Involvement of human Topoisomerase II isoforms in strand transfer events of HIV-1 reverse transcription Lokeswara Bala

1. Involvement of human Topoisomerase II isoforms in strand transfer events of HIV-1 reverse transcription LokeswaraBalakrishna S and Anand K. Kondapi Department of Biotechnology, School of Life Sciences, University of Hyderabad Hyderabad 500 046, India. GFP HIV-1 – veControl + ve Control Mt DNA FLMS SS SST FST siRNA + -- -- + Topo IIα Topo IIβ Control Infected -- + -- + + + -- + Autoradiogram pTopo IIβ -- -- + + Topo IIβ -- -- -- -- -- -- -- + RESULTS AZT INTRODUCTION Heat inactivated virus • Reverse transcription (RTn) is crucial step in HIV-1 life cycle. In cytoplasm viral genomic RNA is reverse transcribed to double strand DNA (dsDNA). • The dsDNAtranslocatesinto 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. • Fig.3 • Fig.4 Co-localization of Topo II isoforms with HIV-1 RT Effect of Topo II knockdown on HIV-1 RTn intermediates 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.4. HIV-1 RT tagged with AF 594 secondary antibody (Magenta). Topo IIαand Topo IIβwere directly tagged with TRITC (Red) and FITC (Green) respectively. DAPI (Blue) used as nuclear counter stain. HIV LIFE CYCLE • Fig.5 • Fig.5 Analysis of in vitro phosphorylation of Topo IIβby Topo IIβkinase during HIV-1 infection. Western Blot • Fig.7 • Fig.6 Inhibition of HIV-1 replication Analysis of inhibition of in vitro phosphorylation A) A) http://www.topnews.in http://t1.gstatic.com/images METHODOLOGY • 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 +vecontrol, pNL4-3 used as PCR +vecontrol & no template used as -vecontrol. • 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. B) B) Fig.6. A&B) Analysis of inhibition of Topo IIβphosphorylation 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. Marker PCR -ve Control +ve Control -ve Control PCR +ve Control 94 DMSO 114 151 113 117 AZT 157 • Fig.8 Effect of Topo IIβKinase inhibitors on RTn intermediates SS FST FLMS Fig.8. Kinase inhibitors effected RTn intermediate formation i.e., SS, FST & FLMS. DMSO used as vehicle, Uninfected cellsas -ve control, infected cells as +vecontrol, AZT as internal control. No template as PCR –ve control and pNL4-3 used as PCR +vecontrol. CONCLUSIONS • 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 IIβkinase 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. HIV-1 Infection RESULTS 0 1 2 4 6 8 Time in hours Topo IIα ~ 170 kDa Topo IIβ ~180 kDa • Fig.1 Fig.2 β-actin ~43 kDa LITERATURE CITED Topo IIα+β Cells +HIV1 Topo IIβ Topo IIα GFP Topo IIα ~170 kDa • 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. 2005Biochemical 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. 2006A study of the topoisomerase II activity in HIV-1 replication using the ferrocene derivatives as probes. Archives of Biochemistry and Biophysics 450:23-132. Topo IIβ ~180 kDa A) A) siRNA β- actin ~43 kDa Topo IIa+b Heat inactivated DNA Ladder AZT Control -ve Control Cells + HIV-1 GFP pNL 4-3 Topo IIb Topo IIa siRNA Proviral DNA ~150 bp β– actin ~460 bp B) B) C) C) ACKNOWLEDGEMENTS http://upload.wikimedia.org/wikipedia/commons/thumb/3/3b 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. 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 4thday. 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. • Presented at Towards an HIV Cure Symposium and IAS 2013 Conference–Kuala Lumpur, Malaysia