HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress

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HIV-1 and Ebola virus encode small peptide motifs
thatrecruit Tsg101 to sites of particle assembly
to facilitate egress

• JUAN MARTIN-SERRANO, TRINITY ZANG PAUL D.
  BIENIASZ
• Presented by Manjari Dani

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The paper deals with

• 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 .

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Background information

• 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

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What is budding?

• Budding is a step in the life cycle of enveloped
  viruses.
• It is the separation or release of nascent virion
  from host cell through membrane fusion.

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Life cycle of enveloped virus
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Introduction

• 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 .

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Investigated that

• 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.

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Methods

• Plasmid construction
• ePTAP,hPTAP,pL,pG2A and more .
• Yeast two-hybrid assays- yeast cell transformed
  with tagged Tsg101and tagged Gag or Vp40
  plasmids. HA,p24,myc are monoclonal antibodies
  used as tags.
• Western-blot analysis- yeast cell , mammalian
  cell ,HIV-1 virions and EbVp40 virus particles
  were separated on gel . Expressionof wt and
  mutant viral proteins is measured.
• Infectivity assays
• Immunofluorescence- for detection of
  relocalization of Tsg101.

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Experiments 1What is the significance of the
HIV-1 Gag Tsg101 interaction?

• Introduced 7 missense mutation (M1-M7) over PTAP
  sequence and residue flanking PTAP in p6
  protein.

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• examinedeach mutant Gag ability to interact
  with Tsg101 and to generate extracellular
  virion

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Another way of examining virion production

• Is by Gag expression.
• Ratio of gag protein present in virion and cell
  is determined

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2 What is the significance of PTAP motif in
Ebola virus budding ?

• 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

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Cont

• examined the ability of mutant EbVp40 protein to
  generate extracellular particles

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3. To observe relocalization of Tsg 101

• 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 .

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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.

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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.

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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.

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6.Whether L-domain function in trans?

• Contructed plasmids
• pL- with defective L domain
• pG2A- with functional L-domain but defective
  membrane binding domain.
• Cotransfection of pL and pG2A result in complex
  formation (due to multimerization) which contains
  both functional L-domain and membrane binding
  domain and able to form virions.

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Cont

• 1 Constructed another plasmid-
• pENX-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 pL- restored virion
  formation

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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.

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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.

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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

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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
  doesnt 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.

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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.

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Discussion

• 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.

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Cont

• The PTAP motif of viral proteins and Tsg101 or
  associated proteins play a general role in
  budding. But It is unclear whether the
  PTAPTsg101 interactions are analogous to
  host-cell proteinTsg101 interaction or exclusive
  for viruses to recruit Tsg101.
• 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

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Cont

• 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