes presentation USEMP15A

RECORDING DURATION: 18 min 50 sec

RECORDING DATE: 10.5.2007

NUMBER OF PARTICIPANTS: 35

NUMBER OF SPEAKERS: 3

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

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

SU: unidentified speaker>


<S5> okay then good afternoon everyone i am <NAME S5> and i will talk today about the packaging [of bacteriophage PRD1] </S5>
<SU-1> [can you talk a bit louder] </SU-1>
<S5> i'll try but i cannot shout </S5>
<SU-2> you can </SU-2>
<S5> okay so packaging of bacteriophage PRD1 , we had a very nice introduction on PRD1 from <NAME S1> and er a lot of about the structure and nice pictures and er just to remind you of the important things for the packaging it has a double-stranded DNA genome with terminal proteins and we have a capsid and and and a membrane inside the capsid erm so as PRD1 is double-stranded DNA bacteriophage what in general do we know about packaging of er double-stranded DNA bacteriophages we know that er it is necessary to have a preformed capsid for for the packaging er it is ATP dependent process and er ATP is needed for packaging ATPase which actually er does the work which er translocates DNA inside the capsid and er packaging ATPases usually are composed of of two domains the the big and small one one is responsible for binding er and recognition of DNA and another one is responsible for binding the capsid er er , ATPase function itself and and also sometimes er cutting the DNA if we have a packaging of er for example T4 which packages in in (xx) manner and er then we need a portal a portal is a ring-like structure through which DNA is er this is er the portal through which DNA gets into the particle and er also in some phages it is important to have scaffolding proteins so how does it all go er we have coat protein and scaffolding protein in the portal and er i- er in some phages it is known that portal is the initiator of the assembly of the whole capsid so first we have the portal then we have the whole erm capsid and the scaffolding protein inside as the the procapsid is not a stable structure yet erm then er comes the ATPase which packages the the DNA inside the capsid er usually we have expansion of the capsid and it turns into the shape of of mature head and er which is sealed and then the tail attached erm it is important that for PRD1 we don't see any expansion of the capsid and we don't know anything about the portal and of course there is no tail , erm so about PRD1 er preformed er we have a preformed capsid with a membrane and er we heard a lot of about the vertices and the structures of the vertex in a very nice presentation and now i will say something about one vertex which was not mentioned which is unique it is unique in a protein structure it has erm several proteins in o- on the basis of that er in a membrane we have proteins P20 and P22 and we have proteins erm protein P6 which is a s- a small structure of protein needed for efficient packaging and er we have packaging ATPase P9 er which is unlike in other phages a structural protein so it does the packaging and then stays er with the capsid er but unfortunately we don't know how all of these proteins are assembled in that unique vertex er er we know only that if we have a mutant for example of P22 er the other proteins of the vertex er will be missing , and er er , all the components of of the unique vertex were were found out by using some mhm immunology methods and er immuno-EM erm so about packaging the best way to analyse er er this process is to create the in vitro packaging system and er it was created long time before i joined this project by <NAME> and er what do we need for that we need procapsids er er which we get from the mut- ATPase mutant so we have an empty particles er then we need packaging ATPase which comes from the extract er then we have a DNA with terminal protein and what happens if we add some ATP and salt we get packaged particles so everything works er nicely here but how d- do we analyse the results how do we see that those particles are packaged er the original system was created so that we in our ASCII we use special DNA which has er (xx) alpha insert and if we (try) tho- those packaged particles on the appropriate host we get blue plaques on a plate and er it's it works nicely and we still use it very much but the problem in this er system was that in order to get the plaques those particles have to be packaged have to be fully assembled and infective and go several rounds of replication to to in order to see the plaques and if anything happens at some other point than actual translocation of DNA we don't see the plaques so we needed to find out how to show the only actual translocation of DNA erm we tried also to purify the whole system because there is another problem that particles come from the cell extract and ATPase was not purified and er a lot of effort was was made to purify it and it is so that er particles can be purified the DNA was was purified like in in the initial system but packaging ATPase is is a very difficult protein and there were at least three persons trying to purify that it didn't work but we have to survive with what we have so we tried something new something actually very very simple , erm we know from the purification of PRD1 that if we load erm , particles on the er sucrose gradient and then use the (xx) centrifugation we get two bands band of the (build) particles lower and er then there is another band of the antiparticles er which are of course lighter and and less dense and er which is higher so the same system should work for the products of packaging reaction the particles which are packaged they should migrate lower in the gradient and the empty ones should stay up , so we get er exactly that we we made the packaging reaction as it is we took the product of of this packaging reaction and loaded it on a gradient and erm then fractionated it and analysed the fractions , er so what can we say about those fractions as PRD1 is mostly componed of er composed of protein DNA and some lipids first we tried to analyse the protein contents of those fractions and here in those gels we see fractions from top to the bottom and this is the position where empty particles should be and the position where er wild type fo- er fold packaged particles should be so basically those fractions are very dilute we need to concentrate er them mhm by (xx) precipitation and er practically the only thing which can see which we can see on the gel is er a major capsid protein P3 which has that that those distinct double bands so erm in a panel in the graph A we see the normal packaging reaction with all bits and pieces which were shown before erm and in the panel B we see the packaging reaction exactly the same just without ATP which means that packaging shouldn't work so , in here we see a slight shift of the capsid major capsid protein towards the bottom of of the gradient erm and we analysed those bands er by densitometry using DNA program and we see that the the peak of of the protein band in in the controlled reaction is in a fraction six approximately where the empties should be and er er and then it shifts in a normal packaging reaction to fraction seven and there is that hump here in fractions eight and nine so er , it should mean that some of the empty er of the empty particles which were added to the reaction er got filled with the with the DNA and migrated lower in the gradient , what else can could we do we could erm test the DNA contents of those fractions and we er i have to mention that in packaging reaction we have a lot of extracts and of course in those extracts we have a lot of DNA so we had to get rid of that so we didn't get the background which is which is more than our DNA which is packaged so all those erm er packaging reactions are stopped by adding DNA so basically the DNA which got inside the particle should be protected and the other DNA which is outside which was not packaged or it was cellular DNA should be er cleaved er so then we run those reactions on the gradient and we fraction them fractionate them and then we extract the DNA which is protected from those erm fractions and in in er gel D we see that the most of the DNA is in the fraction eight and nine and in the mhm packaging reaction control reaction without ATP we basically don't see anything actually there should be very faint band in in the fraction eight but it it comes from the background but er the this is the huge difference i would say between this and this so erm we know that this er DNA which we see here is is protected from DNAs which means that it was packaged , and erm again to be 100 per cent sure we turned back to the initial packaging system and er checked the infectivity and what did we see we see that , the infective er infectious particles are mostly in the fraction eight and nine and if er we look at the control there is no infectivity at all except for the background so this result was very nice it and it nicely correlated with with the DNA in fraction eight and nine because th- this is the the peak and er this is also the that hump in in our er in our graph here er i- increased density in in er fractions eight and nine er so from here we see that it's it's really happening that there is packaging and we can we cannot only see the shift in in protein but also extract the DNA from those particles , er so what could we do with this system now , erm there were some questions which bothered er us for a very long time for example is er protein P8 the terminal protein necessary for the packaging or is it just necessary for replication , so we tried to add the DNA which is er without terminal proteins to our reaction and what did we get we didn't get any packaged particles so from here we we see that this terminal protein is not just important in replication it's also important in the packaging , erm , so what else we need the terminal protein but what about the DNA what about the specificity of DNA because in all other phages there are se- DNA sequences er which er serve as a signal for packaging what about PRD1 but as from this experiment we saw that it needs terminal proteins so we had to find the DNA which would have a different sequence but have the terminal protein , so we didn't look too far we took another bacteriophage of tectiviridae family bam-35 which has a double-stranded DNA genome and er the length is approximately the same as in PRD1 and it has terminal proteins but er the interesting part is that morphologically it's it's indistinguishable from PRD1 but there is very low sequence similarity if you compare nucleotide sequence , er but there is the er similarity in the genome organisation , so we took the DNA from this phage and added it to our system and what happens , we get the packaged particles , and from these two results we can say that the packaging is most dependent on the sequence of the DNA but it is dependent on terminal proteins . so which was qui- quite a , huge finding for us , what else can we do with that erm as we have that similar phage which which doesn't have the same sequence but the same for example protein fold er of the of the major capsid protein we took ATPase from bam-35 and tried it in our system it was quite a crazy idea to use the the packaging ATPase from another virus and think that anything will happen and what actually happened not much we didn't get packaging but er it's er . it would be quite strange that the protein from another virus would recognise the foreign particle and foreign DNA and actually do the job but anyway we tested that now we know how it all goes erm so this is our system and we have a lot of plans for using it in in other experiments for example we want to know what's the maximum length which can be packaged into the particle and what happens if we add pieces of DNA if for example a sh- shorter piece of DNA with a terminal protein would the capsid take only like one bit or will it take another one after that we don't know that but we are planning to test all those things and also for example which end of DNA goes first into the particle if if there is a difference which one , er so we have a lot of plans and i want to thank a couple of people <NAME> who joined our project and did er quite a lot to that and is a a very useful and thinking @@ thank you and of course <NAME> , and thanks for your time </S5>
<APPLAUSE>
