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 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 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? 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.

Life cycle of enveloped virus

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 .

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.

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.

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.

examined each mutant Gag ability to interact with Tsg101 and to generate extracellular virion–

Another way of examining virion production 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 ? 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’ examined the ability of mutant EbVp40 protein to generate extracellular particles

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 .

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. 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.

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

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 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’ 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 host-cell protein–Tsg101 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

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