Human Immunodeficiency Virus Type 1 Integrase Protein Promotes Reverse Transcription through Specific Interactions with the Nucleoprotein Reverse Transcription Complex

Abstract

The human immunodeficiency virus type 1 (HIV-1) integrase protein (IN) is essential for integration of the viral DNA into host cell chromosomes. Since IN is expressed and assembled into virions as part of the 160-kDa Gag-Pol precursor polyprotein and catalyzes integration of the provirus in infected cells as a mature 32-kDa protein, mutations in IN are pleiotropic and may affect virus replication at different stages of the virus life cycle in addition to integration. Several different phenotypes have been observed for IN mutant viruses, including defects in virion morphology, protein composition, reverse transcription, nuclear import, and integration. Because the effects of mutations in the IN domain of Gag-Pol can not always be distinguished from those of mutations in the mature IN protein, there remains a significant gap in our understanding of IN function in vivo. To directly analyze the function of the mature IN protein itself, in the context of a replicating virus but independently from that of Gag-Pol, we used an approach developed in our laboratory for incorporating proteins into HIV virions by their expression in trans as fusion partners of either Vpr or Vpx. By providing IN intrans as a Vpr-IN fusion protein, our analysis revealed, for the first time, that the mature IN protein is essential for the efficient initiation of reverse transcription in infected cells and that this function does not require the IN protein to be enzymatically (integration) active. Our findings of a direct physical interaction between IN and reverse transcriptase and the failure of heterologous HIV-2 IN protein to efficiently support reverse transcription indicate that this novel function occurs through specific interactions with other viral components of the reverse transcription initiation complex. Studies involving complementation between integration- and DNA synthesis-defective IN mutants further support this conclusion and reveal that the highly conserved HHCC motif of IN is important for both activities. These findings provide important new insights into IN function and reverse transcription in the context of the nucleoprotein reverse transcription complex within the infected cell. Moreover, they validate a novel approach that obviates the need to mutate Gag-Pol in order to study the role of its individual mature components at the virus replication level.

The HIV-1 Reverse Transcription (RT) Process as Target for RT Inhibitors

“…This suggests an effect of the integrase enzyme on events proximal to the reverse transcription that determine proper and efficient initiation of viral DNA synthesis. Very recently, Wu et al 55 demonstrated the importance of the highly conserved HisHisCysCys-motif of the mature integrase protein for efficient initiation of reverse transcription.…”

Mechanistic aspects of HIV‐1 reverse transcription initiation

“…Like Tat, IN is required for efficient RTN initiation [39,106]. Interestingly, IN enzymatic activity is not required for this function since mutations in the highly conserved D 64 D 116 E 152 catalytic domain permit RTN, and inhibitors that block IN activity do not inhibit initiation in NERT [59].…”

“…It is likely that an early RTN complex associates with cytoskeletal proteins [36], and during trafficking to the nucleus reorganises into a pre-integration complex containing proviral DNA, IN and Vpr. As IN directly binds to RT [106], IN may be a legitimate component of the RTNIC.…”

Biochemical interactions between HIV‐1 integrase and reverse transcriptase

“…There have been several pieces of evidence suggesting the existence of direct and indirect interactions between HIV-1 IN and RT [4,5]. Direct physical interactions occur as RT binds to the Cterminal domain (CTD) of IN [6,7] genome yields non-integrated DNA [8]; indeed, non-integrated DNA levels can reach 99% of total viral DNA at the asymptomatic phase of infection [9]. This non-integrated DNA has full capacity to synthesize all classes of viral transcripts, both spliced and non-spliced, prior to integration [8,10,11].…”

“…Indirect interaction between IN and RT occurs as the product of RT-catalyzed reactions binds to the IN protein. In vitro studies with purified IN and RT have shown that HIV-1 IN can indirectly stimulate both the initiation and elongation modes of RT-catalyzed reverse transcription by enhancing RT processivity[7]. However, how these two key viral enzymes coordinate viral DNA production by affecting each other activity is still poorly understood.…”