cell entry

[gsgs]

http://quizlet.com/15669054/virology-bms2012-flash-cards/
Negative-Strand RNA-unsegmented-enveloped-replicate in cytoplasm-long filamentous morphology, Filovirus
ss-RNA , What kind of genome does Ebola have?
1967 , When was Marburg virus found?
1976 , When was Ebola found?
-zaire-sudan-cote d'ivoire-reston[-bundibugyo] , What are the four [five] types of Ebola?
50-90% , Ebola mortality:
-incubation of 3-16 days-non-specific flu like-diarrhoea&vomiting-macropapular rash (50%,day7)
-Severe bleeding-major organ failure (1-2 weeks) , Ebola symptoms:
Heammoragic fever , Ebola causes:
-fluid replacement-serum injection-barrier nursing , Ebola treatment:
enveloped , Ebola is enveloped or non-enveloped?
helical , Ebola nucleocapsid
-L protein-NP-VP30-VP35 , What is associated with Ebola genome?
Receptor mediated endocytosis , Ebola enters the cell by?
-budding -VP24, VP40 and matrix protein binding Ebola exits the cell by_, using_
-VP40 and GPs , Ebola virus like particles contain:
-Vesicular stomatitis virus -Ebola Gp-GFP labeled G protein , Ebola pseudo-type viruses are made with:
pantropic , Filoviruses are:
macrophages, monocytes &dendritic cells (later endothelial cells) , Ebola initially infects
TNF alpha, IFN gamma , Cytokines released in ebola
IFN alpha, IFN beta , cytokines not released in ebola
Inappropriate coagulation , What type of coagulopathies occur in Ebola
-CD8 Tcells-high T cell count-10^7 genome per ml serum-detectable abs-low NO , A good response to Ebola
-low CD8 Tcells-low T cells-10^10 genome per ml serum-high NO , A bad response to Ebola

-------------------------------------------------------------------
several groups have confirmed that Axl/Gas6, as well as other phosphatidylserine receptors,
facilitate entry of dengue, West Nile, and Ebola viruses.
Virus binding by viral envelope phosphatidylserine
http://www.ncbi.nlm.nih.gov/pubmed/24478428
-----------------------------------------------------------------------------
Recombinant lentogenic Newcastle disease virus expressing Ebola virus GP infects cells
independently of exogenous trypsin and uses macropinocytosis as the major pathway for cell entry.
-------------------------------------------------------------------------
Lectin-dependent enhancement of Ebola virus infection via soluble and transmembrane
C-type lectin receptors. [gof ?]
-----------------------------------------------------------------------
A mutation in the Ebola virus envelope glycoprotein restricts viral entry in a host species-
and cell-type-specific manner.
---------------------------------------------------------------------
Ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis.
----------------------------------------------------------------------
The Ebola virus glycoprotein mediates entry via a non-classical dynamin-dependent
macropinocytic pathway.
----------------------------------------------------------------------
Zaire Ebola virus (EBOV) is a zoonotic pathogen that causes severe hemorrhagic fever in humans.
A single viral glycoprotein (GP) mediates viral attachment and entry.
GP mutant, GP-F88A, which is defective for entry into a variety of human cell types, including
antigen-presenting cells (APCs), such as macrophages and dendritic cells, can mediate viral
entry into mouse CD11b(+) APCs. Like that of wild-type GP (GP-wt), GP-F88A-mediated entry occurs via a
macropinocytosis-related pathway and requires endosomal cysteine proteases and an intact
fusion peptide. Several additional hydrophobic residues lie in close proximity to GP-F88,
including L111, I113, L122, and F225. GP mutants in which these residues are mutated
to alanine displayed preferential and often impaired entry into several cell types, although
not in a species-specific manner.

===========================================
https://en.wikipedia.org/wiki/Pinocytosis
https://en.wikipedia.org/wiki/Macropinosome
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2940741/
https://en.wikipedia.org/wiki/Endocytosis

[macro-]pinocytosis : no receptors involved
[clathryn mediated] endocytosis : receptors (flu-HAs alpha 2-3,alpha 2-6 and such)



we have A82V in the glycoprotein as compared to previous outbreaks

   00000000000000000000000000000000000000000000000000000000000000000000
   00000022222222222333333333333333333333333333444444444444444444455556
   03446814668999999111111333344556677777788889011122334444555778902457
   37675296023156789045789136729596835678924898514729050136357453937426
   --------------------------------------------------------------------
   VVSDSVRSIATWKNLTRAGPNISENNEMASKTLTPQPPTTPTHIGADAPPLTSASLQYEASTTVITDF
>  .....A..RT........RA...G.T....E...LR..IP....ETAT....GTLP.H.....A.I..
>  .....A...T........RA...G.T....E...LR..IP....ETAT....GTLP.H.....A....
>  .....A...T........RA...G.T....E...L....P....ETAT....GTLP.H.....A....
>e .....A...T........RA...G.T....E...L....P....ETAT....GTLP.H.....A....
>  .....A...T........RA...G.T....E...L....P....ETAT....GTLP.H.....A....
>  .....AK..T........RA...G.T....E.P.L....P....ETAT....GTLP.H.T...A....
>  .....AK..T........RA...G.T....E.P.L....P....ETAT....GTLP.H.T...A....
>  .....AK..T........RA...G.T....E.P.L....P....ETAT....GTLP.H.T...A....
>  .....A...T........RA...G.T....E...L....P....ETAT....GTLP.H.....A....
>  .....A...T........RA...G.T....E........P....ET......GTLP.H....AA....
>  .....A...T........RA...G.T....E........P....ET......GTLP.H....AA....
>  .....A...T........RA...G.T....E........P....ET......GTLP.H....AA....
>  ...X.A...T........RA...G.T....E........P....ET......GTLP.H....AA....
>  ...E.A...T........RA...G.T....E........P....ET....P.GTLP.H....AA....
>  ..N..A...T.......V.A...G.T....E.....SL.P....ET..S.P..TFP.H.....A..N.
>  .....A...T.......V.A...G.T....E.....SL.P....ET..S.P..TFP.H.....A....
>  .....A...T.......V.A...G.T....E.....SL.P....ET..S.P..TFP.H.....A....
>  .....A...T.......V.A...G.T....E.....SL.P....ET..S.P..TFP.H.....A.I..
>  .....A...T.......V.A...G.T....E.....SL.P....ET..S.P..TFP.H.....A.I..
>  .....A...T.......V.A...G.T....E.....SL.P....ET..S.P..TFP.H.....A.I..
>  .....A...T.......V.A...G.T....E.....SL.P....ET..S.P..TFP.H.....A.I..
>  .....A...T.......V.A...G.T....E.....SL.P....ET..S.P..TFP.H.....A.I..
>  .....A...T.......V.A...G.T....E.....SL.P....ET..S.P..TFP.H.....A.I..
>  .....A...T.......V.A...G.T....E.....SL.P....ET..S.P..TFP.H.....A.I..
>  .....A...T.......V.A...G.T....E.....SL.P....ET..S.P..TFP.H.....A....
>  ....PA.P.T.......V.A...G.T....E.....SL.P....ET..S.P..TFP.H.....A....
>  .....A.............A.L.K.T.V..E........P..YTET...HP....PPH.....A....
>  .....A.............A.L.K.T.V..E........P..YTET...HP....PPH.....A....
>  .....A.............A.L.K.T.V..E........P..YTET...HP....PPH.....A....
>  .....A.............A.L.K.T.V..E........P..YTET...HP....PPH.....A....
>  .....A.............A.L.K.T.V..E........P..YTET...HP....PPH.....A....
>  .....A.............A.L.K.T.V..E........P..YTET...HP....PPH.....A....
>  .....A.............A.L.K.T.V..E........P..YTET...HP....PPH.....A....
>  .....A.............A.L.K.T.V..E........P..YTET...HP....PPH.....A....
>  .....A.............A.L.K.T.V..E........P..YTET...HP....PPH.....A....

>  .....A..............................................................
>  ....................................................................
...
>  ....................................................................
>  .....A..............................................................
>  .....A.....R........................................................

>  I....A.............AD...YT...NEI....S..PQ...ET....PI...P....GA.A....
>  I....A.......TSLG..AD...YT...NEI....S..PQ...ET....PI...P..X.GA.A....
>  II...A......NTSLG..AD...YSG..NEIF...S..PQ...ET....PI...P....GA.A....
>  II...A.......TSLG..AD...YTG..NEIF...S..PQRA.ET....PI...P....G..A....
>  I....A....A..TSLG..AD...YT...NEI....S..PQ...ET....PI...P....G..A....
>  I....A.......TSLG..AD...YT..TNEI.A..S..PQ...ET....PI...P....G..AT...
>  I....A.......TSLG..AD.G.YT..TNEI....S..PQ...ET....PI...P....G..A...L
>  ....................................................................
   00000000000000000000000000000000000000000000000000000000000000000000
   00000022222222222333333333333333333333333333444444444444444444455556
   03446814668999999111111333344556677777788889011122334444555778902457
   37675296023156789045789136729596835678924898514729050136357453937426
   --------------------------------------------------------------------
   VVSDSVRSIATWKNLTRAGPNISENNEMASKTLTPQPPTTPTHIGADAPPLTSASLQYEASTTVITDF

http://virologytidbits.blogspot.de/2014/08/ebola-role-of-glycoprotein-in-viral.html


[Attached Images ebow1.GIF (4.8 KB, 2 views) --> replaced with ebow4.gif, see below]

 

[Emily]

Re: cell entry

http://www.pnas.org/content/96/6/2662.full

Vladimir N. Malashkevich,
Brian J. Schneider,
Margaret L. McNally,
Michael A. Milhollen,
James X. Pang,
and Peter S. Kim

Core structure of the envelope glycoprotein GP2 from Ebola virus at 1.9-? resolution PNAS 1999 96 (6) 2662-2667; doi:10.1073/pnas.96.6.2662
Abstract

Ebola virions contain a surface transmembrane glycoprotein (GP) that is responsible for binding to target cells and subsequent fusion of the viral and host-cell membranes. GP is expressed as a single-chain precursor that is posttranslationally processed into the disulfide-linked fragments GP1 and GP2. The GP2 subunit is thought to mediate membrane fusion. A soluble fragment of the GP2 ectodomain, lacking the fusion-peptide region and the transmembrane helix, folds into a stable, highly helical structure in aqueous solution. Limited proteolysis studies identify a stable core of the GP2 ectodomain. This 74-residue core, denoted Ebo-74, was crystallized, and its x-ray structure was determined at 1.9-? resolution. Ebo-74 forms a trimer in which a long, central three-stranded coiled coil is surrounded by shorter C-terminal helices that are packed in an antiparallel orientation into hydrophobic grooves on the surface of the coiled coil. Our results confirm the previously anticipated structural similarity between the Ebola GP2 ectodomain and the core of the transmembrane subunit from oncogenic retroviruses. The Ebo-74 structure likely represents the fusion-active conformation of the protein, and its overall architecture resembles several other viral membrane-fusion proteins, including those from HIV and influenza.

x-ray crystallography
membrane fusion
filovirus
oncogenic retroviruses

Full text at link. :tiphat:

 

[gsgs]

Re: cell entry

GP2 contains the fusion peptide (FP, residues 524–539)

the fusion peptide

(524-539)
GAAIGLAWIPYFGPAA (16 google hits)

spike glycoprotein precursor


flu : RGLFGAIAGFIEGGW (highly preserved, even in flu-B)
http://www.flutrackers.com/forum/showthread.php?t=199974&highlight=fusion+peptide

reston:
GAAVGLAWIPYFGPAA

bundibugyo:
GAAIGLAWIPYFGPAA

Tai Forest:
GAAIGLAWIPYFGPAA

Sudan:
HNAAGIAWIPYFGPGA

common in all : AWIPYFGP


no google hits for that


how many different fusion proteins


now, is all this research more useful or more dangerous to examine ? maybe we should consult the
ethics commission before posting about it

--------------------------------------------------------
16 google hits for GAAIGLAWIPYFGPAA
including this post which google quickly grasped before I edited it

Structure of the Ebola fusion peptide in a membrane-mimetic ...
Patent WO2007056153A2 - Peptide-dicer substrate rna conjugates ...
WO2010053320A2 - Google
Molecular Dynamics Simulations of Folding and Insertion of the ...
edoc.hu-berlin.de/dissertationen/eifart-patricia.../chapter4.html‎Im Cache
Measuring the Strength of Interaction between the Ebola Fusion ...
Brevetto US20030072794 - Encapsulation of plasmid DNA ... - Google
[PDF] Compounds and methods for peptide ribonucleic acid condensate ...
[PDF] Dicer substrate rna peptide conjugates and methods for rna ...
PDF] Compositions and methods for delivery of double-stranded rna
PDF] Pharmaceutical compositions for delivery of ribonucleic acid to a cell
Spike Protein - Tulane University

 

[gsgs]

Re: cell entry

http://www.ncbi.nlm.nih.gov/pubmed/21667336
We identified that four residues appear to be involved in protein folding/structure
and four residues are important for viral entry. An improved entry interference assay
was developed and used to study the role of these residues that are important for viral entry.
It was found that R64 and K95 are involved in receptor binding. In contrast, some residues
such as I170 are important for viral entry, but do not play a major role in receptor binding
as indicated by entry interference assay and/or protein binding data, suggesting that these
residues are involved in post-binding steps of viral entry. Furthermore, our results also
suggested that Ebola and Marburg viruses share a common cellular molecule for entry.

R64(GP),K95(GP),I170(GP)
----------------------------------------------------------------------------------------
highly glycosylated mucin domain in Ebola virus GP
http://jvi.asm.org/content/81/24/13378.full

CatL-treated virions not only are more infectious but also exhibit greater cell binding activity
than untreated virions. In contrast, Ebola virus GP virions lacking the mucin domain do not
demonstrate an overall enhancement of cell binding or infectivity.

------------------------------------------------------------------------------
Analysis of more than 100 deletion and amino acid substitution mutants of GP1 with
respect to protein expression, processing, viral incorporation, and viral entry has
allowed us to map the region of GP1 responsible for viral entry to the N-terminal 150 residues.
http://jvi.asm.org/content/79/8/4793.full
Deletions of GP residues between positions 266 and 476 do not greatly affect viral entry

N-terminal 230-residue region of GP1, which is conserved among different Ebola virus species



receptor binding domain :
in GP1 ,57L,63L,64R,88F,95K , 57-64

RBD, position 56-65 in GP1 is pretty constant in ebola Zaire at genbank ,
I found one KLSSTNQLRP and 267 KLSSTNQLRS

KLSSTNQLRS zaire
KLSSTSQLKS reston
KLSSTSQLKS bundibugyo
HLASTDQLKS sudan
KLSSTSQLKS tai forest

 

[Emily]

Re: cell entry

now, is all this research more useful or more dangerous to examine ?

http://www.nature.com/nsmb/journal/v9/n3/full/nsb0302-164.html

 

[gsgs]

Re: cell entry

I didn't mean dangerous for the researchers themselves, but dangerous for mankind.
We are (obviously ... anyone doubts this ??) going to be able to design new viruses
or modify existing viruses which may be used as weapons or threats or just escape.
I wonder why researchers who are able to put so much effort into their reseach like Don
so rarely talk about this important danger.
Maybe it's just because they don't want to get it into the headlines since it
interferes with they normal work, people would start questioning its usefulness.

 

[gsgs]

Re: cell entry

ebola could be a good opportunity to discuss the idea of making a virus
less virulent and then letting it spread so to increase immunity against
the virulent one.
Not much money can be made with a "vaccine" that spreads like a virus
(in fact, that _is_ a virus) maybe that's the reason why we see no such
research and discussion.
Also maybe ethical problems ... you would release a pathogen -although
attenuated- into nature. Who knows what might happen ...
But then, who knows what might happen if you don't
Or lets put it this way, if we had the chance to change an existing virus
that is circulating and spreading directly and make it less virulent,
should we do it ? In practice, if we had that chance we would probably also
have the chance to stop it in the first place and avoid the spread, in that person
in that location.
Some sort of "vaccine" that reassorts with the circulating virus and makes it
less virulent but allows it to spread instead of just killing it.

 

[Emily]

Re: cell entry

Maybe it's just because they don't want to get it into the headlines since it interferes with they normal work, people would start questioning its usefulness.

http://www.cidrap.umn.edu/news-perspective/2013/08/scientists-air-topics-h7n9-gain-function-research

He said that though he fully supports H7N9 research work, he still sees signs that researchers are overselling the benefits of the findings for public health uses such as supporting better flu surveillance systems.

Shortly after that, he and his supporters were off NSABB. Nobody wants it known they are doing 'basic research.'

Or lets put it this way, if we had the chance to change an existing virus that is circulating and spreading directly and make it less virulent,
should we do it ?

It would be a wonderful way to control disease if it were predictable.

The low virulence of attenuated vaccines can
be reversed by evolution. If the vaccine strain is
again allowed to transmit between hosts, natural
selection may promote the evolution of variants
that grow better and re-acquire high virulence.
Reversion to virulence, often observed for the
Sabin vaccine, has caused several local polio epidemics
(Kew et al. 2004).
CHAPTER 12
The evolution and expression
of virulence
Dieter Ebert and James J. Bull

 

[gsgs]

Re: cell entry

while attenuated virulence is (usually) "gain of function" , I assume
that gof-critics won't be against that kind of research ?!

>> if we had the chance to change an existing virus that is circulating
>> and spreading directly and make it less virulent, should we do it ?
>
> It would be a wonderful way to control disease if it were predictable.

would it be considered ethical to make it less virulent but not kill it ?
(which would have been way easier)
So, lets say I swap doorknobs in random hospitals, then genetically change the viruses that I find
on them with the intend to make them less virulent, and then I put them back onto the same
doorknob again. Would that be considered ethical ? legal ?

 

[Emily]

Re: cell entry

while attenuated virulence is (usually) "gain of function

The way I understand attenuation is that it reduces rather than intensifying a characteristic.

Would that be considered ethical ? legal ?

How much money do you have for a defense lawyer?

http://www.oregonlive.com/rajneesh/

 

[gsgs]

Re: cell entry

usually, long time adapted viruses are mild. Only the newcomers are virulent.
It's better for the virus to be mild, [better=gain of function]

 

[Emily]

Re: cell entry

I've been thinking that only loss of function would be good for a vaccine strain, but maybe not.

http://www.jbc.org/content/274/25/17567.short