Investigation of N-terminal domain charged residues on the assembly and stability of HIV-1CA

ABSTRACT: The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) plays a crucial role in both assembly and maturation of the virion as well as viral infectivity. Previous in vivo experiments generated two N-terminal domain charge change mutants (E45A and E128A/R132A) that showed an increase in stability of the viral core. This increase in core stability resulted in decreased infectivity, suggesting the need for a delicate balance of favorable and unfavorable interactions to both allow assembly and facilitate uncoating following infection. Purified CA protein can be triggered to assemble into tubelike structures through the use of a high salt buffer system. The requirement for high salt suggests the need to overcome charge/charge repulsion between subunits. The mutations mentioned above lie within a highly charged region of the N-terminal domain of CA, away from any of the proposed protein/protein interaction sites. We constructed a number of charge mutants in this region (E45A, E45K, E128A, R132A, E128A/ R132A, K131A, and K131E) and evaluated their effect on protein stability in addition to their effect on the rate of CA assembly. We find that the mutations alter the rate of assembly of CA without significantly changing the stability of the CA monomer. The changes in rate for the mutants studied are found to be due to varying degrees of electrostatic repulsion between the subunits of each mutant

A P22 Scaffold Protein Mutation Increases the Robustness of Head Assembly in the Presence of Excess Portal Protein

Bacteriophage with linear, double-stranded DNA genomes package DNA into preassembled protein shells called procapsids. Located at one vertex in the procapsid is a portal complex composed of a ring of 12 subunits of portal protein. The portal complex serves as a docking site for the DNA packaging enzymes, a conduit for the passage of DNA, and a binding site for the phage tail. An excess of the P22 portal protein alters the assembly pathway of the procapsid, giving rise to defective procapsid-like particles and aberrant heads. In the present study, we report the isolation of escape mutant phage that are able to replicate more efficiently than wild-type phage in the presence of excess portal protein. The escape mutations all mapped to the same phage genome segment spanning the portal, scaffold, coat, and open reading frame 69 genes. The mutations present in five of the escape mutants were determined by DNA sequencing. Interestingly, each mutant contained the same mutation in the scaffold gene, which changes the glycine at position 287 to glutamate. This mutation alone conferred an escape phenotype, and the heads assembled by phage harboring only this mutation had reduced levels of portal protein and exhibited increased head assembly fidelity in the presence of excess portal protein. Because this mutation resides in a region of scaffold protein necessary for coat protein binding, these findings suggest that the P22 scaffold protein may define the portal vertices in an indirect manner, possibly by regulating the fidelity of coat protein polymerization

Kinetic Analysis of the Role of Intersubunit Interactions in Human Immunodeficiency Virus Type 1 Capsid Protein Assembly In Vitro

The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) plays a crucial role in both assembly and maturation of the virion. Numerous recent studies have focused on either the soluble form of CA or the polymer end product of in vitro CA assembly. The CA polymer, in particular, has been used to study CA-CA interactions because it is a good model for the CA interactions within the virion core. However, analysis of the process of in vitro CA assembly can yield valuable insights into CA-CA interactions and the mechanism of core assembly. We describe here a method for the analysis of CA assembly kinetics wherein the progress of assembly is monitored by using turbidity. At pH 7.0 the addition of either of the isolated CA domains (i.e., the N or the C domain) to an assembly reaction caused a decrease in the assembly rate by competing for binding to the full-length CA protein. At pH 8.0 the addition of the isolated C domain had a similar inhibitory affect on CA assembly. However, at pH 8.0 the isolated N domain had no affect on the rate of CA assembly but, when mixed with the C domain, it alleviated the C-domain inhibition. These data provide biochemical evidence for a pH-sensitive homotypic N-domain interaction, as well as for an N- and C-domain interaction

Proteolysis assay and gel filtration of Ca²⁺/CaM–FasDD digests.

<p>(<b>A</b>) Gel filtration and SDS-PAGE of the proteolysis product of a 2:1 Ca²⁺/CaM:FasDD sample left at 4°C for one week. The SDS-PAGE data show that the degraded FasDD protein gave rise to ~5 kDa fragment(s). The Ca²⁺/CaM protein, however, remained stable. Analysis of the digestion products by mass spectrometry confirmed the identity of the FasDD peptides that are resistant to proteolysis. The most abundant peptides are located in the N-terminus (205–238, 205–239, and 205–240) and in the C-terminus (251–288, 259–288 and 262–288). (<b>B</b>) SDS-PAGE of FasDD, Ca²⁺/CaM and Ca²⁺/CaM:FasDD complex without or with added subtilisin at 1:500 (enzyme:protein) 24 hours after initiation of the reaction. Unbound FasDD and Ca²⁺/CaM were stable in the presence of subtilisin. (<b>C</b>) Gel filtration and SDS-PAGE data of the proteolysis product of a 2:1 Ca²⁺/CaM:FasDD sample treated with subtilisin for 24 hours. Similar to our observation in A, the SDS-PAGE data show a band at ~5 kDa eluting with Ca²⁺/CaM. FasDD fragments were identified by mass spectrometry (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146493#pone.0146493.s011" target="_blank">S1 Table</a>).</p

Role of Ca²⁺/CaM Met residues in binding of FasDD peptides.

<p>(<b>A</b>) Surface representation of the Ca²⁺/CaM structure (PDB ID: 3CLN) showing the nine Met residues localized in the N- and C-terminal lobes (red sticks). Ca²⁺ ions are colored in green. (<b>B</b>) Overlay of a selected region of 2D ¹H-¹³C HMQC spectra obtained for ¹³C-labeled Ca²⁺/CaM as a function of added Fas-Pep1 (top) or Fas-Pep2 (bottom). [peptide:Ca²⁺/CaM = 0:1 (black), 0.5:1 (red), 1:1 (blue), 1.5:1 (green)]. Only the ¹H-¹³C signals for the Met methyl groups are shown.</p

ITC data of Ca²⁺/CaM binding to FasDD peptides.

<p>ITC data obtained for titration of (<b>A</b>) Ca²⁺/CaM at 450 μM into Fas-Pep1 at 25 μM, and (<b>B</b>) Ca²⁺/CaM (195 μM) into Fas-Pep2 at 17 μM. Data fitting afforded <i>K</i>_d values of 0.3 and 1.1 μM for Fas-Pep1 and Fas-Pep2, respectively.</p

2D HSQC NMR data of Ca²⁺/CaM:Fas-Pep1 complex.

<p>Overlay of 2D ¹H-¹⁵N HSQC spectra obtained for ¹⁵N-labeled Ca²⁺/CaM in the free state (black) and in complex with Fas-Pep1 (red) at 1.5:1 peptide: Ca²⁺/CaM. No chemical shift changes were observed in the HSQC spectra with further addition of Fas-Pep1, indicating saturation at this ratio. Signal labels correspond to residues of CaM in the bound form. Signals labeled in blue are folded in the spectrum by 20 ppm.</p

FasDD sequence and structure.

<p>Protein sequence, secondary structure and a cartoon representation of the three-dimensional structure of FasDD (PDB ID: 1DDF). The CaM-binding regions (Fas-Pep1 and Fas-Pep2) are highlighted in red and green, respectively.</p

2D HSQC NMR data for Ca²⁺/CaM:Fas-Pep2 complex.

<p>Overlay of 2D ¹H-¹⁵N HSQC spectra obtained for ¹⁵N-labeled Ca²⁺/CaM in the free state (black) and in complex with Fas-Pep2 (green) at 1.5:1 peptide:CaM ratio. No chemical shift changes were observed in the HSQC spectra with further addition of Fas-Pep2, indicating saturation at this ratio. Signal labels correspond to residues of CaM in the bound form. Signals labeled in blue are folded in the spectrum by 20 ppm.</p