ABSTRACT
HIV-1 reverse transcription (RTn) involves synthesis of double strand DNA (dsDNA) from viral genomic RNA. Topoisomerase II (Topo II) alpha and beta maintains topological reorganization of dsDNA regions and catalytic inhibition of these isoforms repressed viral replicative cycle. Present study is aimed to understand the role of Topo II isoforms in HIV-1 early replication. Topo IIα and ß showed differential expression in SupT1 cells and PBMCs during early hours of HIV-1 infection where Topo IIα expression increased after 4h, while Topo IIß showed relatively higher expression at 1 and 4h. In Topo IIα and/or ß down regulated cells, transcription of viral genes gag, pol and env as well as proviral DNA synthesis was abolished. In Topo IIα and/or ß down regulated cells, strong stop DNA synthesis was unaffected while other downstream events of reverse transcription such as first strand transfer, full length minus strand synthesis, and second strand transfer were completely inhibited, which affects HIV-1 replication. Further, co-localization of Topo II isoforms with HIV-1 reverse transcriptase was observed in SupT1 cells and PBMCs by immunofluorescence. These results collectively suggest a role of Topo II isoforms during HIV-1 RTn probably by promoting the alignment of viral RNA-DNA hybrids.
Subject(s)
DNA Topoisomerases, Type II/metabolism , HIV Infections/enzymology , HIV-1/physiology , Host-Pathogen Interactions , Cell Line , Cells, Cultured , DNA Topoisomerases, Type II/analysis , DNA Topoisomerases, Type II/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Viral , Gene Knockdown Techniques , HIV Infections/genetics , HIV Infections/metabolism , HIV Reverse Transcriptase/analysis , HIV Reverse Transcriptase/metabolism , Humans , Leukocytes, Mononuclear/virology , Protein Isoforms/analysis , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Small Interfering/genetics , Virus ReplicationABSTRACT
Human Topoisomerase II is present in two isoforms, 170KDa alpha and 180KDa beta. Both the isoforms play a crucial role in maintenance of topological changes during DNA replication and recombination. It has been shown that Topoisomerase II activity is required for HIV-1 replication and the enzyme is phosphorylated during early time points of HIV-1 replication. In the present study, we have studied the molecular action of Topoisomerase II inhibitors, azalactone ferrocene (AzaFecp), Thiomorpholide amido methyl ferrocene (ThioFecp), and Ruthenium benzene amino pyridine (Ru(ben)Apy) on cell proliferation and also on various events of HIV-1 replication cycle. The Topoisomerase II beta over-expressing neuroblastoma cell line shows a higher sensitivity to these compounds compared to the Sup-T1 cell line. All the three Topoisomerase II inhibitors show significant anti-HIV activity at nanomolar concentrations against an Indian isolate of HIV-1(93IN101) in Sup-T1 cell line. An analysis of action of these compounds on proviral DNA synthesis at 5h of post-infection shows that they inhibit proviral DNA synthesis as well as the formation of pre-integration complexes completely. Further analysis, using polymerase chain reaction and western blot, showed that both the Topoisomerase II alpha and beta isoforms are present in the pre-integration complexes, suggesting their significant role in HIV-1 replication.
