Key Findings
♦ Lenacapavir is a novel long-acting anti-HIV drug with extremely potent antiviral activity.
♦ Its known mode of action is to block infection by viruses entering target cells. A second mechanism of action had been predicted to explain its potent antiviral effect, but its nature remained unknown.
♦ This study revealed that lenacapavir causes abnormalities in HIV particle formation, and that this is its second antiviral mechanism of action.
♦ Analyses using particle size measurement technology and electron microscopy showed that lenacapavir causes virus particles to become abnormally large, reducing the infectivity of the virus.
Overview
Lenacapavir (hereafter LEN) is a novel long-acting therapeutic agent that potently suppresses the replication of HIV-1, the virus that causes AIDS (acquired immunodeficiency syndrome). It targets the capsid protein*1, which forms the framework of the virus particle. LEN strongly inhibits the production of progeny virus by blocking the series of steps from viral entry into the target cell to the integration of the viral genes into the genome of the target cell (the early phase of replication). However, this inhibition of the early phase alone could not explain the potent antiviral activity of LEN. A second mechanism of action was therefore assumed to exist, but its nature remained unknown. Now, a research group led by Dr. Kazuaki Monde, Senior Lecturer at the Faculty of Life Sciences, Kumamoto University, and Dr. Yasumasa Iwatani, Professor at Hamamatsu University School of Medicine, has shown for the first time, through analyses making full use of particle size measurement technology and electron microscopy, that LEN binds to the capsid precursor protein*2 during virus particle formation and promotes the formation of giant virus-like particles. The group found that these abnormal particles enlarged by LEN are still able to enter target cells, but are dysfunctional and have lost their infectivity. These findings not only deepen our understanding of the mechanism of action of LEN, a drug with a novel principle of action, but are also expected to provide clues for applying this principle to the development of even more potent next-generation antiviral drugs.
Background and Objectives
Human immunodeficiency virus type 1 (HIV-1), the causative virus of AIDS, has a cone-shaped core (a cage-like structure that encloses the viral genes) inside the virus particle, which is indispensable for the virus to infect target cells. The core is composed of approximately 200 molecules of the viral capsid protein (CA). Lenacapavir (hereafter LEN), a novel HIV-1 drug recently developed by Gilead Sciences, Inc. (USA), is a long-acting therapeutic agent that targets this CA and has extremely high antiviral activity (see reference figure). It had previously been shown that LEN acts by strongly inhibiting the production of progeny virus through blocking the series of steps from viral entry into the target cell to the integration of the viral genes into the chromosomal genome of the cell (referred to as the early phase of replication). In other words, LEN is thought to inhibit either the transport of the virus particle into the cell nucleus or the release of the viral genes from the core, both of which are essential for establishing infection. However, the inhibition of the early phase alone could not fully explain the potent antiviral effect of LEN, and a second mechanism of action was assumed to exist, but its nature remained unknown. Elucidating this second mechanism is necessary to advance the development of drugs that surpass LEN. This study therefore aimed to clarify the nature of a second mechanism distinct from that acting in the early phase.
Research Details
In this study, the effects of LEN on the late phase of HIV-1 replication were analyzed using a variety of methods. The group found that the inhibitory effect of LEN differed markedly depending on the method used to measure the amount of virus. LEN treatment reduces the solubility of CA derived from the capsid precursor protein (hereafter Gag); as a result, the CA ELISA (enzyme-linked immunosorbent assay) overestimates the inhibitory effect of LEN on virus release. Furthermore, the group discovered that in the presence of LEN, the processing (maturation) of intracellular Gag by the viral protease*3 is excessively accelerated, and determined that this leads to impaired formation and release of normal virus particles.
The group also found that LEN treatment induces the formation of numerous abnormal Gag clusters on the cell membrane. Investigations combining mass photometry*4, a particle size measurement technology (see reference figure A: particle size analysis), with transmission electron microscopy (TEM)*5 analysis (see reference figure B: electron microscopy analysis) revealed that LEN treatment leads to the formation of heterogeneous, giant virus-like particles exceeding 200 nm in diameter (LEN-induced virus-like particles, hereafter LENiVLPs). These LENiVLPs contained Gag as well as envelope glycoproteins (proteins covering the viral surface), showing that they are not mere Gag aggregates but structures derived from the virus particle formation process.
In addition, LENiVLPs were found to retain the ability to fuse with the cell membrane, yet to show no infectivity. This is because, while LENiVLPs maintain the ability to enter target cells, the viral replication steps following entry are impaired. Taken together, these results show that LEN acts not only on mature CA but also on the capsid precursor protein Gag during particle formation, inducing abnormal Gag maturation and particle formation, and ultimately causing the formation of giant, non-infectious LENiVLPs. This study reveals a new principle of action in the late phase of replication as the second mechanism of action of LEN, and provides new insights into the mechanisms of action of CA-targeting anti-HIV drugs.
Future Outlook
This study showed that LEN causes virus particles to become giant and functionally defective as viruses. Although it is now understood that abnormalities occur in the assembly of the precursor protein during particle formation, the detailed mechanism has not yet been clarified. For example, the differences in the binding mode (pharmacophore*6) of LEN to its drug target pocket that give rise to the two mechanisms of action remain unclear, and structural biology analyses are required. Going forward, the group plans to identify the pharmacophores for both mechanisms of action with greater precision. This is expected to advance the creation of compounds that further enhance each of the inhibitory effects, and to accelerate the improvement and development of more potent therapeutic drugs that are less prone to drug resistance.
Glossary
*1 Capsid (CA)
A protein that constitutes the protein shell (core) inside the HIV-1 virus particle (see reference figure). It protects the genetic information of the virus and plays an important role in viral replication within the infected target cell. LEN acts by targeting this capsid.
*2 Capsid precursor protein (Gag)
The precursor protein from which the proteins required to form the shape of the HIV-1 particle are derived. During virus particle formation, it gathers at the cell membrane and forms the basic structure of the particle. It is then sequentially cleaved by the viral protease, resulting in the formation of mature virus particles.
*3 Viral protease
A virus-derived enzyme required to cleave HIV-1 proteins at the appropriate sites and to produce infectious, mature virus particles.
*4 Mass photometry
A technique for measuring the size of ultrafine particles. It uses light scattering to measure, one particle at a time, the size and mass of particles in solution, such as virus particles. In this study, this technique, previously used in other fields, was applied to the detection and analysis of giant virus-like particles.
*5 Transmission electron microscope (TEM)
An instrument that uses an electron beam to observe very small structures, such as virus particles, at high resolution. It enables the analysis of fine structures that cannot be resolved with an ordinary optical microscope.
*6 Pharmacophore
A concept describing the molecular features and their arrangement required for a drug to bind to and act on its target protein; that is, the three-dimensional features of a drug when it binds. Refining the pharmacophore increases the precision with which inhibitory compounds can be designed and improved. This study aims to contribute to more effective drug development by clarifying the differences in the binding modes of LEN when it acts at different stages of HIV-1 replication.
Journal
Proceedings of the National Academy of Sciences of the United States of America (PNAS)
DOI: https://doi.org/10.1073/pnas.2609634123
Paper Title
Lenacapavir binding to immature Gag induces giant virions and causes protease-dependent inhibition of viral release
Authors
Wright Andrews Ofotsu Amesimeku, Yoshihiro Nakata, Nami Monde, Hiromi Terasawa, Hirotaka Ode, Hiroyuki Sasaki, Consolata Elias Rukondo, Takeshi Matsui, Perpetual Nyame, Md. Jakir Hossain, Akatsuki Saito, Tomohiro Sawa, Terumasa Ikeda, Yosuke Maeda, Yasumasa Iwatani, Kazuaki Monde
Research Group
This research was conducted as a collaboration among the Department of Microbiology, Faculty of Life Sciences, Kumamoto University; the Department of Microbiology and Immunology, Hamamatsu University School of Medicine; the Laboratory for Viral Infection Control, Frontier Science Research Center, University of Miyazaki; the Department of Infectious Diseases and Immunology, Clinical Research Center, National Hospital Organization Nagoya Medical Center; the Laboratory for Evolutionary Cell Biology of the Skin, Cosmetics Course, School of Bioscience and Biotechnology, Tokyo University of Technology; and the Joint Research Center for Human Retrovirus Infection, Kumamoto University.
Funding
This research was supported by the Japan Agency for Medical Research and Development (AMED) Research Program on HIV/AIDS (25fk0410058h9903 and 26fk0410080h0001: Principal Investigator Iwatani, Co-Investigator Monde; 19fk0410026h0001 and 24fk0410065h0001: Principal Investigator Monde), among others.
Contact for Research Inquiries
Kazuaki Monde, Senior Lecturer
Department of Microbiology, Faculty of Life Sciences, Kumamoto University (National University Corporation)
1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan
Tel: +81-96-373-5129 / E-mail: monde@kumamoto-u.ac.jp
Contact for Media Inquiries
Kumamoto University (National University Corporation)
Public Relations Strategy Office, General Affairs Division, General Affairs Department
Tel: +81-96-342-3269
E-mail: sos-koho@jimu.kumamoto-u.ac.jp
