The NTD-CTD intersubunit interface plays a critical role in assembly and stabilization of the HIV-1 capsid

BACKGROUND: Lentiviruses exhibit a cone-shaped capsid composed of subunits of the viral CA protein. The intrinsic stability of the capsid is critical for HIV-1 infection, since both stabilizing and destabilizing mutations compromise viral infectivity. Structural studies have identified three intersubunit interfaces in the HIV-1 capsid, two of which have been previously studied by mutational analysis. In this present study we analyzed the role of a third interface, that which is formed between the amino terminal domain (NTD) and carboxyl terminal domain (CTD) of adjacent subunits. RESULTS: We provided evidence for the presence of the NTD-CTD interface in HIV-1 particles by engineering intersubunit NTD-CTD disulfide crosslinks, resulting in accumulation of disulfide-linked oligomers up to hexamers. We also generated and characterized a panel of HIV-1 mutants containing substitutions at this interface. Some mutants showed processing defects and altered morphology from that of wild type, indicating that the interface is important for capsid assembly. Analysis of these mutants by transmission electron microscopy corroborated the importance of this interface in assembly. Other mutants exhibited quantitative changes in capsid stability, many with unstable capsids, and one mutant with a hyperstable capsid. Analysis of the mutants for their capacity to saturate TRIMCyp-mediated restriction in trans confirmed that the unstable mutants undergo premature uncoating in target cells. All but one of the mutants were markedly attenuated in replication owing to impaired reverse transcription in target cells. CONCLUSIONS: Our results demonstrate that the NTD-CTD intersubunit interface is present in the mature HIV-1 capsid and is critical for proper capsid assembly and stability

Protease Cleavage Leads to Formation of Mature Trimer Interface in HIV-1 Capsid

During retrovirus particle maturation, the assembled Gag polyprotein is cleaved by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. To form the mature viral capsid, CA rearranges, resulting in a lattice composed of hexameric and pentameric CA units. Recent structural studies of assembled HIV-1 CA revealed several inter-subunit interfaces in the capsid lattice, including a three-fold interhexamer interface that is critical for proper capsid stability. Although a general architecture of immature particles has been provided by cryo-electron tomographic studies, the structural details of the immature particle and the maturation pathway remain unknown. Here, we used cryo-electron microscopy (cryoEM) to determine the structure of tubular assemblies of the HIV-1 CA-SP1-NC protein. Relative to the mature assembled CA structure, we observed a marked conformational difference in the position of the CA-CTD relative to the NTD in the CA-SP1-NC assembly, involving the flexible hinge connecting the two domains. This difference was verified via engineered disulfide crosslinking, revealing that inter-hexamer contacts, in particular those at the pseudo three-fold axis, are altered in the CA-SP1-NC assemblies compared to the CA assemblies. Results from crosslinking analyses of mature and immature HIV-1 particles containing the same Cys substitutions in the Gag protein are consistent with these findings. We further show that cleavage of preassembled CA-SP1-NC by HIV-1 protease in vitro leads to release of SP1 and NC without disassembly of the lattice. Collectively, our results indicate that the proteolytic cleavage of Gag leads to a structural reorganization of the polypeptide and creates the three-fold interhexamer interface, important for the formation of infectious HIV-1 particles. © 2012 Meng et al

Mature Hiv-1 Capsid Structure By Cryo-Electron Microscopy And All-Atom Molecular Dynamics

Retroviral capsid proteins are conserved structurally but assemble into different morphologies. The mature human immunodeficiency virus-1 (HIV-1) capsid is best described by a \u27fullerene cone\u27 model, in which hexamers of the capsid protein are linked to form a hexagonal surface lattice that is closed by incorporating 12 capsid-protein pentamers. HIV-1 capsid protein contains an amino-terminal domain (NTD) comprising seven α-helices and a β-hairpin, a carboxy-terminal domain (CTD) comprising four α-helices, and a flexible linker with a 3 10-helix connecting the two structural domains. Structures of the capsid-protein assembly units have been determined by X-ray crystallography; however, structural information regarding the assembled capsid and the contacts between the assembly units is incomplete. Here we report the cryo-electron microscopy structure of a tubular HIV-1 capsid-protein assembly at 8 Å resolution and the three-dimensional structure of a native HIV-1 core by cryo-electron tomography. The structure of the tubular assembly shows, at the three-fold interface, a three-helix bundle with critical hydrophobic interactions. Mutagenesis studies confirm that hydrophobic residues in the centre of the three-helix bundle are crucial for capsid assembly and stability, and for viral infectivity. The cryo-electron-microscopy structures enable modelling by large-scale molecular dynamics simulation, resulting in all-atom models for the hexamer-of-hexamer and pentamer-of-hexamer elements as well as for the entire capsid. Incorporation of pentamers results in closer trimer contacts and induces acute surface curvature. The complete atomic HIV-1 capsid model provides a platform for further studies of capsid function and for targeted pharmacological intervention. © 2013 Macmillan Publishers Limited. All rights reserved

Performance evaluation of the Asante Rapid Recency Assay for verification of HIV diagnosis and detection of recent HIV-1 infections: Implications for epidemic control.

We previously described development of a rapid test for recent infection (RTRI) that can diagnose HIV infection and detect HIV-1 recent infections in a single device. This technology was transferred to a commercial partner as Asante Rapid Recency Assay (ARRA). We evaluated performance of the ARRA kits in the laboratory using a well-characterized panel of specimens. The plasma specimen panel (N = 1500) included HIV-1 (N = 570), HIV-2 (N = 10), and HIV-negatives (N = 920) representing multiple subtypes and geographic locations. Reference diagnostic data were generated using the Bio-Rad HIV-1-2-O EIA/Western blot algorithm with further serotyping performed using the Multispot HIV-1/2 assay. The LAg-Avidity EIA was used to generate reference data on recent and long-term infection for HIV-1 positive specimens at a normalized optical density (ODn) cutoff of 2.0 corresponding to a mean duration of about 6 months. All specimens were tested with ARRA according to the manufacturer's recommendations. Test strips were also read for line intensities using a reader and results were correlated with visual interpretation. ARRA's positive verification line (PVL) correctly classified 575 of 580 HIV-positive and 910 of 920 negative specimens resulting in a sensitivity of 99.1% (95% CI: 98.0-99.6) and specificity of 98.9% (95% CI: 98.1-99.4), respectively. The reader-based classification was similar for PVL with sensitivity of 99.3% (576/580) and specificity of 98.8% (909/920). ARRA's long-term line (LTL) classified 109 of 565 HIV-1 specimens as recent and 456 as long-term compared to 98 as recent and 467 as long-term (LT) by LAg-Avidity EIA (cutoff ODn = 2.0), suggesting a mean duration of recent infection (MDRI) close to 6 months. Agreement of ARRA with LAg recent cases was 81.6% (80/98) and LT cases was 93.8% (438/467), with an overall agreement of 91.7% (kappa = 0.72). The reader (cutoff 2.9) classified 109/566 specimens as recent infections compared to 99 by the LAg-Avidity EIA for recency agreement of 81.8% (81/99), LT agreement of 9% (439/467) with overall agreement of 91.9% (kappa = 0.72). The agreement between visual interpretation and strip reader was 99.9% (95% CI: 99.6-99.9) for the PVL and 98.1% (95% CI: 96.6-98.9) for the LTL. ARRA performed well with HIV diagnostic sensitivity >99% and specificity >98%. Its ability to identify recent infections is comparable to the LA-Avidity EIA corresponding to an MDRI of about 6 months. This point-of-care assay has implications for real-time surveillance of new infections among newly diagnosed individuals for targeted prevention and interrupting ongoing transmission thus accelerating epidemic control

A Stable Dried Tube Specimen for Quality Assurance and Training Programs for HIV Rapid Test for Recent Infection

The HIV epidemic is still one of the world's most serious public health challenges, affecting about 38 million people worldwide, especially in sub-Saharan African and Southeast Asian countries. In recent years, tests have been developed to discriminate recent from long-term infection in HIV-infected populations, and these tools can help identify new outbreaks and networks of transmission and target prevention and treatment plans. New rapid tests for recent infection are being deployed in point-of-care settings; however, quality assurance programs need to be implemented to ensure consistency and reliability of the results. We have developed a dried tube specimen (DTS) stabilized with disaccharide trehalose as a quality control reagent for rapid recency testing that can be stored unrefrigerated prior to reconstitution at temperatures up to 37°C for up to 12 weeks. Analysis of 10 trehalose-stabilized DTSs showed that they maintained the same recency classification in all of the samples stored at 4°C and 37°C up to 12 weeks and at 56°C for 2 weeks, while the DTSs prepared without trehalose changed their classification from long-term to recent or recent to negative after storage at 37°C for 12 weeks. Development of DTS quality control reagents will facilitate proficiency and training programs, particularly in settings without cold chain capability in field environments. IMPORTANCE Implementation of stabilized dried tube specimens (DTSs) for quality control and training would facilitate HIV recency programs, especially in point-of-care settings without cold chain availability. This study shows that addition of the disaccharide trehalose to DTSs prior to drying the samples increased stability of the samples across a range of temperatures. This finding provides an affordable way to increase the availability of these key reagents for quality control in resource-constrained settings

A model depicting formation of the mature capsid trimer interface upon protease cleavage.

<p>(A) Structure model of the CA-SP1-NC protein was composed using the CA model derived from molecular docking (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002886#ppat-1002886-g002" target="_blank">Fig. 2</a>), combined with a SP1 partial helix (hypothetical) and the NC model (PDB 1f6u). The linker between CA-CTD and SP1 is in close contact with the MHR region of CA-CTD (in red). Scissors point to the two protease cleavage sites, Site 1 and Site 2, respectively. (B) Superposition of the CA hexameric structural models from CA-SP1-NC (blue) and CA structures (yellow) viewed from top and side. The two CA models were aligned at the CA-NTD region (light blue). (C) Comparison of the trimer interfaces (red-dashed circles) between CA-SP1-NC (blue) and CA structures (yellow).</p

Molecular docking of the CA-SP1-NC density map.

<p>(A–C) Docking of the CA-NTD (PDB 3h47) and CA-CTD (PDB 2kod) domain models, independently, into the CA region (gold) of the density map contoured at 1.22σ, enclosing 100% volume. Shown are a view onto the tube surface (A) and slab views to highlight the NTD (B) and CTD regions (C). Three neighbouring CA hexamer models are in cyan, magenta and pink. The red star indicates the pseudo-three fold axis where the trimer interface is found in the CA assembly <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002886#ppat.1002886-Byeon1" target="_blank">[20]</a>. (D) Placing of the SP1 segment (PDB 1u57) into the CA-SP1-NC tubular density map. The CA-NTD in gold, CTD in light blue and SP1 in orange are shown for a single hexamer in the assembly. The density contour at the NC/DNA region is shown in white. The inset (top right), magnified from the white box, shows the overlapped region of the CA-CTD and SP1 models.</p

Comparison of the intermolecular interfaces and chemical crosslinking patterns between CA and CA-SP1-NC structures.

<p>(A) Superposition of the CA monomer model from the assembled CA structure (gold) and that from the assembled CA-SP1-NC structure (blue). (B–D) Superposition of the CA-NTD models at the NTD interfaces (B&C) and CA-CTD models at the trimer interface (D). The color scheme is identical to (A). Space-filling representation highlights the specific residue pairs; 17/19 in (B), 14/45 in (C) and 207/216 in (D), with red corresponding to those from CA and cyan to those from CA-SP1-NC. (E) Non-reducing SDS-PAGE analysis of <i>in vitro</i> disulfide crosslinked CA (lanes 2, 4 and 6) and CA-SP1-NC assemblies (lanes 3, 5 and 7), stained with Coomassie Blue. Lanes were rearranged from original gels shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002886#ppat.1002886.s003" target="_blank">Fig. S3</a>. (F) Non-reducing SDS-PAGE analysis of crosslinking of mature (lanes 2, 4 and 7) and cleavage-defective HIV-1 CA6 particles (lanes 3, 5 and 6). Proteins were detected by immunoblotting with a CA-specific antibody.</p