evaluation and prediction of the hiv-1 central polypurine ... · researcharticle evaluation and...

Research ArticleEvaluation and Prediction of the HIV-1 CentralPolypurine Tract Influence on Foamy Viral Vectors toTransduce Dividing and Growth-Arrested Cells

Sergey Shityakov1 Carola Foumlrster1 Axel Rethwilm2 and Thomas Dandekar3

1 Department of Anesthesia and Critical Care University of Wurzburg 97080 Wurzburg Germany2Department of Virology University of Wurzburg 97074 Wurzburg Germany3Department of Bioinformatics University of Wurzburg 97074 Wurzburg Germany

Correspondence should be addressed to Sergey Shityakov shityakoffmailru

Received 25 November 2013 Accepted 18 February 2014 Published 9 June 2014

Academic Editors E Carrillo and E Martinez

Copyright copy 2014 Sergey Shityakov et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Retroviral vectors are potent tools for gene delivery and various biomedical applications To accomplish a gene transfer tasksuccessfully retroviral vectors must effectively transduce diverse cell cultures at different phases of a cell cycle However verypromising retroviral vectors based on the foamy viral (FV) backbone lack the capacity to efficiently transduce quiescent cells It ishypothesized that this phenomenon might be explained as the inability of foamy viruses to form a pre-integration complex (PIC)with nuclear import activity in growth-arrested cells which is the characteristic for lentiviruses (HIV-1) In this process the HIV-1central polypurine tract (cPPT) serves as a primer for plus-strand synthesis to produce a ldquoflaprdquo element and is believed to be crucialfor the subsequent double-stranded cDNA formation of all retroviral RNA genomes In this study the effects of the lentiviral cPPTelement on the FV transduction potential in dividing and growth-arrested (G

1S phase) adenocarcinomic human alveolar basal

epithelial (A549) cells are investigated by experimental and theoretical methodsThe results indicated that the HIV-1 cPPT elementin a foamy viral vector background will lead to a significant reduction of the FV transduction and viral titre in growth-arrested cellsdue to the absence of PICs with nuclear import activity

1 Introduction

Retroviral vectors are efficient gene transfer vehicles thatdeliver transgenes by integration of the viral genome into thegenome of host cells This remarkable ability has been widelyexploited for many biomedical applications including genetherapy In order to replicate all retroviruses pursue a verycomplex process which is known as reverse transcriptionDuring this event they transcribe their single-stranded RNAgenomes into double-stranded complementary DNA (dscDNA) prior to its integration into the host genome [1 2]Theoutcome of reverse transcription is a linear DNA with longterminal repeats (LTRs) at the 31015840 and 51015840 ends of the viralgenome As an example lentiviruses (HIV-1) have evolvedinto new forms with a complex reverse transcription strategyincluding a strand displacement event controlled by thecentral polypurine tract (cPPT) which serves for plus-strandinitiation and priming [3 4]

The HIV-1 reverse transcription produces a linear DNAmolecule bearing a ldquopeculiar formationrdquo known as the centralDNA flap element because the reverse transcription complexstops at the central termination sequence (CTS) (Figure 1(a))This flap element is believed to be essential for a preintegra-tion complex (PIC) assembly which is composed of both viraland cellular proteins [5ndash7] and possess the ability to cross thenuclear pore to enter the nucleus It was previously reportedthat some constituents of PIC such as the viral matrixprotein contained a nuclear localization signal (NLS) forPIC-mediated nuclear import [8 9] However the detailedmechanisms of this process are not yet fully elucidated

The other retroviruses such as foamy viruses (FV) havea replication strategy and probably a PIC formation mecha-nism different from that present in lentiviruses FVs belongto the Spumavirinae retroviral subfamily and are knownas nonpathogenic retroviral species which are endemic to

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 487969 11 pageshttpdxdoiorg1011552014487969

2 The Scientific World Journal

Flap

cPPT CTS

(a)

Gap

cPPT

(b)

Figure 1 Different reverse transcription outcomes in lenti- (a) and foamy viruses (b) cPPT and CTS abbreviations stand for the centralpolypurine tract and central termination sequence respectively

a number of mammalians such as non-human primates catsand cows [10]

The genomic organization of the FVs including theprototype foamy virus (PFV) which is a molecular clonedsimian foamy virus SFVcpz(hu) is similar to that of othercomplex retroviruses with several additional open readingframes located in 31015840 of the canonical Gag Pol and Env genesincluding the transcriptional transactivator gene (tas) [11ndash15]

The foamy viruses have a very broad host range and infecta variety of cell lines including fibroblastoid epithelioid andlymphatic lineages [16] There are currently a few cell linesthat are resistant to FV infection such as the zebrafish PAC2and human erythroid precursor cell lines [17] FV infectionleads to massive cell death via apoptosis in vitro and withoutany overt pathogenic effect in vivo [18]

Essentially foamy viruses seem to have diverse outcomesfor the plus-strand priming events in contrast to lentivirusesIn FV reverse transcription the strong stop plus-strand DNAis displaced by DNA elongating from cPPT This DNA canfurther be transferred to the 31015840 end of the minus-strand DNAor serve as a template for synthesis of a double-stranded LTRConsequently cPPT is degraded after completion of theminus-strand cDNA synthesis producing a single-strandedgap (Figure 1(b)) [19ndash21]

There are a number of FV-based vectors for gene therapythat have been developed [22ndash24]These constructs have sev-eral desirable properties in comparison to lentiviral vectorsThe main characteristics of foamy viral vectors are (i) safetyFVs have not been linked to any established pathology [1825] (ii) wide tissue tropism [10 26ndash28] (iii) viral particlesstable and resilient to ultracentrifugation [29] (iv) packagingefficacy for foreign DNA that is at least 9 kb and offers avaluable tool for the transfer of long genetic elements andtherapeutic transgenes [30] (v) potential for production ofhigh-titre FV vectors suitable for ex vivo gene therapyapplications [24 30 31]

However these very promising foamy viral vectors haveone limitation in their ability to efficiently transduce ter-minally differentiated and growth-arrested mammalian cells[32] potentially because of their mitosis-dependent integra-tion [22] and the deficiency in forming PICs with nuclearimport activity from the absence of the lentivaral cPPTelement and central termination sequence

Therefore the role of HIV-1 cPPT on FV replication andits mechanism of action is still to be determined To pursue

this goal the transduction rates of cPPT (HIV-)modifiedFV vectors in dividing and growth-arrested adenocarcinomichuman alveolar basal epithelial (A549) cells were accessedalong the investigation of underlying molecular mechanismsand evaluation of their potential for FV-based gene therapy

2 Materials and Methods

Recombinant DNA techniques standard molecular cloningtechniques were used for the generation of constructs [33] Aseries of plasmid cloning vectors were preceded by the letterldquoprdquo In brief the pUC19-based intermediate was first gener-ated by ligating a 1259 kb EcoRI-KpnI fragment from pMD9(9940 kb) with a 2674 kb EcoRI-KpnI fragment from thepUC19 vector to use the proper restriction sites Then fourpUC19-based plasmids such as pUC19-cPPT (HIV)-CTSpUC19-cPPT (PFV)-CTS pUC19-cPPT (HIV) and pUC19-CTS were generated by PCR amplification and insertion ofthe fragments bearing the viral structural elements (Table 1)using relevant oligonucleotides (Table 2) with MfeI-StuIrestriction sites The lentiviral pWPXL vector (10510 kb) wasused as a template for the cPPT (HIV) cPPT (HIV)-CTSand CTS amplifications All pMD9-based FV vectors con-tained the gene encoding enhanced green fluorescent protein(eGFP) under the control of a constitutively active heterolo-gous retroviral U3 promoter to enable the quantification ofvector transfer rates (Figure 2)

Cell transfection and purification of supernatant humanembryonic kidney (HEK 293T) cells [34] were seeded in 2 times106 density into 6 cm dishes a day before the transfectionThe cells were transfected with plasmid DNA using a poly-ethyleneimine transfection reagent (Polysciences EuropeGmbH Eppelheim Germany) [35] The PFV transfectionmixture contained 25120583g of modified pMD9 vectors 125 120583gof pCIgag2 05120583g of pCpol-2 and pCenv-1 packaging plas-mids [35] The HIV transfection system contained 25120583g ofpWPXL 19 120583g of psPAX2 and 08 120583g of pMD2G (VSV-G)vectors (gifts from Professor Didier Trono) respectively ThepMD9 plasmid without Env (pMD9-woEnv) and pcDNA(Invitrogen GmbH Karlsruhe Germany) as an empty vectorwere used as negative controls without a production of viralparticles One day after transfection the cellular transcriptionwas induced by addition of 10mM sodium butyrate for8 hrs [36] After two days the supernatant was harvestedpassed through a 045120583mfilter (MerckMillipore Darmstadt

The Scientific World Journal 3

Table 1 Sequences of the viral structural elements used in the construction of modified expression vectors

Element Length (bp) SequencecPPT (PFV) 9 AGGAGAGGGcPPT (HIV) 33 ATCCACAATTTTAAAAGAAAAGGGGGGATTGGGCTS 16 AAAAATTCAAAATTTT

Table 2 Forward and reverse primers for a PCR amplification of the fragments used in the construction of modified expression vectors

Fragments Primers

cPPT (PFV)-CTS 51015840-TATACAATTGCAGGAGAGGGATTGGGGGGTACAGTGCAG-31015840

51015840-TATAAGGCCTCTGTCCCTGTAATAAACC-31015840

cPPT (HIV)-CTS 51015840-TATACAATTGATGGCAGTATCCAC-31015840


cPPT (HIV) 51015840-TATACAATTGATGGCAGTATCCAC-31015840

51015840-TATAAGGCCTGTAATTTGTTTTTGTAATTCT-31015840

CTS 51015840-TATATACAATTGGGGGGTACAGTGCAGGGG-31015840

51015840-TATATAAGGCCTTCCCTGTAAACCCGAAAATTTTG-31015840

Germany) and layered onto 6mL of sucrose cushion (20in DMEM) The supernatant was centrifuged in a SureSpin630 rotor (Thermo Scientific Sorvall Waltham MA USA)at 25000 rpm at 4∘C for 3 hrs

Cell transduction and cell cycle experiments after clarifi-cation the vector-containing supernatants from HEK 293Tcells were functionally assayed to transduce the A549 cell-line whichwere incubatedwith them for two days at differentconditionsThe conditions were created in the experiment totest the generated pMD9-based plasmids to efficiently trans-duce dividing and nondividing cells (G

1S phase of a cell

cycle) in Dulbeccorsquos modified Eagles medium (DMEM) as acontrol in the presence of dimethyl sulfoxide (DMSO) and inthe presence of aphidicolin (Figure 3) The drug waspurchased from Sigma-Aldrich (Germany Sigma-AldrichChemie GmbH Germany) as a potent antiviral antimitoticagent and DNA polymerase inhibitor which is used to stopa cell cycle at G

1S phase The drug was dissolved in pure

DMSO and added to cells at 5 120583gmL concentration for 24 hrs[32] The final concentration of DMSO was approximately01 in the medium

Immunoblotting of viral proteins analysis of viral proteinexpression was done essentially as described elsewhere [37]In brief the lysates were prepared from the partially purifiedvector supernatant and from transfected cells by suspensionin a detergent-containing buffer Viral proteins were reactedwith anti-Gag [23] and anti-Pol [38] mouse monoclonalantibodies (mAbs) after separation in 8 SDS-PAGE andsemidry blotting onto Hybond ECL membranes (AmershamPharmacia Biotech Freiburg Germany) Protein bands weredetected by using horseradish peroxidase-coupled secondaryantibodies (Dako Hamburg Germany) and employing theenhanced chemiluminescence detection system (AmershamPharmacia Biotech FreiburgGermany)The ImageJ software(National Institute of Health Bethesda MD USA) was usedto analyse quantitatively the immunoblotting results

Vector transfer technique after clarification the vector-containing supernatants were also assayed functionally bytransfer to 15 times 104 recipient A549 cells purchased fromAmerican Type Culture Collection (ATCC Manassas VAUSA)The expression of eGFP per 104 cells was monitored byflow cytometry (FCM) for 48 hrs after the transduction Thevector transfer assays were repeated at least three times

Viral transduction dynamics and viral titre predictionthe MATLAB R2012a software (MathWorks Natick MAUSA) was used to predict the viral transduction dynamics(119891(119909)td) and viral titre (119891(119909)vt) for the analysed plasmidsSigmoid dose-response logistic function was implemented inthe MATLAB script M-files using the following equations

119891(119909)td =001119886

001 + 119890(minus119887119909) (1)

119891(119909)vt =119886

001 + 119890119909 (2)

where 119909 is any independent variable described by linspacefunction 119886 is a transduction rate (percentage of eGFP cells) 119890is an exponential constant (119890 = 2718) and 119887 integer is aHill Slope The linspace function (119909 = linspace(0 48)) wasused to generate linearly spaced vectors for 48-hour intervalcurve The 119887 integer (119887 = 021) was adjusted to the 119891(119909)tdlogistic function for pWPXL (119910

1= 1(001 + exp(minus119909 lowast

021))) as a reference curve reaching the upper plateau levelwith maximal effect after 48 hrs (Supplementary Material 1in Supplementary Material available online at httpdxdoiorg1011552014487969) To determine the viral titre dyna-mics seven dilutions were inspected in the range from thehighest (01) to the lowest (10minus7) concentrations of the virus(119909 = linspace(0 6))


LTR RRE

CMV RU5CASI CASII

cPPT-CTS

CTS

EF1-120572

SFFV eGFP

U3

pMD9

pWPXL

cPPT-CTS

cPPT

cPPT

pMD9

pMD9

pMD9

pMD9

cPPT-CTS eGFP

ΔU3

Figure 2 The structural elements of pWPXL and pMD9 parental plasmid backbones are shown long terminal repeat (LTR) with U3 Rand U5 regions ref-responsive element (RRE) human elongation factor alpha promoter (EF1-120572) gene encoding enhanced green fluorescentprotein (eGFP) enhancerpromoter of the human cytomegalovirus immediate early gene (CMV) cis-acting sequences (CASI and CASII)constitutively active spleen focus forming virus U3 promoter (SFFV U3) and internally deleted U3 region of the 31015840 LTR (ΔU3) Foamy viralcPPT was replaced with cPPT (HIV) in different variations CTS was deleted or inserted in accordance with the scheme shown above

Control

DMSO

Aphidicolin

Add virus 0hrs

OH

HO

CH3

OH

OH

+Seed cells minus24hrs Add drug 24hrs FCM +48hrs

CH3

Figure 3The timescale representation of the transduction experiments with aphidicolin substance to stop a cell cycle in G1S phase DMSO

was added to the A549 cells for a toxicity evaluation and as a solvent for the drug

3 Results and Discussion

To be functional the retroviral system for gene therapy mustcomprise two principal elements these being as truncatedviral backbones and packaging plasmids The PFV systemtherefore includes the Cas I and Cas II (cPPT) backboneelements as a part of pMD9 and pCIgag2 pCpol-2 andpCenv-1 packaging plasmids On the contrary the HIV sys-tem comprises the120595 packaging signal ref-responsive element(RRE) cPPT-CTS and posttranscriptional cis-acting regula-tory element (WPRE) as a part of the pWPXL vector togetherwith psPAX2 (Gag Pol rev and tat) and pMD2G (VSV-G)packaging vectors to produce G-glycoprotein pseudotypedretroviral particles

To address the question of the influence of cPPT (HIV)together with the CTS element on the foamy viral cycle wecompared the cell cycle requirements for efficient cell trans-duction by the pMD9- and pWPXL-based plasmids express-ing the same transgene (eGFP) under control of different pro-moters (SFFVU3 in pMD6 and EF1-120572 in pWPXL) in dividingand growth-arrested (G

1S) A549 cells Although there are

the previously published reports that have already focusedon the cell cycle aspects of retroviral infection [39 40]the investigation is the first direct attempt to assess the role ofcPPTmodifications for the two genera of viruses lentiviruses(HIV-1) and spumaviruses (PFV)

The analysis of the viral transduction rates revealed thatboth untreated or DMSO-treated cells experienced minor


0

50

100

150eG

FP ce

lls (

)

pWPX

L

pMD9

pMD9

-(w

oEn

v)

pcD

NA

pMD9

-cPP

T (H

IV)-

CTS

pMD9

-cPP

T (P

FV)-

CTS

pMD9

-cPP

T (H

IV)

pMD9

-CTS

(a)

0

50

100

150

pWPX

L

pMD9

pMD9

-(w

oEn

v)

pcD

NA

pMD9

-cPP

T (H

IV)-

CTS

pMD9

-cPP

T (P

FV)-

CTS

pMD9

-cPP

T (H

IV)

pMD9

-CTS

eGFP

cells

()

(b)

pWPX

L

pMD9

pMD9

-(w

oEn

v)

pcD

NA

pMD9

-cPP

T (H

IV)-

CTS

pMD9

-cPP

T (P

FV)-

CTS

pMD9

-cPP

T (H

IV)

PMD9

-CTS

0

50

150

100

eGFP

cells

()

(c)

Figure 4The summarized statistics of retroviral replication efficiency (transduction rate) in the A549 cells The pMD9-based plasmids weretested to efficiently transduce dividing and growth-arrested cells (G

1S phase of cell cycle)Three conditions were analysed in the experiment

control (a) DMSO (b) aphidicolin (c) The mean plusmn SD values from three independent assays are shown

decreases in transduction rates for the pMD9-based plasmidswith different cPPT modifications in comparison to pMD9and pWPXL controls (Figures 4(a) and 4(b)) These resultsindicated that the productive infection was observed only iftarget cells were allowed to pass throughmitosis On the otherhand there was a huge difference in the relative transductionefficiencies of the vectors when the cells were G

1S-arrested

for 24 hours after transduction while the efficiency ofpWPXL was approximately twenty- (pMD9-cPPT (HIV)-CTS) to fivefold (pMD9-CTS) higher than that of the pMD9-based vectors (Figure 4(c)) The short inserts such as CTS

and cPPT (HIV) also displayed an insignificant reduction inthe transduction rates observed in dividing cells The longerinsert (cPPT (HIV)-CTS) resulted in an almost 50 reduc-tion in transduction rates compared to the parental vectorcontrols (Figures 4(a) and 4(b)) One point to note is that thedifferent promoters directed the eGFP transgene expressionin the vectors In this regard it is always inaccurate tocompare the levels of transgene expression under control ofthe different promoters (SFFV U3 versus EF1-120572)

FV replication strategy differs in many aspects from thatof orthoretroviruses with the structural proteins displaying


Cellularlysates

Gag

Pol

pMD9

pMD9

-cPP

T

127

71

(kDa)

68

(HIV

)-CT

S

(a)

0

2000

4000

6000

8000

pMD9

pMD9

GagPol

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS

(b)

0

2000

4000

6000

8000

pMD9

pMD9

p71p68

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS

(c)Figure 5 Analysis of pMD9 parental vector and its modification (pMD9-cPPT (HIV)-CTS) Detection of PFV proteins in HEK 293T cells(a) contransfected with the retroviral vectors was measured quantitatively for GagPol (b) and Gag fractions (p71 p68) (c) The mean plusmn SDvalues from three independent assays are shown The area is measured in arbitrary units which are abbreviated as au

many unique functions not found for the correspondingorthoretroviral proteins The FV structural proteins GagPrt-RT and Env are initially translated in polyprotein formsthat are subsequently cleaved by cellular proteases (Gag andPrt-RT proteins) while Env polyprotein is cleaved by viralprotease [15] Interestingly the protein analysis of the pMD9-cPPT (HIV)-CTS plasmid compared to its parental formindicated that the A549 transduction for the modified vectorwas reduced when judged by the GagPol decrease in theHEK 293T cellular lysates (Figures 5(a) and 5(b)) Howeverthis was offset by a significant increase in the capacity to

encapsidate Gag and Pol producing a large pool of defectivevirus-like particles (Figures 6(a) and 6(b))

The organization of Gag matrix protein into MA (p17matrix) CA (capsid p24) and NC (p6 nucleocapsid) withdistinct cleavage sites observed in HIV-1 is absent in FVsThe only processing of FV Gag observed in the course of FVparticle morphogenesis in vivo occurs at the C terminus ofthemolecule removing a 3 kDa peptide (p3) and producing ashortened protein of 68 kDa However three internal sec-ondary cleavage sites have been characterized in vitro thatseem to be important during steps of the FV replication


Particlepreparation

Gag

Pol

pMD9

127

71

(kDa)

68pM

D9

-cPP

T(H

IV)-

CTS

(a)

0

5000

20000

pMD9

pMD9

GagPol

15000

10000

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS

(b)

0

5000

15000

pMD9

pMD9

p71p68

10000

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS

(c)Figure 6 Analysis of pMD9 parental vector and its modification (pMD9-cPPT (HIV)-CTS) Detection of PFV proteins in partially purifiedviral particles produced by HEK 293T cells (a) contransfected with the retroviral vectors was measured quantitatively for GagPol (b) andGag fractions (p71 p68) (c) The mean plusmn SD values from three independent assays are shown The area is measured in arbitrary units whichare abbreviated as au

cycle upon entry into target cells [15] Thereby in the 293Tcellular lysates the domination of the cleaved versions ofGag (p68) for either the pMD9 or pMD9-cPPT (HIV)-CTSconstructs (Figure 5(c)) was observed However the proteinanalysis of the particle preparation revealed the prevalenceof an uncleaved Gag fraction (p71) for pMD9 indicating theincomplete proteolytic cleavage of the FV capsid proteinThesame fractions were detected in equal amounts in partiallypurified particles produced by HEK 293T cells and cotrans-fected with the modified plasmid (Figure 6(c))

To assess the viral transduction dynamics and viral titrethe logistic dose-response function was transformed accord-ing to (1) and (2) to model the experimentally determined

efficiency of the retroviral replication as percentage of eGFPcells observed in the A549 cells Both parameters demon-strated a correlation to the aforementioned data shown inFigure 4 indicating some decrease of 119891(119909)td and 119891(119909)vt individing cells (Figures 7(a) 7(b) 8(a) and 8(b)) Similarly theFV inability to transduce at a significant extent the growth-arrested cells provided very low 119891(119909)td and 119891(119909)vt values(Figures 7(c) and 8(c))

Overall the results might be in conflict with a publishedreport claiming that simian FV vectors can efficiently trans-duce aphidicolin-treated cells [40] While the results cannotrule out strain-specific differences in the vector systems usedit is highly likely that simian FV vectors require mitosis for


0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

pWPXLpMD9pMD9-(woEnv)pcDNA

pMD9-cPPT (HIV)-CTSpMD9-cPPT (PFV)-CTSpMD9-cPPT (HIV)pMD9-CTS

eGFP

cells

()

(a)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

eGFP

cells

()

(b)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

Aphidicolin

eGFP

cells

()

(c)

Figure 7 Dynamic 119891(119909)td function logistic dose-response curve to predict andmodel viral infection cycle of retroviral replication efficiencyin the A549 cellsThe pMD9-based plasmids were tested to efficiently transduce dividing and growth-arrested cells (G

1S phase of cell cycle)

Three conditions were analysed in the experiment control (a) DMSO (b) aphidicolin (c)

their efficient transduction Moreover they require a break-down of the nuclear envelope for a successful nuclear entry[39 41] Lentiviral vectors also enter the nucleus using specificnuclear localization signals [8 42]

It is more complicated that after viral disassembly in acytoplasm some retroviral NLS sequences are recognized byhost-cell import factors such as transportins that mediatenuclear targeting of PICs via the nuclear pore complex[7] Several similar sequences are present in HIV-1 most

significantly in the virus integrase [8 43] Foamy viral Gagand Pol proteins also include NLS sequences [38] but theywere recently shown to be nonfunctional [44]

Like HIV-1 PIC formation is also present in FVs beforethe transport of double-stranded cDNAof the virus to the cel-lular chromatinTherefore it is an essential part of replicationof FVs [45] However the lower transduction efficiency of FVvectors in growth-arrested cells in comparisonwith lentiviralforms could be due to the fact that the foamy viral NLSs




20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(a)



20

40

60

80

100

eGFP

cells

()

0

Dilution10

minus110

minus210

minus310

minus410

minus510

minus610

minus7

(b)



20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(c)

Figure 8 Dynamic 119891(119909)vt function logistic dose-response curve to predict and model the viral titreThe pMD9-based vectors were tested toefficiently transduce dividing and growth-arrested cells (G

1S phase of cell cycle) Three conditions were analysed in the experiment control

(a) DMSO (b) aphidicolin (c)

are being partially occluded in PIC [46] showing that PFVgenerates 2-LTR circles in nondividing cells However foamyvirus vector preparations already contain 2-LTR circles sotheir presence is not useful for monitoring PFV DNA entryinto the nucleus although it is still possible that there arefurther functional blocks for successful PFV integration intoa host genome after the nuclear translocation of its DNA

In addition some retroviruses (HIV-1) have a tendencyto package some cellular proteins nonspecifically and DNAligase activity has been previously demonstrated for viralparticles [47] Therefore these steps might also take place in

intracellular or extracellular FV particles that contain cDNAmolecules Some studies even detected additional FVLTRcir-cles by PCR in aphidicolin-arrested but not serum-deprivedcultures [46]There is a possibility that FV vectors use distinctmechanisms for nuclear import as both the viral genome andGag proteins accumulate near the centrosomeMTOC andwait for cell division They then disassemble releasing PIC[46]

The presence of cPPT that serves as a primer for plus-strand synthesis to produce the HIV-1 ldquoflaprdquo element has notbeen reported so far for FVs But plus-strand synthesis that


ultimately leads to the double-stranded cDNA the molecularmoiety that actually integrates into the host chromatin is auniversal feature of all retroviruses including spumavirusesthe subfamily of FVs The mechanism of formation of dscDNA in FV might have a different mechanism

Taken all together the cell cycle dependences of foamyvirus infection have demonstrated that vectors based onthese viral genomes are not sufficient for specific targetingof nondividing cells Nonetheless the neurotropism of foamyviruses is worth testing in animal models Further studieswill be needed to directly compare foamy viral and lentiviralvectors in preclinical gene therapy experiments anddefine thenature of the stable FV vector transduction intermediate inquiescent cells

4 Conclusion

In the experiments modified foamy viral vectors showed areduction in transduction rates of dividing especially G

1S-

arrested A549 cells in comparison to lentiviral forms Thefindings have confirmed that mitosis is a critical phase in thecell cycle for FV transduction whichwas previously observedby Bieniasz and coauthors as an absence of the FV proteinexpression in G

1S- and G

2-arrested cells [22]

From the data it is clear that foamy viruses are not ableto infect nondividing cells efficiently most likely because oflower integration efficiency compared to lentiviruses unlesscells are undergoing mitosis The results are in accordancewith the previous findings of Trobridge and Russell butare contrary to the observations published by Mergia andcoauthors [32 40] Hence the current study leads to the factthat foamy viral vectors could be further improved to beeffective in gene therapy to target nondividing cells

It is shown how foamy viral vectors can infect the A549cells in dividing and growth-arrested states cell-type specificexperiments and molecular cloning strategies were includedto clearly demonstrate that HIV-1 cPPT cannot be substitutedfor PFV cPPT without loss of functionality This might bebecause the cognate reverse transcription enzyme is requiredfor recognition of cPPT and CTS sequences Furthermorevalidated simulation data and curves were provided for thedynamic FV replication potential

Conflict of Interests

Theauthors do not have any conflict of interests regarding thepublication of this paper

Acknowledgments

Special thanks are extended to Professor George Vassilopou-los from the University of Thessaly Medical School DrJayashree S Nandi from the Albert Einstein College ofMedicine and Anna Poon from the City College of New Yorkfor their assistance in the paperrsquos writingThe authors are alsograteful to Drs Otto Erlwein and Nathan Sweeney from theImperial College of Science Technology and Medicine fortheir valuable suggestions They also thank Interdisziplinares

Zentrum fur Klinische Forschung (IZKF) for funding Somedata from this study were used in the doctoral thesis titledldquoMolecular modelling and simulation of retroviral proteinsand nanobiocompositesrdquo by Sergey Shityakov This publica-tion was financially supported by Deutsche Forschungsge-meinschaft (DFG) and the University of Wurzburg in thefunding program for Open Access Publishing

References

[1] P Charneau and F Clavel ldquoA single-stranded gap in humanimmunodeficiency virus unintegrated linear DNA defined bya central copy of the polypurine tractrdquo Journal of Virology vol65 no 5 pp 2415ndash2421 1991

[2] P Charneau M Alizon and F Clavel ldquoA second origin of DNAplus-strand synthesis is required for optimal human immunod-eficiency virus replicationrdquo Journal of Virology vol 66 no 5 pp2814ndash2820 1992

[3] P Charneau G Mirambeau P Roux S Paulous H Buc and FClavel ldquoHIV-1 reverse transcription A termination step at thecenter of the genomerdquo Journal of Molecular Biology vol 241 no5 pp 651ndash662 1994

[4] M Lavigne P Roux H Buc and F Schaeffer ldquoDNA curvaturecontrols termination of plus strand DNA synthesis at the centreof HIV-1 genomerdquo Journal of Molecular Biology vol 266 no 3pp 507ndash524 1997

[5] B Bowerman P O Brown J M Bishop and H E VarmusldquoA nucleoprotein complex mediates the integration of retroviralDNArdquo Genes amp Development vol 3 no 4 pp 469ndash478 1989

[6] P O Brown B Bowerman H E Varmus and J M BishopldquoRetroviral integration structure of the initial covalent productand its precursor and a role for the viral IN proteinrdquoProceedingsof the National Academy of Sciences of the United States ofAmerica vol 86 no 8 pp 2525ndash2529 1989

[7] S Shityakov A Rethwilm and T Dandekar ldquoStructural anddocking analysis of HIV-1 integrase and transportin-SR2 inter-action is this a more general and specific route for retroviralnuclear import and its regulationrdquo Online Journal of Bioinfor-matics vol 11 no 1 pp 19ndash33 2010

[8] M I Bukrinsky S Haggerty M P Dempsey et al ldquoA nuclearlocalization signal within HIV-1 matrix protein that governsinfection of non-dividing cellsrdquo Nature vol 365 no 6447 pp666ndash669 1993

[9] U Von Schwedler R S Kornbluth and D Trono ldquoThe nuclearlocalization signal of the matrix protein of human immunod-eficiency virus type 1 allows the establishment of infection inmacrophages and quiescent T lymphocytesrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 91 no 15 pp 6992ndash6996 1994

[10] J J Hooks and C J Gibbs Jr ldquoThe foamy virusesrdquo Bacteriolog-ical Reviews vol 39 no 3 pp 169ndash185 1975

[11] P Brown G Nemo and D C Gajdusek ldquoHuman foamy virusfurther characterization seroepidemiology and relationship tochimpanzee foamy virusesrdquo Journal of Infectious Diseases vol137 no 4 pp 421ndash427 1978

[12] G J Nemo P W Brown C J Gibbs Jr and D C GajdusekldquoAntigenic relationship of human foamy virus to the simianfoamy virusesrdquo Infection and Immunity vol 20 no 1 pp 69ndash721978

[13] O Herchenroder R Renne D Loncar et al ldquoIsolation cloningand sequencing of simian foamy viruses from chimpanzees


(SFVcpz) high homology to human foamy virus (HFV)rdquoVirology vol 201 no 2 pp 187ndash199 1994

[14] M Schweizer and D Neumann-Haefelin ldquoPhylogenetic analy-sis of primate foamy viruses by comparison of pol sequencesrdquoVirology vol 207 no 2 pp 577ndash582 1995

[15] O Delelis J Lehmann-Che and A Saıb ldquoFoamy virusesmdashaworld apartrdquo Current Opinion in Microbiology vol 7 no 4 pp400ndash406 2004

[16] J A Mikovits P M Hoffman A Rethwilm and F W RuscettildquoIn vitro infection of primary and retrovirus-infected humanleukocytes by human foamy virusrdquo Journal of Virology vol 70no 5 pp 2774ndash2780 1996

[17] K Stirnnagel D Luftenegger A Stange et al ldquoAnalysis of pro-totype foamy virus particle-host cell interaction with autofluo-rescent retroviral particlesrdquoRetrovirology vol 7 article 45 2010

[18] C D Meiering and M L Linial ldquoHistorical perspective offoamy virus epidemiology and infectionrdquo Clinical MicrobiologyReviews vol 14 no 1 pp 165ndash176 2001

[19] H E Huber andC C Richardson ldquoProcessing of the primer forplus strand DNA synthesis by human immunodeficiency virus1 reverse transcriptaserdquoThe Journal of Biological Chemistry vol265 no 18 pp 10565ndash10573 1990

[20] G Luo L Sharmeen and J Taylor ldquoSpecificities involved in theinitiation of retroviral plus-strand DNArdquo Journal of Virologyvol 64 no 2 pp 592ndash597 1990

[21] K A Pullen A J Rattray and J J Champoux ldquoThe sequencefeatures important for plus strand priming by human immun-odeficiency virus type 1 reverse transcriptaserdquo The Journal ofBiological Chemistry vol 268 no 9 pp 6221ndash6227 1993

[22] P D Bieniasz O Erlwein A Aguzzi A Rethwilm and MO McClure ldquoGene transfer using replication-defective humanfoamy virus vectorsrdquo Virology vol 235 no 1 pp 65ndash72 1997

[23] M Heinkelein M Dressler G Jarmy et al ldquoImproved primatefoamy virus vectors and packaging constructsrdquo Journal ofVirology vol 76 no 8 pp 3774ndash3783 2002

[24] D Lindemann and A Rethwilm ldquoFoamy virus biology and itsapplication for vector developmentrdquo Viruses vol 3 no 5 pp561ndash585 2011

[25] M Linial ldquoWhy arenrsquot foamy viruses pathogenicrdquo Trends inMicrobiology vol 8 no 6 pp 284ndash289 2000

[26] A Mergia N J Leung and J Blackwell ldquoCell tropism ofthe simian foamy virus type 1 (SFV-1)rdquo Journal of MedicalPrimatology vol 25 no 1 pp 2ndash7 1996

[27] DW Russell and A D Miller ldquoFoamy virus vectorsrdquo Journal ofVirology vol 70 no 1 pp 217ndash222 1996

[28] C L Hill P D Bieniasz and M O McClure ldquoProperties ofhuman foamy virus relevant to its development as a vector forgene therapyrdquo Journal of General Virology vol 80 no 8 pp2003ndash2009 1999

[29] G Vassilopoulos G Trobridge N C Josephson and D WRussell ldquoGene transfer into murine hematopoietic stem cellswith helper-free foamy virus vectorsrdquo Blood vol 98 no 3 pp604ndash609 2001

[30] G Trobridge N Josephson G Vassilopoulos J Mac and DWRussell ldquoImproved foamy virus vectors with minimal viralsequencesrdquoMolecular Therapy vol 6 no 3 pp 321ndash328 2002

[31] G D Trobridge and D W Russell ldquoHelper-free foamy virusvectorsrdquo Human Gene Therapy vol 9 no 17 pp 2517ndash25251998

[32] G Trobridge and D W Russell ldquoCell cycle requirements fortransduction by foamy virus vectors compared to those of

oncovirus and lentivirus vectorsrdquo Journal of Virology vol 78no 5 pp 2327ndash2335 2004

[33] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory New York NY USA2001

[34] R B DuBridge P Tang andH CHsia ldquoAnalysis ofmutation inhuman cells by using an Epstein-Barr virus shuttle systemrdquoMolecular and Cellular Biology vol 7 no 1 pp 379ndash387 1987

[35] A Stange I Mannigel K Peters et al ldquoCharacterization ofprototype foamy virus Gag late assembly domain motifs andtheir role in particle egress and infectivityrdquo Journal of Virologyvol 79 no 9 pp 5466ndash5476 2005

[36] KGartner TWiktorowicz J ParkAMergia A Rethwilm andC Scheller ldquoAccuracy estimation of foamy virus genome copy-ingrdquo Retrovirology vol 6 article 32 2009

[37] K Peters T Wiktorowicz M Heinkelein and A RethwilmldquoRNA and protein requirements for incorporation of the Polprotein into foamy virus particlesrdquo Journal of Virology vol 79no 11 pp 7005ndash7013 2005

[38] H Imrich M Heinkelein O Herchenroder and A RethwilmldquoPrimate foamy virus Pol proteins are imported into thenucleusrdquo Journal of General Virology vol 81 no 12 pp 2941ndash2947 2000

[39] T Roe T C Reynolds G Yu and P O Brown ldquoIntegration ofmurine leukemia virus DNA depends on mitosisrdquo The EMBOJournal vol 12 no 5 pp 2099ndash2108 1993

[40] A Mergia S Chari D L Kolson M M Goodenow and TCiccarone ldquoThe efficiency of simian foamy virus vector type-1(SFV-1) in nondividing cells and in human PBLsrdquo Virology vol280 no 2 pp 243ndash252 2001

[41] D Lindemann I Steffen and S Pohlmann ldquoCellular entry ofretrovirusesrdquo in Viral Entry into Host Cells vol 790 of Advancesin Experimental Medicine and Biology pp 128ndash149 2013

[42] S PopovM Rexach G Zybarth et al ldquoViral protein R regulatesnuclear import of the HIV-1 pre-integration complexrdquo TheEMBO Journal vol 17 no 4 pp 909ndash917 1998

[43] M Bouyac-Bertoia J D Dvorin R AM Fouchier et al ldquoHIV-1 infection requires a functional integrase NLSrdquoMolecular Cellvol 7 no 5 pp 1025ndash1035 2001

[44] E Mullers K Stirnnagel S Kaulfuss and D Lindemann ldquoPro-totype foamy virus gag nuclear localization a novel pathwayamong retrovirusesrdquo Journal of Virology vol 85 no 18 pp9276ndash9285 2011

[45] U Berka M V Hamann and D Lindemann ldquoEarly eventsin foamy virus-host interaction and intracellular traffickingrdquoViruses vol 5 no 4 pp 1055ndash1074 2013

[46] A Saıb F Puvion-DutilleulM Schmid J Peries andH deTheldquoNuclear targeting of incoming human foamy virus Gag pro-teins involves a centriolar steprdquo Journal of Virology vol 71 no2 pp 1155ndash1161 1997

[47] H M Temin and D Baltimore ldquoRNA-directed DNA synthesisand RNA tumor virusesrdquoAdvances in Virus Research vol 17 noC pp 129ndash186 1972


Flap

cPPT CTS

(a)

Gap

cPPT

(b)

Figure 1 Different reverse transcription outcomes in lenti- (a) and foamy viruses (b) cPPT and CTS abbreviations stand for the centralpolypurine tract and central termination sequence respectively

a number of mammalians such as non-human primates catsand cows [10]

The genomic organization of the FVs including theprototype foamy virus (PFV) which is a molecular clonedsimian foamy virus SFVcpz(hu) is similar to that of othercomplex retroviruses with several additional open readingframes located in 31015840 of the canonical Gag Pol and Env genesincluding the transcriptional transactivator gene (tas) [11ndash15]

The foamy viruses have a very broad host range and infecta variety of cell lines including fibroblastoid epithelioid andlymphatic lineages [16] There are currently a few cell linesthat are resistant to FV infection such as the zebrafish PAC2and human erythroid precursor cell lines [17] FV infectionleads to massive cell death via apoptosis in vitro and withoutany overt pathogenic effect in vivo [18]

Essentially foamy viruses seem to have diverse outcomesfor the plus-strand priming events in contrast to lentivirusesIn FV reverse transcription the strong stop plus-strand DNAis displaced by DNA elongating from cPPT This DNA canfurther be transferred to the 31015840 end of the minus-strand DNAor serve as a template for synthesis of a double-stranded LTRConsequently cPPT is degraded after completion of theminus-strand cDNA synthesis producing a single-strandedgap (Figure 1(b)) [19ndash21]

There are a number of FV-based vectors for gene therapythat have been developed [22ndash24]These constructs have sev-eral desirable properties in comparison to lentiviral vectorsThe main characteristics of foamy viral vectors are (i) safetyFVs have not been linked to any established pathology [1825] (ii) wide tissue tropism [10 26ndash28] (iii) viral particlesstable and resilient to ultracentrifugation [29] (iv) packagingefficacy for foreign DNA that is at least 9 kb and offers avaluable tool for the transfer of long genetic elements andtherapeutic transgenes [30] (v) potential for production ofhigh-titre FV vectors suitable for ex vivo gene therapyapplications [24 30 31]

However these very promising foamy viral vectors haveone limitation in their ability to efficiently transduce ter-minally differentiated and growth-arrested mammalian cells[32] potentially because of their mitosis-dependent integra-tion [22] and the deficiency in forming PICs with nuclearimport activity from the absence of the lentivaral cPPTelement and central termination sequence

Therefore the role of HIV-1 cPPT on FV replication andits mechanism of action is still to be determined To pursue

this goal the transduction rates of cPPT (HIV-)modifiedFV vectors in dividing and growth-arrested adenocarcinomichuman alveolar basal epithelial (A549) cells were accessedalong the investigation of underlying molecular mechanismsand evaluation of their potential for FV-based gene therapy

2 Materials and Methods

Recombinant DNA techniques standard molecular cloningtechniques were used for the generation of constructs [33] Aseries of plasmid cloning vectors were preceded by the letterldquoprdquo In brief the pUC19-based intermediate was first gener-ated by ligating a 1259 kb EcoRI-KpnI fragment from pMD9(9940 kb) with a 2674 kb EcoRI-KpnI fragment from thepUC19 vector to use the proper restriction sites Then fourpUC19-based plasmids such as pUC19-cPPT (HIV)-CTSpUC19-cPPT (PFV)-CTS pUC19-cPPT (HIV) and pUC19-CTS were generated by PCR amplification and insertion ofthe fragments bearing the viral structural elements (Table 1)using relevant oligonucleotides (Table 2) with MfeI-StuIrestriction sites The lentiviral pWPXL vector (10510 kb) wasused as a template for the cPPT (HIV) cPPT (HIV)-CTSand CTS amplifications All pMD9-based FV vectors con-tained the gene encoding enhanced green fluorescent protein(eGFP) under the control of a constitutively active heterolo-gous retroviral U3 promoter to enable the quantification ofvector transfer rates (Figure 2)

Cell transfection and purification of supernatant humanembryonic kidney (HEK 293T) cells [34] were seeded in 2 times106 density into 6 cm dishes a day before the transfectionThe cells were transfected with plasmid DNA using a poly-ethyleneimine transfection reagent (Polysciences EuropeGmbH Eppelheim Germany) [35] The PFV transfectionmixture contained 25120583g of modified pMD9 vectors 125 120583gof pCIgag2 05120583g of pCpol-2 and pCenv-1 packaging plas-mids [35] The HIV transfection system contained 25120583g ofpWPXL 19 120583g of psPAX2 and 08 120583g of pMD2G (VSV-G)vectors (gifts from Professor Didier Trono) respectively ThepMD9 plasmid without Env (pMD9-woEnv) and pcDNA(Invitrogen GmbH Karlsruhe Germany) as an empty vectorwere used as negative controls without a production of viralparticles One day after transfection the cellular transcriptionwas induced by addition of 10mM sodium butyrate for8 hrs [36] After two days the supernatant was harvestedpassed through a 045120583mfilter (MerckMillipore Darmstadt





Fragments Primers











1S phase of a cell







119891(119909)td =001119886

001 + 119890(minus119887119909) (1)

119891(119909)vt =119886

001 + 119890119909 (2)





LTR RRE

CMV RU5CASI CASII

cPPT-CTS

CTS

EF1-120572

SFFV eGFP

U3

pMD9

pWPXL

cPPT-CTS

cPPT

cPPT

pMD9

pMD9

pMD9

pMD9

cPPT-CTS eGFP

ΔU3


Control

DMSO

Aphidicolin

Add virus 0hrs

OH

HO

CH3

OH

OH


CH3










0

50

100

150eG

FP ce

lls (

)

pWPX

L

pMD9

pMD9

-(w

oEn

v)

pcD

NA

pMD9

-cPP

T (H

IV)-

CTS

pMD9

-cPP

T (P

FV)-

CTS

pMD9

-cPP

T (H

IV)

pMD9

-CTS

(a)

0

50

100

150

pWPX

L

pMD9

pMD9

-(w

oEn

v)

pcD

NA

pMD9

-cPP

T (H

IV)-

CTS

pMD9

-cPP

T (P

FV)-

CTS

pMD9

-cPP

T (H

IV)

pMD9

-CTS

eGFP

cells

()

(b)

pWPX

L

pMD9

pMD9

-(w

oEn

v)

pcD

NA

pMD9

-cPP

T (H

IV)-

CTS

pMD9

-cPP

T (P

FV)-

CTS

pMD9

-cPP

T (H

IV)

PMD9

-CTS

0

50

150

100

eGFP

cells

()

(c)





1S-arrested





Cellularlysates

Gag

Pol

pMD9

pMD9

-cPP

T

127

71

(kDa)

68

(HIV

)-CT

S

(a)

0

2000

4000

6000

8000

pMD9

pMD9

GagPol

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS

(b)

0

2000

4000

6000

8000

pMD9

pMD9

p71p68

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS






Particlepreparation

Gag

Pol

pMD9

127

71

(kDa)

68pM

D9

-cPP

T(H

IV)-

CTS

(a)

0

5000

20000

pMD9

pMD9

GagPol

15000

10000

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS

(b)

0

5000

15000

pMD9

pMD9

p71p68

10000

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS







0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)



eGFP

cells

()

(a)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

eGFP

cells

()

(b)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

Aphidicolin

eGFP

cells

()

(c)











20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(a)



20

40

60

80

100

eGFP

cells

()

0

Dilution10

minus110

minus210

minus310

minus410

minus510

minus610

minus7

(b)



20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(c)











4 Conclusion


1S-


1S- and G






Acknowledgments



References























































Fragments Primers











1S phase of a cell







119891(119909)td =001119886

001 + 119890(minus119887119909) (1)

119891(119909)vt =119886

001 + 119890119909 (2)





LTR RRE

CMV RU5CASI CASII

cPPT-CTS

CTS

EF1-120572

SFFV eGFP

U3

pMD9

pWPXL

cPPT-CTS

cPPT

cPPT

pMD9

pMD9

pMD9

pMD9

cPPT-CTS eGFP

ΔU3


Control

DMSO

Aphidicolin

Add virus 0hrs

OH

HO

CH3

OH

OH


CH3










0

50

100

150eG

FP ce

lls (

)

pWPX

L

pMD9

pMD9

-(w

oEn

v)

pcD

NA

pMD9

-cPP

T (H

IV)-

CTS

pMD9

-cPP

T (P

FV)-

CTS

pMD9

-cPP

T (H

IV)

pMD9

-CTS

(a)

0

50

100

150

pWPX

L

pMD9

pMD9

-(w

oEn

v)

pcD

NA

pMD9

-cPP

T (H

IV)-

CTS

pMD9

-cPP

T (P

FV)-

CTS

pMD9

-cPP

T (H

IV)

pMD9

-CTS

eGFP

cells

()

(b)

pWPX

L

pMD9

pMD9

-(w

oEn

v)

pcD

NA

pMD9

-cPP

T (H

IV)-

CTS

pMD9

-cPP

T (P

FV)-

CTS

pMD9

-cPP

T (H

IV)

PMD9

-CTS

0

50

150

100

eGFP

cells

()

(c)





1S-arrested





Cellularlysates

Gag

Pol

pMD9

pMD9

-cPP

T

127

71

(kDa)

68

(HIV

)-CT

S

(a)

0

2000

4000

6000

8000

pMD9

pMD9

GagPol

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS

(b)

0

2000

4000

6000

8000

pMD9

pMD9

p71p68

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS






Particlepreparation

Gag

Pol

pMD9

127

71

(kDa)

68pM

D9

-cPP

T(H

IV)-

CTS

(a)

0

5000

20000

pMD9

pMD9

GagPol

15000

10000

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS

(b)

0

5000

15000

pMD9

pMD9

p71p68

10000

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS







0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)



eGFP

cells

()

(a)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

eGFP

cells

()

(b)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

Aphidicolin

eGFP

cells

()

(c)











20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(a)



20

40

60

80

100

eGFP

cells

()

0

Dilution10

minus110

minus210

minus310

minus410

minus510

minus610

minus7

(b)



20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(c)











4 Conclusion


1S-


1S- and G






Acknowledgments



References




















































LTR RRE

CMV RU5CASI CASII

cPPT-CTS

CTS

EF1-120572

SFFV eGFP

U3

pMD9

pWPXL

cPPT-CTS

cPPT

cPPT

pMD9

pMD9

pMD9

pMD9

cPPT-CTS eGFP

ΔU3


Control

DMSO

Aphidicolin

Add virus 0hrs

OH

HO

CH3

OH

OH


CH3










0

50

100

150eG

FP ce

lls (

)

pWPX

L

pMD9

pMD9

-(w

oEn

v)

pcD

NA

pMD9

-cPP

T (H

IV)-

CTS

pMD9

-cPP

T (P

FV)-

CTS

pMD9

-cPP

T (H

IV)

pMD9

-CTS

(a)

0

50

100

150

pWPX

L

pMD9

pMD9

-(w

oEn

v)

pcD

NA

pMD9

-cPP

T (H

IV)-

CTS

pMD9

-cPP

T (P

FV)-

CTS

pMD9

-cPP

T (H

IV)

pMD9

-CTS

eGFP

cells

()

(b)

pWPX

L

pMD9

pMD9

-(w

oEn

v)

pcD

NA

pMD9

-cPP

T (H

IV)-

CTS

pMD9

-cPP

T (P

FV)-

CTS

pMD9

-cPP

T (H

IV)

PMD9

-CTS

0

50

150

100

eGFP

cells

()

(c)





1S-arrested





Cellularlysates

Gag

Pol

pMD9

pMD9

-cPP

T

127

71

(kDa)

68

(HIV

)-CT

S

(a)

0

2000

4000

6000

8000

pMD9

pMD9

GagPol

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS

(b)

0

2000

4000

6000

8000

pMD9

pMD9

p71p68

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS






Particlepreparation

Gag

Pol

pMD9

127

71

(kDa)

68pM

D9

-cPP

T(H

IV)-

CTS

(a)

0

5000

20000

pMD9

pMD9

GagPol

15000

10000

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS

(b)

0

5000

15000

pMD9

pMD9

p71p68

10000

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS







0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)



eGFP

cells

()

(a)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

eGFP

cells

()

(b)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

Aphidicolin

eGFP

cells

()

(c)











20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(a)



20

40

60

80

100

eGFP

cells

()

0

Dilution10

minus110

minus210

minus310

minus410

minus510

minus610

minus7

(b)



20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(c)











4 Conclusion


1S-


1S- and G






Acknowledgments



References




















































0

50

100

150eG

FP ce

lls (

)

pWPX

L

pMD9

pMD9

-(w

oEn

v)

pcD

NA

pMD9

-cPP

T (H

IV)-

CTS

pMD9

-cPP

T (P

FV)-

CTS

pMD9

-cPP

T (H

IV)

pMD9

-CTS

(a)

0

50

100

150

pWPX

L

pMD9

pMD9

-(w

oEn

v)

pcD

NA

pMD9

-cPP

T (H

IV)-

CTS

pMD9

-cPP

T (P

FV)-

CTS

pMD9

-cPP

T (H

IV)

pMD9

-CTS

eGFP

cells

()

(b)

pWPX

L

pMD9

pMD9

-(w

oEn

v)

pcD

NA

pMD9

-cPP

T (H

IV)-

CTS

pMD9

-cPP

T (P

FV)-

CTS

pMD9

-cPP

T (H

IV)

PMD9

-CTS

0

50

150

100

eGFP

cells

()

(c)





1S-arrested





Cellularlysates

Gag

Pol

pMD9

pMD9

-cPP

T

127

71

(kDa)

68

(HIV

)-CT

S

(a)

0

2000

4000

6000

8000

pMD9

pMD9

GagPol

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS

(b)

0

2000

4000

6000

8000

pMD9

pMD9

p71p68

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS






Particlepreparation

Gag

Pol

pMD9

127

71

(kDa)

68pM

D9

-cPP

T(H

IV)-

CTS

(a)

0

5000

20000

pMD9

pMD9

GagPol

15000

10000

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS

(b)

0

5000

15000

pMD9

pMD9

p71p68

10000

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS







0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)



eGFP

cells

()

(a)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

eGFP

cells

()

(b)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

Aphidicolin

eGFP

cells

()

(c)











20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(a)



20

40

60

80

100

eGFP

cells

()

0

Dilution10

minus110

minus210

minus310

minus410

minus510

minus610

minus7

(b)



20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(c)











4 Conclusion


1S-


1S- and G






Acknowledgments



References




















































Cellularlysates

Gag

Pol

pMD9

pMD9

-cPP

T

127

71

(kDa)

68

(HIV

)-CT

S

(a)

0

2000

4000

6000

8000

pMD9

pMD9

GagPol

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS

(b)

0

2000

4000

6000

8000

pMD9

pMD9

p71p68

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS






Particlepreparation

Gag

Pol

pMD9

127

71

(kDa)

68pM

D9

-cPP

T(H

IV)-

CTS

(a)

0

5000

20000

pMD9

pMD9

GagPol

15000

10000

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS

(b)

0

5000

15000

pMD9

pMD9

p71p68

10000

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS







0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)



eGFP

cells

()

(a)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

eGFP

cells

()

(b)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

Aphidicolin

eGFP

cells

()

(c)











20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(a)



20

40

60

80

100

eGFP

cells

()

0

Dilution10

minus110

minus210

minus310

minus410

minus510

minus610

minus7

(b)



20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(c)











4 Conclusion


1S-


1S- and G






Acknowledgments



References




















































Particlepreparation

Gag

Pol

pMD9

127

71

(kDa)

68pM

D9

-cPP

T(H

IV)-

CTS

(a)

0

5000

20000

pMD9

pMD9

GagPol

15000

10000

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS

(b)

0

5000

15000

pMD9

pMD9

p71p68

10000

Are

a (au

)

pMD9

-cPP

T(H

IV)-

CTS

pMD9

-cPP

T(H

IV)-

CTS







0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)



eGFP

cells

()

(a)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

eGFP

cells

()

(b)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

Aphidicolin

eGFP

cells

()

(c)











20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(a)



20

40

60

80

100

eGFP

cells

()

0

Dilution10

minus110

minus210

minus310

minus410

minus510

minus610

minus7

(b)



20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(c)











4 Conclusion


1S-


1S- and G






Acknowledgments



References




















































0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)



eGFP

cells

()

(a)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

eGFP

cells

()

(b)



0

20

40

60

80

100

0 8 16 24 32 40 48

Time (hrs)

Aphidicolin

eGFP

cells

()

(c)











20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(a)



20

40

60

80

100

eGFP

cells

()

0

Dilution10

minus110

minus210

minus310

minus410

minus510

minus610

minus7

(b)



20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(c)











4 Conclusion


1S-


1S- and G






Acknowledgments



References






















































20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(a)



20

40

60

80

100

eGFP

cells

()

0

Dilution10

minus110

minus210

minus310

minus410

minus510

minus610

minus7

(b)



20

40

60

80

100

0

Dilution

eGFP

cells

()

10minus1

10minus2

10minus3

10minus4

10minus5

10minus6

10minus7

(c)











4 Conclusion


1S-


1S- and G






Acknowledgments



References






















































4 Conclusion


1S-


1S- and G






Acknowledgments



References



















































evaluation and prediction of the hiv-1 central polypurine ... · researcharticle evaluation and...

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