Involvement of human Topoisomerase II isoforms in strand transfer events of HIV-1

reverse transcriptionLokeswara Balakrishna S and Anand K. Kondapi

Department of Biotechnology, School of Life Sciences, University of Hyderabad Hyderabad 500 046, India.

Involvement of human Topoisomerase II isoforms in strand transfer events of HIV-1

reverse transcriptionLokeswara Balakrishna S and Anand K. Kondapi

Department of Biotechnology, School of Life Sciences, University of Hyderabad Hyderabad 500 046, India.

HIV-1 infected SupT1 cells were collected at different time points & used for Topo II isoforms expression analysis.

Topo II isoforms were down regulated using siRNA in SupT1 cells & were infected with HIV-193IN101 for 8 h. A part was used for Topo II expression analysis.

Virus replication was measured in culture supernatant after 96 h by quantifying by p24 ELISA.

Total RNA was isolated from above cells & used for transcript analysis using primers specific to Gag, Pol & Env of HIV-1.

Genomic DNA was isolated to study viral integration using SK38/39 primers.

RTn intermediates were analyzed in 1 h HIV-1 infected cells with primers specific RTn events.

GFP siRNA treated cells were used as internal control, HIV-1 infected cells as +ve control, pNL4-3 used as PCR +ve control & no template used as -ve control.

Co-localization analysis of Topo IIα & β with HIV-1 RT at different time points after HIV-1 infection are performed using different fluorescent tags.

Infection was conducted in the presence of p32 labelled inorganic phosphate & phosphorylation of Topo IIβ was analyzed by immunoprecipitation of Topo IIβ.

Effect of pyridine derivatives on Topo IIβ kinase was analyzed by in vitro phosphorylation of Topo IIβ. Topo IIβ, Topo IIβ kinase & hot ATP32 were incubated in presence or absence of drug. TCA precipitated mixture was dried on filter paper & radioactivity was measured in liquid scintillation counter.

Effect of kinase inhibitors on HIV-1 replication was analyzed collecting culture supernatants on 4th day of infection for viral quantity by p24 ELISA.

Cytosolic DNA was analyzed for effect of kinase inhibitors on HIV-1 RTn events using primers specific to each event by adding them to cells 1 h prior to infection.

Fig.1. A) Expression of Topo II isoforms in HIV-1 infected cells showed Topo IIα up-regulation at 4 h & Topo IIβ is up-regulated at 1 & 4 h. B) Topo II siRNAs efficiently down-regulated Topo II isoforms levels . C) Viral replication impaired in Topo II isoforms down regulated cells when analyzed by p24 ELISA at 4th day.

Fig.3Reverse transcription (RTn) is crucial step in HIV-1 life cycle. In cytoplasm viral genomic RNA is reverse transcribed to double strand DNA (dsDNA).

The dsDNA translocates into nucleus & integrates into host genome. In each stage of HIV-1 life cycle, many viral & cellular proteins are involved.

Topoisomerase II is present as two isoforms Topo IIα (~170 kDa) & Topo IIβ (~180 kDa), & have role in different viral infections: Simian Virus 40 infection in TC7 cells & HCMV infection in HEL cells.

Reports from our laboratory showed HIV-1 replicative cycle can be blocked by Topo II inhibitors.

Present study explores the role of Topo II isoforms in early events of HIV-1 infection. We report distinct expression profile of Topo II isoforms in early hours of HIV-1 infection & evaluated ability of Topo II deficient cells in HIV-1 replication.

RTn events in Topo II and/or deficient cells showed affect on first strand transfer and other downstream events through interaction with RT.

In early hours of infection Topo II undergoes phosphorylation by Topo II kinase.

Pyridine derivatives were synthesized & screened for their action on Topo IIβ kinase activity which inhibited Topo IIβ phosphorylation & HIV-1 replication.

Presented at Towards an HIV Cure Symposium and IAS 2013 Conference –Kuala Lumpur, Malaysia

Fig.3. Topo II knockdown effected HIV-1 RTn intermediates after SSDNA synthesis. Strong stop (SS), first strand transfer (FST), full length minus strand (FLMS) and second strand transfer (SST).

Fig.8. Kinase inhibitors effected RTn intermediate formation i.e., SS, FST & FLMS. DMSO used as vehicle, Uninfected cells as -ve control, infected cells as +ve control, AZT as internal control. No template as PCR –ve control and pNL4-3 used as PCR +ve control.

Results provide evidence that Topo II isoforms were up-regulated & physically interacted with HIV-1 RT. Topo II isoforms are required for HIV-1 RTn & are involved in strand transfer events resulting in complete viral dsDNA synthesis.

To complete RTn process, phosphorylation of Topo IIβ by Topo IIβ kinase is essential. Our studies on inhibition of Topo IIβ kinase affected the reverse transcription. This signifies the role of phosphorylated Topo IIβ in promoting RTn.

Further studies on the mechanism through which Topo II isoforms regulate the viral dsDNA synthesis can be probed.

Isel, C., Ehresmann, C., and Marquet, R. 2010. Initiation of HIV reverse transcription, Viruses 2:213-243.

Drake, F.H., Zimmerman, J.P., McCabe, F.L., Bartus, H.F., Per, S.R., Sullivan, D., Ross, M.W.E., Mattern, M.R., Johnson, R.K., Crooke, S.T., Mirabelli, C.K. 1987. Purification of topoisomerase II from amsacrine-resistant P388 leukemia cells. Evidence for two forms of the enzyme. Journal of Biological Chemistry 262:16739-47.

Rainwater, R., Mann, K. 1990. Differential increase in topoisomerase II in simian virus 40-infected cells. Journal of Virology 64:918-921.

Kondapi, A.K., Padmaja, G., Satyanarayana, N., Mukhopadyaya, R. M. S., Reitz, A. 2005 Biochemical analysis of topoisomerase IIα and β kinase activity found in HIV-1 infected cells and virus. Archives of Biochemistry and Biophysics 441:41-55.

Kondapi, A.K., Satyanarayana, N., Saikrishna, A.D. 2006 A study of the topoisomerase II activity in HIV-1 replication using the ferrocene derivatives as probes. Archives of Biochemistry and Biophysics 450:23-132.

Grants-in-Aid from Department of Science and Technology (DST), India to AKK supported this work completely. Infrastructure for this work is provided under University Grants Commission (UGC)-XI plan and Department of Biotechnology (DBT)-Centre for Research and Education in Biology and Biotechnology (CREBB) program of University of Hyderabad (UoH). SLB acknowledges CSIR for PhD fellowship.

RESULTS

INTRODUCTION

CONCLUSIONS

Fig.1

0 1 2 4 6 8

HIV-1 Infection

Topo IIα ~ 170 kDa

Topo IIβ ~180 kDa

β-actin ~43 kDa

Time in hours

Topo IIα+β

Topo IIαTopo IIβ

GFPCells +

HIV1

Topo IIα ~170 kDa

Topo IIβ ~180 kDa

β- actin ~43 kDa

B)

A)

C)

siRNA

+ -- -- +

-- + -- +

+ + -- +

-- -- + +

-- -- -- --

-- -- -- +

siRNA

GFP

Topo

IIβ

Topo

IIα

HIV

-1

SS FLMS

FST

SST

Mt D

NA

+ ve

Con

trol

– ve

Con

trol

AZT

Heat inactivated virus

Fig.4 Co-localization of Topo II isoforms with HIV-

1 RTEffect of Topo II knockdown on HIV-1 RTn intermediates

Autoradiogram

Topo IIβ

pTopo IIβ

Control

Infec

ted

Marker

94 113 117

+ve Contro

l

157 PCR +ve Control

AZT

114 151

DMSO

-ve C

ontrol

PCR -ve Control

Effect of Topo IIβ Kinase inhibitors on RTn intermediates

Western Blot

Fig.5 Analysis of in vitro phosphorylation of Topo IIβ by Topo IIβ kinase during HIV-1 infection.

Fig.5

Fig.6

Fig.7

Fig.8

Fig.6. A&B) Analysis of inhibition of Topo IIβ phosphorylation by different pyridine derivatives showed higher activity in a dose dependent manner.

Fig.7. A& B) Molecules 94, 113, 114,117, 151 & 157 showed high inhibitory activity on HIV-1 replication.

B)

A)

B)

A)

Analysis of inhibition of in vitro phosphorylation

Inhibition of HIV-1 replication

SS

FLMS

FST

Fig.2

DNA Ladder

Topo IIa

Topo IIb

Topo IIa+b

Cells + H

IV-1

GFP pNL 4-3

Heat inacti

vated

ProviralDNA ~150 bp

β– actin ~460 bp

siRNA

-ve C

ontrol

AZT C

ontrol

B)

A)

C)

Fig.2. Effect of Topo II knockdown on viral gene expression showed absence of viral gag, pol & env transcripts. B) Proviral DNA synthesis was effected in Topo II knockdown cells when compared to control. C) Schematic representation of RTn events.

RESULTS

Fig.4. HIV-1 RT tagged with AF 594 secondary antibody (Magenta). Topo IIα and Topo IIβ were directly tagged with TRITC (Red) and FITC (Green) respectively. DAPI (Blue) used as nuclear counter stain. HIV LIFE

CYCLE

METHODOLOGY

LITERATURE CITED

ACKNOWLEDGEMENTS

http://www.topnews.in

http://t1.gstatic.com/images

http://upload.wikimedia.org/wikipedia/commons/thumb/3/3b