The N-Terminal Zinc Finger and Flanking Basic Domains Represent the Minimal Region of the Human Immunodeficiency Virus Type-1 Nucleocapsid Protein for Targeting Chaperone Function

Abstract

The human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC) protein is a chaperone that facilitates nucleic acid conformational changes to form the most thermodynamically stable arrangement. The critical role of NC in many steps of the viral life cycle makes it an attractive therapeutic target. The chaperone activity of NC depends on its nucleic acid aggregating ability, duplex destabilizing activity and rapid on/off binding kinetics. During the minus-strand transfer step of reverse transcription, NC chaperones the annealing of highly structured transactivation response region (TAR) RNA to the complementary TAR DNA. In this work, the role of different functional domains of NC in facilitating 59-nucleotide TAR RNA/DNA annealing was probed by using chemically-synthesized peptides derived from full-length (55 amino acids) HIV-1 NC: NC(1-14), NC(15-35), NC(1-28), NC(1-35), NC(29-55), NC(36-55) and NC(11-55). Most of these peptides displayed significantly reduced annealing kinetics, even when present at much higher concentrations than wild-type (WT) NC. In addition, these truncated NC constructs generally bind more weakly to single-stranded DNA and are less effective nucleic acid aggregating agents than full-length NC, consistent with the loss of both electrostatic and hydrophobic contacts. However, NC(1-35) displayed annealing kinetics, nucleic acid binding, and aggregation activity that were very similar to that of WT NC. Thus, we conclude that the N-terminal zinc finger, flanked by the N-terminus and linker domains, represents the minimal sequence that is necessary and sufficient for chaperone function in vitro. In addition, covalent continuity of the N-terminal 35 amino acids of NC is critical for full activity. Thus, although the hydrophobic pocket formed by residues proximal to the C-terminal zinc finger has been a major focus of recent anti-NC therapeutic strategies, NC(1-35) represents an alternative target for therapeutics aimed at disrupting NC’s chaperone function.

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

“…TAR destabilization in our conditions was limited because NC destabilizes only three consecutive base pairs with high efficiency. However, the NC:nt ratio was significantly different in the two studies (1:7.5 versus 1:59) (Bernacchi et al 2002) and NC(1-55) has a greater destabilization capacity that NC(12-55) (Levin et al 2005;Vo et al 2009b;Mitra et al 2013). Finally, the extensive broadening of imino proton resonance does not necessarily indicate a loss of the corresponding base pair.…”

“…The relationship between binding polarity and the polarized destabilization of stem-loops may result from the lack of equivalence of the two zinc fingers, ZF1 and ZF2, with ZF1 more involved in the destabilization than ZF2. In contrast, ZF2 is involved in the specific recognition of guanine residues (Guo et al 2002;Heath et al 2003;Beltz et al 2005;Narayanan et al 2006;Hergott et al 2013;Mitra et al 2013). Binding polarity may therefore be responsible for the localization of ZF1 close to a double-stranded region.…”

“…Nucleic acid aggregation is thought to be dependent on the N-terminal basic part of NC, whereas duplex destabilization is associated with the zinc fingers (Beltz et al 2005;Levin et al 2005Levin et al , 2010Vo et al 2009a,b;Godet and Mély 2010). However, some studies have suggested a role of zinc fingers in nucleic acid aggregation (Krishnamoorthy et al 2003;Levin et al 2005;Mirambeau et al 2006;Mitra et al 2013) and others have implicated basic residues in nucleic acid destabilization .…”

Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone

“…1). The highly basic nature of HIV-1 NC stems primarily from its a-helical N-terminal domain, and is the driving force behind NC's strong NA aggregation activity [4][5][6][7]. The Zn fingers each contain one or two aromatic residues that can interact with nucleobases via stacking interactions, and have been shown to promote duplex destabilization [8][9][10][11][12][13][14].…”

Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly

“…Indeed, the NC mutant in which ZF1 has been duplicated (ZF1+ZF1) is more replication competent than mutants with zinc finger-swap (ZF2+ZF1) or with duplicated ZF2 (ZF2+ZF2)6. Several studies of ZF mutants revealed that ZF1 was more critical than ZF2 for the NC chaperone activity789. In addition, nucleic acid binding abilities of different NC domains have been analyzed and revealed that the absence of ZF2 (NC 1-35) did not reduce NC binding affinity, demonstrating that ZF1 provides the major contribution to DNA binding9.…”