Transcript HIV-1 and Ebola virus encode small peptide motifs that recruit

HIV-1 and Ebola virus encode small
peptide motifs that
recruit Tsg101 to sites of particle
assembly to facilitate egress
JUAN MARTIN-SERRANO, TRINITY ZANG & PAUL D. BIENIASZ
Presented by – Manjari Dani
The paper deals with
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Viral and cellular factors involved in budding process of
enveloped viruses
Viruses- HIV-1 ,ebola virus
HIV- AIDS
Ebola- haemorrhagic fever .There is no specific treatment and
death can occur within 10 days .
Background information
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Enveloped virus - A virus consisting of nucleic acid
within capsid and surrounded by a lipoprotein layer
called envelope.
HIV – is a enveloped single strand + sense RNA
virus- retroviridiae
Ebola – is an enveloped ss – sense RNA virus filoviridae
What is budding?
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Budding is a step in the life cycle of enveloped
viruses.
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It is the separation or release of nascent virion from
host cell through membrane fusion.
Life cycle of enveloped virus
Introduction
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Retroviral Gag protein is the protein of the capsid
shell around the RNA of a retrovirus.
It encode L or late domains required for particle
budding
L-domains are transferable bn different retroviruses
and position independent .
L domain contains one of the 3 sequence motfis PT/SAP, PPXY or YXXL
These motifs constitute binding site for cellular
proteins involved in budding - Tsg101, Nedd4 .
Investigated that
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HIV-1 L-domain contains
PTAP motif within the p6
Gag protein.
p6 bind to Tsg101 –host cell
protein.
Ebola virus matrix protein Vp40 also contains PTAP
motif .
PTAP motif of both HIV-1
Gag and Ebola virus- Vp40
recruit Tsg 101 to site of
particle assembly.
Methods
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Plasmid construction:
ePTAP,hPTAP,pL,pG2A and more .
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Yeast two-hybrid assays- yeast cell transformed with tagged –
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Western-blot analysis- yeast cell , mammalian cell ,HIV-1 virions
Tsg101and tagged –Gag or Vp40 plasmids. HA,p24,myc are
monoclonal antibodies used as tags.
and EbVp40 virus particles were separated on gel . Expressionof wt
and mutant viral proteins is measured.
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Infectivity assays
Immunofluorescence- for detection of relocalization of Tsg101.
Experiments
1What is the significance of the HIV-1 Gag
& Tsg101 interaction?
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Introduced 7
missense mutation
(M1-M7) over PTAP
sequence and
residue flanking
PTAP in p6 protein.
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examined
each mutant Gag ability to
interact with Tsg101
and to generate
extracellular virion–
Another way of examining virion
production
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Is by Gag expression.
Ratio of gag protein
present in virion and
cell is determined
2 What is the significance of PTAP
motif in Ebola virus budding ?
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The Ebola virus matrix
protein (EbVp40) contains
overlapping PTAP and
PPXY motifs
PPXY motif interacs with
Nedd4 and is essential for
formation of ebola virus like
particle
To determine the role of
PTAP –
Introduced a single
aminoacid substitution within
the PTAP motif (P7L),
keeping the PPXY motif
intact
Cont’
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examined the ability of
mutant
EbVp40 protein to generate
extracellular particles
3. To observe relocalization of Tsg 101
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If PTAP mediates
budding process by
recruitment of Tsg101
then localization of
Tsg101 shuld be
observed.
Examined in the
absence or presence of
wt orP7L mutant
EbVp40 .
4. Which motif PTAP or PPXY imp for
L-domain function?
1 Generated
hPTAP- 10 residues conatining
PTAP motif fromHIV-1 Gag
ePTAP -12 residues containing
PTAP motif from EbVp40
2 Substitued hPTAP in HIV
Gag with ePTAP to form
pHIV/p6/ePTAP.
cont
3 Found pHIV/p6/ePTAP is as
infectious as parental HIV-1
p6 protein.
4 then introduced P7L
mutation in PTAP keeping
PPXY unchanged .
 This reduced ability of
HIV/p6/ePTAP to interact
with Tsg101 and to form
virions.
5.Is EbVp40 derived sequence ePTAP a
true L-domain ?
1 Generated a plasmid dGH
from Gag in which
sequences not required for
particle formation were
replaced with 2 copies of
influenza sequence (2xHA
)as control or 2 copies of
ePTAP (2xePTAP).
2 dGH(2xHA ) forms virion in
presence of Ldomain but not
in absence of Ldomain.
 dGH(2xePTAP) forms
virions even in absence of
Ldomain.
6.Whether L-domain function in trans?
Contructed plasmids
pL- with defective L domain
pG2A- with functional L-domain but
defective membrane binding
domain.
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Cotransfection of pL and pG2A
result in complex formation (due
to multimerization) which
contains both functional Ldomain and membrane binding
domain and able to form virions.
Cont’
1 Constructed another plasmidpENX-Gag is truncated at p6
protein and membrane
domains replaced with
synthetic sequence for
insertion of other sequences
containing L-domain.
2 When p6 or ePTAP or hPTAP
were inserted into ENX and
coexpressed with pLrestored virion formation
cont
3 But in case of mutant p6 with
defective L-domain – there
was no virion formation.
4 Distantly related equine
infectious anemia (EIA)Virus
L-domain within p9 protein
also resulted in virion
formation.
7.Is L-domain dispensable for budding
process?
1 Directly inserted Tsg101 into
pENX and coexpressed with
pL- resulted in virion
formation even in complete
absence of L-domain.
2 In tsg101 protein, C-terminal
half of total 390 residue are
required for budding
process.
Answers /results
1 What is the significance of HIV-Gag interaction with Tsg101?
- if there is no interaction , there is no virion formation.Thus
recuritment of Tsg 101 is required for budding of virions.
2. What is the significance of PTAP motif in Ebola virus Vp40 protein?
- PTAp sequence is required for Ebola virus particles formation. In
both HIV and ebola virus PTAP is the motif which interacts with
Tsg101
3 Is Tsg 101 relocalize to the site of budding?
Yes, in the presence of active or wt Ebola Vp40 Tsg localizes
Answers/results
4.Which motif PTAP or PPXY is important for L domain (HIV Gag
protein) function?
- Ldomain function is entirely due to PTAP .PPXY doesn’t work in
absence of PTAP.
5. Is EbVp40 derived sequence ePTAP a true L-domain ?
- Yes,ePTAP has position independent characteristic of L domain.
6.Whether L domain function in trans ?
- L – domains can function in trans.The viral L domain expressed
in plasmid in which pol is defective,are able to complement a
plasmid that contains defective L domain.
Results
7. Is L domain dispensible?
- Yes, L domain is dispensible for budding of virus particles if Tsg
101 can be recruited to the site of budding by another
mechanism.
Discussion
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The exact mechanism by which Tsg mediates viral budding is not
known.
Predicted is :
a)Tsg101 is a component of a complex,ESCRT-I that is essential for
the sorting of ubiquitinated proteins into the multi-vesicular body
(MVB) in yeast and for endosomal targeting in mammalian Cells.
b)The ‘budding’ and membrane-fusion events that lead to the
formation MVB are equivalent to the budding of enveloped viral
particle except the cellular location.
c) hypothesis is that viral proteins recruit the machinery involved in
MVB formation to sites of virus budding at the plasma membrane.
Cont’
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The PTAP motif of viral proteins and Tsg101 or associated
proteins play a general role in budding. But It is unclear
whether the PTAP–Tsg101 interactions are analogous to hostcell protein–Tsg101 interaction or exclusive for viruses to recruit
Tsg101.
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Short sequence from Gag protein and EbVp40 are sufficient to
bind Tsg101 .This strategy can be used in anti viral activity
against HIV and Ebola by using small inhibitors
Cont’
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The findings of Garrus et al. are entirely consistent.
Reported that PTAP motif of p6 protein directly interacts with
Tsg101 and depletion of Tsg101 from Hiv-1 producing cells
result in defects in budding