Mutations affecting cleavage at the p10-capsid protease cleavage site block Rous sarcoma virus replication

BioMed Cen al Page 1 o 6 (page numbe no o ci a ion pu poses) Re o i ology Open Access Sho epo Mu a ions a ec ing clea age a he p10-capsid p o ease clea age si e block Rous sa coma i us eplica ion Ma cy L Vana1, Aiping Chen1, Pe e Bo oss2,3, I ene Webe 2, Dalbinde Colman4, E ic Ba klis4 and Jona han Leis*1 Add ess: 1Depa men o Mic obiology and Immunology, Feinbe g School o Medicine, No hwes e n Uni e si y, Chicago, Illinois 60611, USA, 2Depa men o Biology, Geo gia S a e Uni e si y, A lan a, GA 30303, USA, 3Biochemis y and Molecula Biology Depa men , Medical and Heal h Sciences Cen e , Uni e si y o Deb ecen, Deb ecen, Hunga y and 4Vollum Ins i u e and Depa men o Mic obiology, O egon Heal h and Science Uni e si y, Po land, OR, 97201, USA Email: Ma cy L Vana - [email p o ec ed]; Aiping Chen - a-chen2@no hwes e n.edu; Pe e Bo oss - biopib@langa e.gsu.edu; I ene Webe - [email p o ec ed]; Dalbinde Colman - [email protected]; E ic Ba klis - ba [email protected]; Jona han Leis* - j- [email p o ec ed]u * Co esponding au ho Abs ac A se ies o amino acid subs i u ions (M239F, M239G, P240F, V241G) we e placed in he p10-CA p o ease clea age si e (VVAM*PVVI) o change he a e o clea age o he junc ion. The e ec s o hese subs i u ions on p10-CA clea age by RSV PR we e con i med by measu ing he kine ics o clea age o model pep ide subs a es con aining he wild ype and mu an p10-CA si es. The e ec s o hese subs i u ions on p ocessing o he Gag polyp o ein we e de e mined by labeling Gag ans ec ed COS-1 cells wi h 35S-Me and -Cys, and immunop ecipi a ion o Gag and i s clea age p oduc s om he media and lysa e ac ions. All subs i u ions excep M239F caused dec eases in de ec able Gag p ocessing and subsequen elease om cells. Se e al o he mu an s also caused de ec s in p oduc ion o he h ee CA p o eins. The p10-CA mu a ions we e subcloned in o an RSV p o i al ec o (RCAN) and in oduced in o a chick emb yo ib oblas cell line (DF-1). All o he mu a ions excep M239F blocked RSV eplica ion. In addi ion, he e ec s o he M239F and M239G subs i u ions on he mo phology o eleased i us pa icles we e examined by elec on mic oscopy. While he M239F pa icles appea ed simila o wild ype pa icles, M239G pa icles con ained co es ha we e la ge and misshapen. These esul s sugges ha mu a ions a ec ing clea age a he p10-CA p o ease clea age si e block RSV eplica ion and can ha e a nega i e impac on i us pa icle mo phology. Findings The s uc u al p o eins o e o i uses a e encoded by he gag gene and a e ansla ed as a single polyp o ein. Du ing o subsequen o i us budding, he Gag polyp o ein is clea ed by he i al p o ease (PR), he eby eleasing he ma u e s uc u al p o eins. Gag p ocessing leads o mo - phological changes in he i us pa icle, including con- densa ion o he capsid co e, and is associa ed wi h he appea ance o in ec ious pa icles [1]. I has p e iously been demons a ed ha p ope p ocessing a se e al p o- ease si es h oughou RSV Gag is equi ed o p oduc ion o in ec ious i us [2,3]. Howe e , he p o ease si e sepa- a ing he C- e minus o p10 and he N- e minus o CA has no been examined. Published: 27 Sep embe 2005 Re o i ology 2005, 2:58 doi:10.1186/1742-4690-2-58 Recei ed: 01 Feb ua y 2005 Accep ed: 27 Sep embe 2005 This a icle is a ailable om: h p://www. e o i ology.com/con en /2/1/58 © 2005 Vana e al; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Re o i ology 2005, 2:58 h p://www. e o i ology.com/con en /2/1/58 Page 2 o 6 (page numbe no o ci a ion pu poses) Mul iple s udies ha e highligh ed he impo ance o clea - age a he N- e minus o e o i us CA p o eins in pa icle assembly and ma u a ion. S uc u al s udies ha e iden i- ied a β hai pin s uc u e a he N- e minus o RSV CA ha is hough o o m a e p o eolysis a he p10-CA si e and libe a ion o he N- e minus o CA [4]. Mo eo e , a con- se ed P o esidue a he ex eme N- e minus o RSV CA o ms a sal b idge wi h an in e nal Asp esidue, he eby s abilizing he β-hai pin s uc u e [4]. These P o and Asp esidues a e highly conse ed among many e o i us CA p o eins, sugges ing ha he β-hai pin is a common s uc- u al ea u e o e o i us CA p o eins [5-8]. Mu a ing he conse ed Asp esidue in HIV-1 CA (Asp51) o mu ine leukemia i us CA (MLV, Asp63) causes a loss in i us in ec i i y [8]. In addi ion, blocking p o ease clea age a he N- e minus o MLV CA esul s in he p oduc ion o i us ha is non-in ec ious [9]. I has also been demon- s a ed ha he N- e minus o CA and he esidues imme- dia ely ups eam o CA ha e a ole in de e mining he shape o assembling e o i us pa icles [8,10-13]. Mo e speci ically o RSV, i has been demons a ed ha he p esence o p10 on he N- e minus o CA-NC con e s he in i o assembly pheno ype om cylind ical pa icles o sphe ical pa icles ha esemble wild ype imma u e RSV pa icles [10,11]. In his s udy, amino acid subs i u ions we e made in he i s wo N- e minal esidues o CA and he las C- e minal amino acid o p10 in o de o al e clea age a he p10-CA si e and examine he ole o p10-CA clea age in Gag p ocessing and RSV eplica ion (Fig. 1A). P e ious s udies ocusing on he RSV NC-PR o HIV-1 MA-CA clea age si es showed ha subs i u ing Gly a any o he P2-P2' posi- ions esul ed in g ea ly educed in i o hyd olysis o he pep ides [14,15]. Phe subs i u ions o P1 p o ided good clea age o he RSV NC-PR o HIV-1 MA-CA pep ides, while Phe subs i u ions o P1' we e ole a ed in he RSV NC-PR pep ide, bu no in he HIV-1 MA-CA pep ide. The abili y o RSV PR o clea e pep ides con aining he p10- CA amino acid subs i u ions compa ed o a pep ide con- aining he wild ype p10-CA si e was es ed using an in i o p o ease assay [16]. All o he subs i u ions excep M239F led o a dec ease in he a e o pep ide clea age (Fig. 1A). Subs i u ing Phe o Me in he P1 posi ion (M239F) had a small s imula o y e ec on pep ide clea - age by PR, while changing he same Me o Gly (M239G) esul ed in a comple e block in pep ide clea age. Simi- la ly, changing he P1' P o o Phe (P240F) caused a se e e i no comple e loss in pep ide clea age and eplacemen o he Val in he P2' posi ion wi h Gly (V241G) esul ed in a 200- old dec ease in pep ide clea age. Thus, mu a ing esidues on ei he side o he clea age junc ion signi i- can ly al e ed p ocessing o he si e. The e ec s o he p10-CA subs i u ions on Gag p ocessing we e es ed by in oduc ion o he mu a ions in o he con- ex o ull-leng h Gag and exp essing he wild ype o mu an Gag p o eins in COS-1 cells [2,3]. Gag and i s clea age p oduc s we e immunop ecipi a ed om he media and lysa e ac ions om ans ec ed cells ollowing me abolic labeling and we e sepa a ed using SDS-PAGE (Fig. 1B, op). By compa ison o wild ype (Fig. 1B, op lanes 2), all o he p10-CA subs i u ions excep M239F caused p ocessing de ec s. The banding pa e n in he lysa e and media ac ions om cells ans ec ed wi h M239F (Fig. 1B, op, lanes 3) was e y simila o wild ype, sugges ing ha he M239F subs i u ion did no a ec Gag p ocessing. In con as , a no el and s able band ep esen - ing a p10-CA usion p o ein was p esen in he lysa e and media ac ions om cells ans ec ed wi h he M239G (Fig. 1B, op, lanes 4) and P240F (Fig. 1B, op, lanes 5) mu an s ha was no p esen in ac ions om cells ans- ec ed wi h wild ype Gag (lanes 2 op). The p esence o a p10-CA usion indica ed ha hese mu a ions esul ed in a educ ion in he abili y o PR o clea e he p10-CA si e wi hin Gag. In cells ans ec ed wi h wild ype Gag, h ee CA species we e de ec ed (CA1, CA2, and CA3) in he media and lysa e ac ions (Fig. 1B, op, lanes 2) [2,3]. These species a e he esul o p ocessing o CA a i s C- e minus a di - e en si es. In con as , in cells ans ec ed wi h he M239G mu an , CA2 and CA3 we e de ec ed in he media ac ion, bu CA1 was no (Fig. 1B, op, lanes 4). Fu he - mo e, ma u e CA p o eins we e no de ec ed in he lysa e. Simila ly, none o he ma u e CA p o eins we e de ec ed in he media o lysa e ac ions om cells ans ec ed wi h he P240F (Fig. 1B, op, lanes 5) mu an , and CA1 made up he majo i y o he CA p o ein in he media and lysa e ac ions om cells ans ec ed wi h he V241G (Fig. 1B, op, lanes 6) mu an . The e also appea ed o be a educ- ion in he amoun o Gag eleased in o he media om cells ans ec ed wi h he V241G mu an compa ed o cells ans ec ed wi h wild ype Gag (Fig. 1B, op, lanes 6 and 2). This e ec was mos appa en when examining he sig- nal o PR in he lysa e and media ac ions. The amoun o PR in he lysa e ac ion om cells ans ec ed wi h he V241G mu an was simila o wild ype, bu he amoun o PR in he media ac ion om cells ans ec ed wi h he V241G mu an was g ea ly educed compa ed o wild ype. In o de o de e mine whe he he educ ion in pa - icle elease obse ed wi h he V241G mu an was due o impai ed Gag p ocessing, a D37S mu a ion in he PR domain was cons uc ed in he con ex o he p10-CA Gag mu an s. COS-1 cells we e ans ec ed wi h he p10-CA/ PR-D37S mu an s and ull-leng h Gag was immunop e- cipi a ed om he media and lysa e ac ions. A simila le el o Gag elease was obse ed wi h all o he p10-CA/ PR-D37S mu an s when compa ed o PR-D37S (Fig. 1B, Re o i ology 2005, 2:58 h p://www. e o i ology.com/con en /2/1/58 Page 3 o 6 (page numbe no o ci a ion pu poses) A. Schema ic diag am o he RSV Gag polyp o ein and he amino acid subs i u ions placed in he p10-CA p o ease clea age si e wi hin GagFigu e 1 A. Schema ic diag am o he RSV Gag polyp o ein and he amino acid subs i u ions placed in he p10-CA p o ease clea age si e wi hin Gag. The ec angle ep esen s he RSV Gag polyp o ein wi h he encoded p o ein sequences indica ed by he s anda d nomencla u e. The ho izon al lines ep esen he PR clea age si es. SP is he space pep ide. The L domain o RSV Gag esides in he p2b pep ide. In he box below, he P4-P1 and P1'-P4' amino acid sequence o he wild ype p10-CA p o ease clea age si e is shown. The p10-CA mu an s (unde lined bold ex ) a e shown below he wild ype sequence. The esul s o in i o p o ease assays examining RSV PR-media ed clea age o pep ides con aining he wild ype (PVVAM*PVVIKRR) and mu an p10-CA si es a e also indica ed. The si e o p10-CA clea age is designa ed wi h an as e isk. B. Top, E ec o p10-CA amino acid subs i u- ions on p ocessing o RSV Gag. COS-1 cells we e ans ec ed wi h wild ype Gag o he p10-CA mu an s in pSV.My 0(HpaI). 48 hou s a e ans ec ion, cells we e labeled wi h [35S]-Me and Cys and Gag p o eins we e immunop ecipi a ed wi h an an i- RSV abbi an ise um om he media ( igh panel) and lysa e (le panel) ac ions. Immunop ecipi a ed p o eins we e sepa- a ed by SDS-PAGE and exposed o ilm. Lane 1, un ans ec ed cells. Cells ans ec ed wi h wild ype, lane 2; M239F, lane 3; M239G, lane 4; P240F, lane 5; V241G, lane 6. B. Bo om. E ec o p10-CA amino acid subs i u ions on Gag elease in he con- ex o a p o ease inac i a ing subs i u ion (PR-D37S). COS-1 cells we e ans ec ed and ull-leng h Gag p o eins we e immu- nop ecipi a ed and sepa a ed by SDS-PAGE as abo e. Cells ans ec ed wi h M239F/PR-D37S, lane 1; M239G/PR-D37S, lane 2; P240F/PR-D37S, lane 3; V241G/PR-D37S, lane 4; un ans ec ed cells, lane 5; PR-D37S, lane 6. Re o i ology 2005, 2:58 h p://www. e o i ology.com/con en /2/1/58 Page 4 o 6 (page numbe no o ci a ion pu poses) bo om), sugges ing ha he pa icle elease de ec obse ed wi h he V241G subs i u ion was due o impai ed Gag p ocessing. Taken oge he , hese esul s indica e ha mu a ions o he p10-CA si e o Gag a ec p ocessing o he C- e minus o CA. In o de o de e mine he e ec s o he p10-CA subs i u- ions on RSV eplica ion, he p10-CA mu a ions we e sub- cloned in o he RCAN p o i al ec o [17]. DF-1 cells we e ans ec ed wi h each o he mu an s, and e e se an- sc ip ase (RT) ac i i y was moni o ed in he media o ans ec ed cells a egula in e als [18]. All o he p10-CA mu a ions excep M239F had a de imen al e ec on RSV eplica ion (Fig. 2). The M239F mu a ion caused an ini ial delay in eplica ion wi h an app oxima e ou - old educ- ion in RT ac i i y bu eached a simila peak in i us p o- duc ion o wild ype by day six. In con as , all o he o he p10-CA mu a ions led o a se e e block in i al eplica ion (Fig. 2). The RT ac i i y o hese mu an s could no be de ec ed abo e con ol le els o 5TE bu e (da a no shown), media om un ans ec ed cells (da a no shown), o media om cells ans ec ed wi h an L domain dele ion mu an (∆PY/RCAN). To be e unde s and he e ec o he p10-CA mu a ions on RSV eplica ion, wild ype and p10-CA i us pa icles we e examined using elec on mic oscopy. Vi us pa icles we e ha es ed h ee days pos ans ec ion and iewed a a magni ica ion o 11,000× (Fig. 3, le ) and 37,000× (Fig. 3, igh ). We we e only able o examine wild ype, M239F and M239G pa icles by EM, as we we e unable o ob ain high enough amoun s o P240F and V241G pa icles. M239F pa icles appea ed o be simila o wild ype pa i- cles in diame e (wild ype; 119 ± 7 nm, MF; 118 ± 11 nm). The a io o he c oss-sec ional a eas o he i us co e and he en i e i us pa icle we e also simila be ween he wild ype (Fig. 3, op le and igh ) and M239F (Fig. 3, middle le and igh ) pa icles (wild ype; 28 ± 3%, MF; 26 ± 3%). In con as , he M239G pa icles (Fig. 3, bo om le and igh ) we e la ge in diame e (MG; 125 ± 5 nm) compa ed o he wild ype and M239F pa icles, and had a highe a io o co e o pa icle c oss-sec ional a ea (MG; 45 ± 5%). I is likely ha he de ec in pa icle mo phology obse ed wi h he M239G mu an played a ole in he loss o eplica ion capaci y o his mu an . Toge he , hese esul s highligh he impo ance o p ope p ocessing a he p10-CA si e in RSV eplica ion, and suppo p e ious indings demons a ing he impo ance o his egion in e o i us eplica ion [4-13]. Compe ing in e es s The au ho (s) decla e ha hey ha e no compe ing in e es s. Au ho s' con ibu ions M. L. V. cons uc ed he p10-CA mu a ions, pe o med he Gag p ocessing and eplica ion assays, pu i ied i us pa - icles o EM analysis, and w o e he pape . A. C. con- s uc ed he D37S mu a ions and pe o med he budding assay wi h he D37S mu an s. P. B. pe o med he in i o p o ease assay. D. C. and E. B. pe o med he EM analysis. E ec o p10-CA subs i u ions on abili y o RSV o eplica e in ans ec ed DF-1 cellsFigu e 2 E ec o p10-CA subs i u ions on abili y o RSV o eplica e in ans ec ed DF-1 cells. DF-1 cells we e ans ec ed wi h wild ype RCAN, RCAN cons uc s con aining he p10-CA mu a ions, o an RCAN cons uc con aining an L domain dele ion (∆PY). A he indica ed imes a e ans ec ion, he RT ac i i y in he cul u e medium was de e mined by quan i- ica ion o [α-32P]-dTTP inco po a ion du ing e e se an- sc ip ion using a polyadenylic acid (poly A) empla e and a oligodeoxy hymidyla e (p(dT)12–18) p ime . Wild ype (), L- domain dele ion (䊐), M239F (X), M239G (*), P240F (-), and V241G (+). Re o i ology 2005, 2:58 h p://www. e o i ology.com/con en /2/1/58 Page 5 o 6 (page numbe no o ci a ion pu poses) E ec o p10-CA mu a ions on i us pa icle mo phologyFigu e 3 E ec o p10-CA mu a ions on i us pa icle mo phology. WT ( op le and igh ), M239F (middle le and igh ), and M239G (bo om le and igh ) i uses om ans ec ed cells we e sedimen ed h ough 20% suc ose cushions, esuspended, and p oc- essed o elec on mic oscopy. A low magni ica ion (le ; op, middle and bo om), WT and M239F co es appea ed conical o bulle -shaped, whe eas M239G co es some imes appea ed conical (le -bo om, le mos i us), bu mo e o en appea ed wi h la ge misshapen co es. A highe magni ica ion ( igh ; op, middle and bo om), in e nal co es we e di icul o disce n wi hou signi ican adjus men o image con as le els. Size ba s o he wo magni ica ions o images appea in bo om le and igh panels, and co espond o 100 nm. Publish wi h BioMed Cen al and e e y scien is can ead you wo k ee o cha ge "BioMed Cen al will be he mos signi ican de elopmen o dissemina ing he esul s o biomedical esea ch in ou li e ime." Si Paul Nu se, Cance Resea ch UK You esea ch pape s will be: a ailable ee o cha ge o he en i e biomedical communi y pee e iewed and published immedia ely upon accep ance ci ed in PubMed and a chi ed on PubMed Cen al you s — you keep he copy igh Submi you manusc ip he e: h p://www.biomedcen al.com/in o/publishing_ad .asp BioMedcen al Re o i ology 2005, 2:58 h p://www. e o i ology.com/con en /2/1/58 Page 6 o 6 (page numbe no o ci a ion pu poses) Acknowledgemen s This wo k was suppo ed in pa by Uni ed S a es Public Heal h Se ice g an CA52047 ( o J.L.), CA58166 ( o I. W.), and GM60170 ( o E.B.), he Hunga ian Science and Resea ch Fund, OTKA F35191 ( o P.B.), and he Cance Biology Fellowship P og am, Chicago Baseball Cance Cha i ies, om he Robe H. Lu ie Comp ehensi e Cance Cen e ( o M.L.V.). Pep- ides we e a gene ous gi o D . Te y Copeland, NCI, F ede ick, Ma yland. Re e ences 1. Wills JW, C a en RC: Fo m, unc ion, and use o e o i al gag p o eins. Aids 1991, 5:639-654. 2. 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