Human immunodeficiency virus type 1 nucleocapsid protein specifically stimulates Mg2+-dependent DNA integration in vitro

Abstract

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؉ .

Retrospective on the all-in-one retroviral nucleocapsid protein

“…Additional results by Gorelick et al also indicated that the integration reaction was impaired (Buckman et al, 2003;Thomas et al, 2006), supporting the view that functional interactions exist between NC and IN as it stands between NC and RT since mutating the ZF strongly diminish NC-RT interactions (Druillennec et al, 1999a;Lener et al, 1998). The existence of critical interactions between NC and IN is further supported by the fact that NC can strongly stimulate concerted LTR DNA integration by IN under physiological conditions in vitro (Buckman et al, 2003;Carteau et al, 1997Carteau et al, , 1999Poljak et al, 2003;Thomas et al, 2006).…”

“…In this context RV NCs represent remarkable examples of multifunctional NA chaperones that drive the necessary structural rearrangements of the genomic RNA during the early and late phases of virus replication (see below; reviewed in Darlix et al, 1995;Levin et al, 2005). In addition RV NCs might well be endowed with protein chaperoning activity by providing assistance to the viral RT and IN enzymes during viral DNA synthesis and integration (Buckman et al, 2003;Carteau et al, 1997;Lapadat-Tapolsky et al, 1993;Poljak et al, 2003;reviewed in Darlix et al, 2011).…”

Branched oligonucleotide‐intercalator conjugate forming a parallel stranded structure inhibits HIV‐1 integrase

“…Different assays were carried out to investigate the effect of the conjugates on integration in vitro: (1) 3′‐end processing of a 21‐bp duplex, (2) strand transfer using a 21‐bp duplex as both DNA substrate and target, and (3) strand transfer of a 492‐bp mini‐viral DNA into a plasmid target [15].…”

“…(b) Heterologous integration into a long DNA fragment. A 492‐bp U3U5 DNA which is generated by Nde I restriction of the pU3U5 vector as previously described [15] was used as donor substrate. Plasmid pSP70 was used as target DNA.…”

Reconciling NMR Structures of the HIV-1 Nucleocapsid Protein (NCp7) using Extensive Polarizable Force Field Free-Energy Simulations

“…16 Moreover, it protects the retroviral genome against cellular nucleases 17 and interacts with viral proteins such as Vpr 18 (Viral Protein R) and integrase. 19 Most of these functions are related to its higher affinity for single-stranded nucleic acids and to its structural features. In fact, NCp7 contains flexible N-terminal and C-terminal domains surrounding two highly conserved zinc finger motives CX2CX4HX4C, where X represents any amino acid residue.…”