Scientists at the Electron Bio-Imaging Centre (eBIC) at Diamond Light Source, in the U.K., have harnessed a new technique, using cryo-electron tomography (cryoET) and subtomogram averaging (STA) to solve the structure of the HIV-1 capsid alone, and in complex with host cell factors. Their work enabled them to use the information gained from electron tomography, to build an atomistic model of the whole HIV capsid. The team believes this could serve as a blueprint for the development of capsid-targeting antivirals.

The researchers, headed by Peijun Zhang, PhD, Director of the eBIC and Professor of Structural Biology at the University of Oxford, reported on their findings in Science Advances, in a paper titled, “Structure of native HIV-1 cores and their interactions with IP6 and CypA.” The reported work involved a collaboration between scientists at the University of Oxford, eBIC—the U.K.’s national cryo-electron microscopy facility within Diamond Light Source—and the University of Delaware.

Retroviruses such as HIV are enveloped single-stranded positive-sense RNA viruses. Study lead author, Tao Ni, at the University of Oxford commented, “Despite the global efforts to combat HIV/AIDS and the achievement of antiviral treatments, there are still approximately 38 million people with HIV/AIDS with no complete cure so far.”

HIV has its RNA genome encapsulated inside a conical-shaped capsid. As well as offering protection from host innate immune responses during the early stage of infection this capsid plays essential roles in HIV replication, and is “a major platform engaging host factors,” the team continued. “The capsid plays multiple roles during the early stage of HIV-1 replication, including protecting the genome from cellular innate immune responses and fostering reverse transcription, as well as regulating intracellular transport and entry into the nucleus.” Many of these functions are affected by its interactions with host cell factors and small molecules. “

When retroviruses infect a cell, the viral RNA genome is reverse-transcribed into a double-stranded DNA, which is then integrated into the host genome. During infection, HIV assembles and buds as immature virions with Gag polyprotein, which undergoes maturation process, a step involving proteolysis and conformational change, which converts from an immature spherical shape to mature conical capsid.

However, because of the metastable property of HIV-1 capsid, isolating fully intact native capsid in quantities and concentrations suitable for high-resolution structural analyses has been challenging. The capsid suffers artefactual dissociation after the membrane is dissolved by detergent, a traditional way for capsid purification.  “To solve this problem, Peijun Zhang’s team devised a novel approach,” Ni noted. “Instead of detergent extraction, we punctuate the membrane of HIV virus-like particles with a pore-forming toxin, which avoids the trauma associated with lysis of the virions and isolation of the cores, but also makes the capsid accessible to external cell factors and small molecules.”

The authors further explained, “… we devised a novel strategy to directly image authentic HIV-1 cores, in the absence or presence of exogenous host factors and small molecules, by treating virus particles with perfringolysin O (PFO). PFO is a cholesterol-dependent cytolysin secreted by the pathogenic Clostridium perfringens that forms giant homo-oligomeric pores with diameter ~40 nm on cholesterol-rich membrane bilayers, such as membranes of HIV-1 particles.”

Having established this experimental approach, the authors investigated the interactions between the authentic HIV capsid and a cellular factor cyclophilin A (CypA), and a small-molecule cofactor IP6 (inositol hexakisphosphate). They then applied electron tomography and subtomogram averaging to these samples. “We imaged PFO-perforated HIV-1 VLPs by cryoET STA and determined the structures of HIV-1 native capsomeres in the apo state and in the presence of IP6.”

Using this new technique, the team solved the structures of HIV capsid alone, and its complex with CypA and IP6 at around 5.4 Å resolution. These structures confirmed the double IP6 binding site in mature HIV capsid and provide new insights into the role of IP6 and CypA in regulating HIV capsid stability. “Zhang further commented, “In collaboration with prof. Juan Perilla’s group in the University of Delaware, using information derived from electron tomography, we also built an atomistic model of the whole HIV capsid which could serve as a blueprint for the development of capsid-targeting antivirals. The perforation of the enveloped virus membrane also provides a novel approach to study host-virus interaction for other viral systems.”

The authors concluded, “Technically, this study established PFO-perforated VLPs as an excellent ex vivo system to study interactions between host cell factors and authentic HIV-1 capsid, circumventing the difficulties inherent with purifying the metastable core from virions. Perforated VLPs enable host cell factors and small molecules to access the native capsid while protecting virus cores.”

Zhang carries out research into HIV and other infectious diseases, and her work, focuses on structural and functional studies of large molecular complexes and assemblies, viruses and cellular machineries, using integrated structural, biochemical and computational approaches to understand biological complexity.

As the director of eBIC, professor Zhang is establishing and leading eBIC to become a world-leading centre for research, expertise and training in cryo-EM and a user facility providing access to cutting-edge cryo-EM technologies. eBIC focuses on using state-of-the-art electron microscopic techniques to determine the 3D structures of molecules, cells and tissues at high resolution, as well as developing new methods and technologies to advance 3D EM imaging.

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