Thermal stressed human immunodeficiency virus type 1 nucleocapsid protein NCp7 maintains nucleic acid-binding activity
Introduction
Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is a small highly basic protein with two conserved CCHC zinc-finger motifs, flanked by basic residues. HIV-1 NC is derived from the Gag polyprotein Pr55Gag which consists of matrix, capsid, NC, p6 domains, as well as two spacer peptides [1]. The NC domain is responsible for the specific recognition of viral packaging signal (Psi), resulting in the encapsidation of two copies of genomic RNA (gRNA) into the viral particle [[2], [3], [4]]. Some mutations affecting the NC domain of Pr55Gag inhibits gRNA packaging and reduces virus infectivity dramatically [[5], [6], [7]]. The NC domain also interacts with cellular factors and promotes viral assembly [8,9].
Mature NC is liberated by viral protease-mediated cleavages of Pr55Gag shortly after virus release from the cell [10,11]. About 1500–2000 NC molecules are present per viral core, extensively coating the viral gRNA, protecting the gRNA via its nucleic acid (NA)-binding and condensing properties, in a histone-like manner [12,13]. As an ATP independent NA chaperone, NC can bind to different NA sequences at varying affinities, and the NC-mediated NA condensing and chaperoning activities are dependent upon the degree of sequence occupancy in the nucleoprotein complex [14,15]. This degree of occupancy from low (the NC:NA ratio about 1:100 nt), to high (1:7–15 nt), and very high (1:1–5 nt), defines the activity, from high affinity binding to specific viral sequences, to NA-binding and driving the remodeling of the NC-NA complexes to reach thermodynamically stable conformation, and ultimately to freezing the compact nucleoprotein complexes [15]. Mature NC also functions in early infection processes, facilitating reverse transcription and the integration of viral DNA into the host genome [16,17]. Therefore, the NC domain of Pr55Gag and the mature NC play critical roles in almost every step of the viral replication cycle.
Previously, we reported that the equine infectious anemia virus NCp11 is thermostable [18]. In this study, the thermostability of HIV-1 NCp7 and its effects on the NA-binding activity of NCp7 were investigated. The results showed that NCp7 is thermostable. Heat-treated NCp7 maintained its abilities to bind to HIV-1 Psi and the stem-loop 3 (SL3) recognition element of the Psi, maintained its inhibition of the first-strand cDNA synthesis at very high degree of sequence occupancy. Moreover, we found that the NA-binding activity of NCp7 at high NC:NA ratios is independent on its zinc-fingers, since EDTA-treated and H23K + H44K double mutant of NCp7 still inhibited first-strand cDNA synthesis. Our findings may benefit further investigations of NC’s properties and functions, which might be helpful to the development of effective and rational therapeutic strategies against HIV.
Section snippets
Plasmids
The coding regions of NCp7, p6, reverse transcriptase p66 and p51 were amplified from HIV-1 NL4-3 genome (GenBank U26942.1) using primers listed in the Supplementary. The DNA sequence of H23K + H44K double mutant of NCp7 was synthesized (BGI, China). Each of the fragments was cloned into pET28 vector (Novagen), respectively. Plasmid pBS-HIV Psi was constructed, which contains the 5’ leader sequence from nt 559 to nt 798 covering the Psi. All the plasmids constructed in this study were verified
Considerable percentage of HIV-1 NCp7 remained soluble after incubated at 100 °C
The HIV-1 NCp7 was expressed in E. coli BL21 (DE3) and analyzed by SDS-PAGE. The apparent molecular weight of IPTG-induced NCp7 is about 14.7 kDa, which was detected in the soluble fraction almost exclusively (lanes 1–3, Fig. 1A). When the clear cell lysate of IPTG-induced E. coli BL21 (DE3) was incubated at 100 °C for 5, 10, 30 or 60 min, respectively, and clarified, bacterial soluble proteins were barely detected, whereas a considerable portion of NCp7 remained in the soluble fraction (lanes
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Acknowledgements
This work was supported by the Natural Science Foundation of China (No. 31870158 and No. 81470095) and the Natural Science Foundation of Tianjin City (No. 16JCYBJC24000).
References (26)
- et al.
The molecular architecture of HIV
J. Mol. Biol.
(2011) - et al.
Mapping of nucleocapsid residues important for HIV-1 genomic RNA dimerization and packaging
Virology
(2008) - et al.
Strict conservation of the retroviral nucleocapsid protein zinc finger is strongly influenced by its role in viral infection processes: characterization of HIV-1 particles containing mutant nucleocapsid zinc-coordinating sequences
Virology
(1999) - et al.
The choreography of HIV-1 proteolytic processing and virion assembly
J. Biol. Chem.
(2012) - et al.
First glimpses at structure-function relationships of the nucleocapsid protein of retroviruses
J. Mol. Biol.
(1995) - et al.
Flexible nature and specific functions of the HIV-1 nucleocapsid protein
J. Mol. Biol.
(2011) - et al.
Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism
Prog. Nucleic Acid Res. Mol. Biol.
(2005) - et al.
Nucleocapsid protein function in early infection processes
Virus Res.
(2008) - et al.
Thermostable properties of the equine infectious anemia virus nucleocapsid protein NCp11
Biochem. Biophys. Res. Co.
(2019) - et al.
Covalent conjugation of the equine infectious anemia virus Gag with SUMO
Biochem. Biophys. Res. Co.
(2017)
Possible roles of HIV-1 nucleocapsid protein in the specificity of proviral DNA synthesis and in its variability
J. Mol. Biol.
Structural characterization of a 39-residue synthetic peptide containing the two zinc binding domains from the HIV-1 p7 nucleocapsid protein by CD and NMR spectroscopy
FEBS Lett.
The reaction of dichlorodiammineplatinum (II), [PtCl2 (NH3) 2], isomers with zinc fingers
J. Inorg. Biochem.
Cited by (2)
Comparison of HIV-1 Gag and NCp7 in their selectivity for package signal, affinity for stem-loop 3, and Zn<sup>2+</sup> content
2020, BiochimieCitation Excerpt :These results will be helpful to elucidate the important roles that Zn2+ played in the viral life cycle, and may benefit further investigations of how the different functions of the NC domain of Gag and NCp7 are switched and regulated. Plasmids pET28-NCp7 and pBS-HIV Psi were described previously [44]. The coding regions of Gag, NCp15, NCp9, and truncated forms of Gag, including Δp6, Δp1p6, ΔMA, ΔMA&p6, CAp2NC, ΔMA&CA, p2NCp1 and p2NC (Supplementary Fig. S1), were amplified from the HIV-1 NL4-3 genome (GenBank U26942.1), using the primers listed in Supplementary Table 1.