High‐level expression of active HIV‐1 integrase from a synthetic gene in human cells

Abstract

A synthetic gene encoding for HIV-1 integrase was designed to circumvent the intrinsic instability and the repressor elements present in the wild-type gene. High-level expression of HIV-1 integrase was obtained in various human cell lines independently of viral accessory proteins. A human 293T cell line was selected that stably expresses HIV-1 integrase and has growth kinetics comparable to the parental cell line. The enzyme was localized in the nucleus and remained stably associated with the chromosomes during mitosis. Lentiviral vector particles carrying the inactivating D64V mutation in the integrase gene were capable of stably transducing 293T cells when complemented in the producer cells with integrase expressed from the synthetic gene. When the cell line that stably expresses integrase was infected with the defective viral particles, complementation of integrase activity was detected as well. Expression of active HIV-1 integrase in human cells will facilitate the study of the interplay between host and viral factors during integration.

The Interaction of LEDGF/p75 with Integrase Is Lentivirus-specific and Promotes DNA Binding

“…The molecular mechanism of this cofactor is apparently based on the stimulation of the binding of IN to the DNA. Our findings support our hypothesis that LEDGF/p75 plays a role in tethering IN to the chromosomal DNA (12,15).We have unambiguously demonstrated the specificity of interaction of LEDGF/p75 with lentiviral integrases both in a pull-down interaction assay and with FCS. While this article was in preparation, Llano et al (43) reported as well on the role of LEDGF/p75 in the nuclear accumulation of HIV-1 and FIV but not Mo-MuLV IN.…”

“…The functional holoprotein required for concerted integration of two long terminal repeat ends is believed to exist as a homodimer of two tetramers (13,14). In cells stably overexpressing IN, the enzyme remains stably associated with condensed chromosomes during mitosis (15,16).…”

“…The functional holoprotein required for concerted integration of two long terminal repeat ends is believed to exist as a homodimer of two tetramers (13,14). In cells stably overexpressing IN, the enzyme remains stably associated with condensed chromosomes during mitosis (15,16).The insight has grown that HIV relies on cellular proteins for completion of its replication cycle. Identification and characterization of these cellular cofactors will increase our understanding of the viral replication cycle and aid in the development of new antiviral drugs.…”

Integrase, LEDGF/p75 and HIV replication

“…The first indication of the protein's significance was the clarification it provided about the vexing issue of the intracellular trafficking of retroviral integrase proteins. When HIV IN is expressed outside the viral context it is karyophilic [118][119][120][121] and chromatin-associated [73][74][75]86,122,123]. This property is characteristic of the entire lentivirus genus since IN proteins from the two nonprimate (feline and ungulate) subgenera behave identically [75,87,100].…”

Lentiviral vectors: optimization of packaging, transduction and gene expression

“…One report has described HIV-1-derived vector production with split packaging elements, one expressing the gag-pro genome and the other expressing the pol gene [41]. In addition, another laboratory has used HIV-1 integrase-expressing cells to transiently deliver only gag, pro and RT genes [42].…”