Integration of retroviral cDNA involves coupled joining of the two ends of the viral genome at precisely spaced positions in the host cell DNA. Correct coupled joining is essential for viral replication, as shown, for example, by the finding that viral mutants defective in coupled joining are defective in integration and replication. To date, reactions with purified human immunodeficiency virus type 1 (HIV-1) integrase protein in vitro have supported mainly uncoupled joining of single cDNA ends. We have analyzed an activity stimulating coupled joining present in HIV-1 virions, which led to the finding that the HIV-1 nucleocapsid (NC) protein can stimulate coupled joining more than 1,000-fold under some conditions. The requirements for stimulating coupled joining were investigated in assays with mutant NC proteins, revealing that mutations in the zinc finger domains can influence stimulation of integration. These findings (i) provide a means for assembling more authentic integrase complexes for mechanistic studies, (ii) reveal a new activity of NC protein in vitro, (iii) indicate a possible role for NC in vivo, and (iv) provide a possible method for identifying a new class of inhibitors that disrupt coupled joining.

Integration of retroviral cDNA into host chromosomal DNA is an essential and distinctive step in viral replication. Despite considerable study, the host determinants of sites for integration have not been fully clarified. To investigate integration site selection in vivo, we used two approaches. (i) We have analyzed the host sequences flanking 61 human immunodeficiency virus type 1 (HIV-1) integration sites made by experimental infection and compared them to a library of 104 control sequences. (ii) We have also analyzed HIV-1 integration frequencies near several human repeated-sequence DNA families, using a repeat-specific PCR-based assay. At odds with previous reports from smaller-scale studies, we found no strong biases either for or against integration near repetitive sequences such as Alu or LINE-1 elements. We also did not find a clear bias for integration in transcription units as proposed previously, although transcription units were found somewhat more frequently near integration sites than near controls. However, we did find that centromeric alphoid repeats were selectively absent at integration sites. The repeat-specific PCR-based assay also indicated that alphoid repeats were disfavored for integration in vivo but not as naked DNA in vitro. Evidently the distinctive DNA organization at centromeres disfavors cDNA integration. We also found a weak consensus sequence for host DNA at integration sites, and assays of integration in vitro indicated that this sequence is favored as naked DNA, revealing in addition an influence of target primary sequence.

The integrase (IN) protein of the human immunodeficiency virus mediates integration of the viral DNA into the cellular genome. In vitro, this reaction can be mimicked by using purified recombinant IN and model DNA substrates. IN mediates two reactions: an endonucleolytic cleavage at each 3 end of the proviral DNA (terminal cleavage) and the joining of the linear viral DNA to 5 phosphates in the target DNA (strand transfer). Previous investigators have shown that purified IN requires Mn 2؉ or Mg 2؉ to promote strand transfer in vitro, although Mg 2؉ is the likely metal cofactor in vivo. IN activity in the presence of Mg 2؉ in vitro requires high IN concentrations and low concentrations of salt. Here, we show that the viral nucleocapsid protein NCp7 allows efficient IN-mediated strand transfer in the presence of Mg 2؉ at low enzyme concentrations. This potentiating effect appears to be unique to NCp7, as other small DNA-binding proteins, while capable of stimulating integration in the presence of Mn 2؉ , all failed to stimulate strand transfer in the presence of Mg 2؉ .

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In an attempt to target short purine sequences in view of pharmacological application, we have synthesized three new TFO (triple-helix-forming oligonucleotide) conjugates in which an intercalating oxazolopyridocarbazole (OPC) chromophore is linked by a pentamethylene linker to a 7-mer oligonucleotide matching the polypurine/polypyrimidine sequence located in the HIV-1 U3 LTR end region. The TFO moiety of conjugates are 5'CCTTCCC, 5'GGGAAGG, and 5'GGGTTGG. Their ability to bind to double-stranded DNA targets was examined. This binding is demonstrated by a footprinting technique using DNase I as a cleaving agent. The complex involved intermolecular pyr-pur*pyr or pur-pur*pyr triple helix. Pyrimidine TFO-OPC binds in a pH-dependent manner, whereas the others do not. The formation of the complex has been investigated at neutral pH and increasing temperature. We observed that the protection due to the purine and mixed TFO-OPC was pH independent and remained identical up to 40 degrees C. To determine the position of the OPC chromophore, molecular modeling was undertaken on the purine-conjugate/target complex. It has been suggested that the complex involved the intercalation of the OPC at the triplex-duplex junction with a small unwinding at the next excluded site.

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