Functional and Structural Characterization of Novel Type of Linker Connecting Capsid and Nucleocapsid Protein Domains in Murine Leukemia Virus

Abstract

The assembly of immature retroviral particles is initiated in the cytoplasm by the binding of the structural polyprotein precursor Gag with viral genomic RNA. The protein interactions necessary for assembly are mediated predominantly by the capsid (CA) and nucleocapsid (NC) domains, which have conserved structures. In contrast, the structural arrangement of the CA-NC connecting region differs between retroviral species. In HIV-1 and Rous sarcoma virus, this region forms a rod-like structure that separates the CA and NC domains, whereas in Mason-Pfizer monkey virus, this region is densely packed, thus holding the CA and NC domains in close proximity. Interestingly, the sequence connecting the CA and NC domains in gammaretroviruses, such as murine leukemia virus (MLV), is unique. The sequence is called a charged assembly helix (CAH) due to a high number of positively and negatively charged residues. Although both computational and deletion analyses suggested that the MLV CAH forms a helical conformation, no structural or biochemical data supporting this hypothesis have been published. Using an in vitro assembly assay, alanine scanning mutagenesis, and biophysical techniques (circular dichroism, NMR, microcalorimetry, and electrophoretic mobility shift assay), we have characterized the structure and function of the MLV CAH. We provide experimental evidence that the MLV CAH belongs to a group of charged, E(R/K)-rich, single α-helices. This is the first single α-helix motif identified in viral proteins.

Keywords: capsid protein (CA); charged assembly helix (CAH); circular dichroism (CD); electron microscopy (EM); murine leukemia virus (MLV); nuclear magnetic resonance (NMR); retrovirus; single alpha-helix (SAH); spacer peptide (SP); virus assembly.

FIGURE 1.

Analysis of CAH importance for MLV CANC assembly. A, SDS-PAGE gel showing produced and purified MLV Δ10CANC and Δ10CANC ΔCAH. Lanes: ST, molecular weight standard; 0, cell lysates before induction; 1 and 2, cell lysates 4 h after induction; 3 and 4, purified and concentrated proteins. B–D, TEM analysis of thin-sectioned E. coli expressing MLV Δ10CANC and Δ10CANC ΔCAH (B); negatively stained lysates of E. coli producing MLV Δ10CANC and Δ10CANC ΔCAH (C); and purified, in vitro assembled, and negatively stained MLV Δ10CANC and Δ10CANC ΔCAH (D). The bottom right bars represent a 200 nm scale.

FIGURE 2.

Design and expression of the CAH mutants. A, positions of the mutations introduced to the CAH region. The plus and minus signs in the right column stand for: (+), mutants assembled into spherical structures similar to those observed for the wild type; (−/+), mutants formed visible electron-dense protein layers without the ability to assemble into spherical particles; and (−), mutants formed no organized protein layer. MA, matrix. B, SDS-PAGE gel showing the production of the MLV Δ10CANC mutants in E. coli (lanes 1–18). Lanes: ST, molecular weight standard; 0, cell lysate before induction; 1–18, cell lysates 4 h after induction. The arrow indicates the position of expressed MLV Δ10CANC mutants.

FIGURE 3.

TEM analysis of thin-sectioned E. coli cells producing the MLV Δ10CANC mutants. The bars represent a 200 nm scale.

FIGURE 4.

Analysis of the CAH-derived peptide by circular dichroism spectroscopy. A, sequence of the synthetic, CAH-derived peptide. B, circular dichroism spectra of the peptide at increasing concentrations (c). deg, degrees. C, temperature dependence of [Θ]₂₂₂.

FIGURE 5.

NMR analysis of CAH. A, sequence of the His-TEV-CAH. The part derived from the CAH region is shown in bold. The helical part is shown in color: uncharged residues in yellow, negatively charged residues in red, and positively charged residues in blue. B, SDS-PAGE gel showing purified and concentrated His-TEV-CAH (lane C). ST, molecular weight standard. C, helical wheel representation of the helical part of the CAH region (residues Pro²²²–His²⁵⁶). D, graphic model of the CAH. E, HN-HSQC spectrum of His-TEV-CAH. The resonances of the backbone amides are marked by residue number. The resonances of Gln and Asn side chain amides are marked by residue number and type. Gln and Asn side chain amide resonances are connected by lines. F, graphical representation of the output from the TALOS+ software. Upper panel, residues with random coil index-derived order parameter (RCI S²) lower than 0.5 are considered dynamic. Lower panel, predicted secondary structure (red, helix; blue, β-sheet). The height of the bars shows the probability of the prediction.

FIGURE 6.

Potential intrahelical ionic interactions stabilizing the MLV CAH predicted by the SCAN4CSAH algorithm on the CSAH server (23, 29). Potential i, i+4 interactions are shown above the sequence, and i, i+3 interactions are shown below the sequence. Positively charged arginine and lysine residues are shown in blue, and negatively charged glutamate and aspartate residues are shown in red. The gray rectangle marks the experimentally confirmed helical part of the region.

FIGURE 7.

Preparation of the proteins for ITC and EMSA. A, schematic representation of the proteins used for ITC and EMSA. B, SDS-PAGE gel showing purified and concentrated proteins used for ITC. Lanes: ST, molecular weight standard; 1, CA; 2, CA ΔCAH; 3, CTD-CA; 4, CTD-CA ΔCAH; 5, CAH.

FIGURE 8.

Isothermal titration calorimetry analysis. Isothermal titration calorimetry data showing the sequential dilution of the MLV CA, CA ΔCAH, CAH, CTD-CA, CTD-CA ΔCAH, and HIV-1 CA are displayed. Upper panel, experimental data; lower panel, fit (line) to the integrated heat (full circles) from each injection. The lines are a result of fit to a dimer dissociation model.

FIGURE 9.

EMSA. The indicated proteins were incubated with a 1-kb DNA ladder, and the sample aliquots were then treated with proteinase K. All the samples were analyzed by agarose gel electrophoresis. Lanes: L: 1-kb DNA ladder; 1: the tested proteins without the addition of the 1-kb DNA ladder; 2: tested proteins incubated with the 1-kb DNA ladder without proteinase K treatment; 3: tested proteins incubated with the 1-kb DNA ladder with proteinase K treatment.