Photocatalytic Inactivation of Viruses Using Graphitic Carbon Nitride-Based Photocatalysts: Virucidal Performance and Mechanism

The prevalence of lethal viral infections necessitates the innovation of novel disinfection techniques for contaminated surfaces, air, and wastewater as significant transmission media of disease. The instigated research has led to the development of photocatalysis as an effective renewable solar-driven technology relying on the reactive oxidative species, mainly hydroxyl (OH●) and superoxide (O2●−) radicals, for rupturing the capsid shell of the virus and loss of pathogenicity. Metal-free graphitic carbon nitride (g-C3N4), which possesses a visible light active bandgap structure, low toxicity, and high thermal stability, has recently attracted attention for viral inactivation. In addition, g-C3N4-based photocatalysts have also experienced a renaissance in many domains, including environment, energy conversion, and biomedical applications. Herein, we discuss the three aspects of the antiviral mechanism, intending to highlight the advantages of photocatalysis over traditional viral disinfection techniques. The sole agenda of the review is to summarize the significant research on g-C3N4-based photocatalysts for viral inactivation by reactive oxidative species generation. An evaluation of the photocatalysis operational parameters affecting viral inactivation kinetics is presented. An overview of the prevailing challenges and sustainable solutions is presented to fill in the existing knowledge gaps. Given the merits of graphitic carbon nitride and the heterogeneous photocatalytic viral inactivation mechanism, we hope that further research will contribute to preventing the ongoing Coronavirus pandemic and future calamities.

The Hepatitis B Virus Nucleocapsid—Dynamic Compartment for Infectious Virus Production and New Antiviral Target

Hepatitis B virus (HBV) is a small enveloped DNA virus which replicates its tiny 3.2 kb genome by reverse transcription inside an icosahedral nucleocapsid, formed by a single ~180 amino acid capsid, or core, protein (Cp). HBV causes chronic hepatitis B (CHB), a severe liver disease responsible for nearly a million deaths each year. Most of HBV’s only seven primary gene products are multifunctional. Though less obvious than for the multi-domain polymerase, P protein, this is equally crucial for Cp with its multiple roles in the viral life-cycle. Cp provides a stable genome container during extracellular phases, allows for directed intracellular genome transport and timely release from the capsid, and subsequent assembly of new nucleocapsids around P protein and the pregenomic (pg) RNA, forming a distinct compartment for reverse transcription. These opposing features are enabled by dynamic post-transcriptional modifications of Cp which result in dynamic structural alterations. Their perturbation by capsid assembly modulators (CAMs) is a promising new antiviral concept. CAMs inappropriately accelerate assembly and/or distort the capsid shell. We summarize the functional, biochemical, and structural dynamics of Cp, and discuss the therapeutic potential of CAMs based on clinical data. Presently, CAMs appear as a valuable addition but not a substitute for existing therapies. However, as part of rational combination therapies CAMs may bring the ambitious goal of a cure for CHB closer to reality.

High Resolution Structure of the Mature Capsid of Ralstonia Solanacearum Bacteriophage ϕRSA1 by Cryo-Electron Microscopy

The ϕRSA1 bacteriophage has been isolated from Ralstonia solanacearum, a gram negative bacteria having a significant economic impact on many important crops. We solved the three-dimensional structure of the ϕRSA1 mature capsid to 3.9 Å resolution by cryo-electron microscopy. The capsid shell, that contains the 39 kbp of dsDNA genome, has an icosahedral symmetry characterized by an unusual triangulation number of T = 7, dextro. The ϕRSA1 capsid is composed solely of the polymerization of the major capsid protein, gp8, which exhibits the typical “Johnson” fold first characterized in E. coli bacteriophage HK97. As opposed to the latter, the ϕRSA1 mature capsid is not stabilized by covalent crosslinking between its subunits, nor by the addition of a decoration protein. We further describe the molecular interactions occurring between the subunits of the ϕRSA1 capsid and their relationships with the other known bacteriophages.

All-atom molecular dynamics study of hepatitis B virus containing pregenome RNA in solution

Immature hepatitis B virus (HBV) captures nucleotides in its capsid for reverse transcription. The nucleotides and nucleotide analogue drugs, which are triphosphorylated and negatively charged in the cell, approach the capsid via diffusion and are absorbed into it. In this study, we performed a long-time molecular dynamics (MD) calculation of the entire HBV capsid containing pregenome RNA to investigate the interactions between the capsid and negatively charged substances. Electric field analysis demonstrated that negatively charged substances can approach the HBV capsid by thermal motion, avoiding spikes. The substances then migrate all over the floor of the HBV capsid. Finally, they find pores through which they can pass through the HBV capsid shell. Free energy profiles were calculated along these pores for small ions to understand their permeability through the pores. Anions (Cl-) showed higher free energy barriers than cations (Na+ and K+) through all pores, and the permeation rate of Cl- was eight times slower than that of K+ or Na+. Furthermore, the ions were more stable in the capsid than in the bulk water. Thus, the HBV capsid exerts ion selectivity for uptake and provides an environment for ions, such as nucleotides and nucleotide analogue drugs, to be stabilized within the capsid.

RNA X-ray footprinting reveals the consequences of an in vivo acquired determinant of viral infectivity

The secondary structures of the bacteriophage MS2 ssRNA genome, frozen in defined states, were determined with minimal perturbation using constraints from X-ray synchrotron footprinting (XRF). The footprints of the gRNA in the virion and as transcript are consistent with single, dominant but distinct conformations, and reveal the presence of multiple Packaging Signals potentially involved in assembly regulation that have not been detected by other techniques. XRF also reveals the dramatic effect of the unique Maturation Protein (MP) on both the capsid lattice, and the gRNA conformation inside the phage compared with a virus-like-particle composed only of coat protein subunits. Aspects of genome organisation in the phage, their impacts on the capsid shell, and the distortion of lattice geometry by MP, are hallmarks of molecular frustration. Phage assembly therefore appears to prepare the particle for the next step of the infectious cycle.

Atomic Structure of the Trichomonas vaginalis Double-Stranded RNA Virus 2

ABSTRACT Trichomonas vaginalis, the causative pathogen for the most common nonviral sexually transmitted infection worldwide, is itself frequently infected with one or more of the four types of small double-stranded RNA (dsRNA) Trichomonas vaginalis viruses (TVV1 to 4, genus Trichomonasvirus, family Totiviridae). Each TVV encloses a nonsegmented genome within a single-layered capsid and replicates entirely intracellularly, like many dsRNA viruses, and unlike those in the Reoviridae family. Here, we have determined the structure of TVV2 by cryo-electron microscopy (cryoEM) at 3.6 Å resolution and derived an atomic model of its capsid. TVV2 has an icosahedral, T = 2*, capsid comprised of 60 copies of the icosahedral asymmetric unit (a dimer of the two capsid shell protein [CSP] conformers, CSP-A and CSP-B), typical of icosahedral dsRNA virus capsids. However, unlike the robust CSP-interlocking interactions such as the use of auxiliary “clamping” proteins among Reoviridae, only lateral CSP interactions are observed in TVV2, consistent with an assembly strategy optimized for TVVs’ intracellular-only replication cycles within their protozoan host. The atomic model reveals both a mostly negatively charged capsid interior, which is conducive to movement of the loosely packed genome, and channels at the 5-fold vertices, which we suggest as routes of mRNA release during transcription. Structural comparison of TVV2 to the Saccharomyces cerevisiae L-A virus reveals a conserved helix-rich fold within the CSP and putative guanylyltransferase domain along the capsid exterior, suggesting conserved mRNA maintenance strategies among Totiviridae. This first atomic structure of a TVV provides a framework to guide future biochemical investigations into the interplay between Trichomonas vaginalis and its viruses. IMPORTANCE Trichomonas vaginalis viruses (TVVs) are double-stranded RNA (dsRNA) viruses that cohabitate in Trichomonas vaginalis, the causative pathogen of trichomoniasis, the most common nonviral sexually transmitted disease worldwide. Featuring an unsegmented dsRNA genome encoding a single capsid shell protein (CSP), TVVs contrast with multisegmented dsRNA viruses, such as the diarrhea-causing rotavirus, whose larger genome is split into 10 dsRNA segments encoding 5 unique capsid proteins. To determine how TVVs incorporate the requisite functionalities for viral replication into their limited proteome, we derived the atomic model of TVV2, a first for TVVs. Our results reveal the intersubunit interactions driving CSP association for capsid assembly and the properties that govern organization and maintenance of the viral genome. Structural comparison between TVV2 capsids and those of distantly related dsRNA viruses indicates conserved strategies of nascent RNA release and a putative viral guanylyltransferase domain implicated in the cytoplasmic maintenance of viral messenger and genomic RNA.

A smart viral vector for targeted delivery of hydrophobic drugs

Targeted delivery of hydrophobic chemotherapeutic drugs to tumor cells remains a fundamental problem in cancer therapy. Effective encapsulation of hydrophobic drugs in nano-vehicles can improve their pharmacokinetics, bioavailability and prevent off-target localization. We have devised a method for easy chemical conjugation and multivalent display of a tumor-homing peptide to virus-like particles of a non-mammalian virus, Flock House Virus (FHV), to engineer it into a smart vehicle for targeted delivery of hydrophobic drugs. This conjugation method provides dual functionalization to the VLPs, first, a 2 kDa PEG spacer arm shields VLPs from immune reactivity, and second, attachment of the tumor homing peptide tLyP-1 chauffeurs the encapsulated hydrophobic drugs to target cells. The fortuitous affinity of the FHV capsid towards hydrophobic molecules, and dependence on Ca2+ for maintaining a stable capsid shell, were utilized for incorporation of hydrophobic drugs—doxorubicin and ellipticine—in tLyP-1 conjugated VLPs. The drug release profile from the VLP was observed to be gradual, and strictly endosomal pH dependent. We propose that this accessible platform empowers surface functionalization of VLP with numerous ligands containing terminal cysteines, for generating competent delivery vehicles, antigenic display and other biomedical applications.

Cryo-Electron Tomography of the Herpesvirus Procapsid Reveals Interactions of the Portal with the Scaffold and a Shift on Maturation

ABSTRACT Herpes simplex virus 1 (HSV-1) requires seven proteins to package its genome through a vertex in its capsid, one of which is the portal protein, pUL6. The portal protein is also thought to facilitate assembly of the procapsid. While the portal has been visualized in mature capsids, we aimed to elucidate its role in the assembly and maturation of procapsids using cryo-electron tomography (cryoET). We identified the portal vertex in individual procapsids, calculated a subtomogram average, and compared that with the portal vertex in empty mature capsids (A-capsids). The resulting maps show the portal on the interior surface with its narrower end facing outwards, while maintaining close contact with the capsid shell. In the procapsid, the portal is embedded in the underlying scaffold, suggesting that assembly involves a portal-scaffold complex. During maturation, the capsid shell angularizes with a corresponding outward movement of the vertices. We found that in A-capsids, the portal translocates outward further than the adjacent capsomers and strengthens its contacts with the capsid shell. Our methodology also allowed us to determine the number of portal vertices in each capsid, with most having one per capsid, but some none or two, and rarely three. The predominance of a single portal per capsid supports facilitation of the assembly of the procapsid. IMPORTANCE Herpes simplex virus 1 (HSV-1) infects a majority of humans, causing mostly mild disease but in some cases progressing toward life-threatening encephalitis. Understanding the life cycle of the virus is important to devise countermeasures. Production of the virion starts with the assembly of an icosahedral procapsid, which includes DNA packaging proteins at a vertex, one of which is the dodecameric portal protein. The procapsid then undergoes maturation and DNA packaging through the portal, driven by a terminase complex. We used cryo-electron tomography to visualize the portal in procapsids and compare them to mature empty capsids. We found the portal located inside one vertex interacting with the scaffold protein in the procapsid. On maturation, the scaffold is cleaved and dissociates, the capsid angularizes, and the portal moves outward, interacting closely with the capsid shell. These transformations may provide a basis for the development of drugs to prevent HSV-1 infections.

HIV-1 uncoating occurs via a series of rapid biomechanical changes in the core related to individual stages of reverse transcription

The HIV core consists of the viral genome and associated proteins encased by a cone-shaped protein shell termed the capsid. Successful infection requires reverse transcription of the viral genome and disassembly of the capsid shell within a cell in a process known as uncoating. The integrity of the viral capsid is critical for reverse transcription, yet the viral capsid must be breached to release the nascent viral DNA prior to integration. We employed atomic force microscopy to study the stiffness changes in HIV-1 cores during reverse transcription in vitro in reactions containing the capsid-stabilizing host metabolite IP6. Cores exhibited a series of stiffness spikes, with up to three spikes typically occurring between 10-30, 40-80, and 120-160 minutes after initiation of reverse transcription. Addition of the reverse transcriptase (RT) inhibitor efavirenz eliminated the appearance of these spikes and the subsequent disassembly of the capsid, thus establishing that both result from reverse transcription. Using timed addition of efavirenz, and analysis of an RNAseH-defective RT mutant, we established that the first stiffness spike requires minus-strand strong stop DNA synthesis, with subsequent spikes requiring later stages of reverse transcription. Additional rapid AFM imaging experiments revealed repeated morphological changes in cores that were temporally correlated with the observed stiffness spikes. Our study reveals discrete mechanical changes in the viral core that are likely related to specific stages of reverse transcription. These reverse-transcription-induced changes in the capsid progressively remodel the viral core to prime it for temporally accurate uncoating in target cells. Importance For successful infection, the HIV-1 genome, which is enclosed inside a capsid shell, must be reverse transcribed into double-stranded DNA and released from the capsid (in a process known as uncoating) before it can be integrated into the target cell genome. The mechanism of HIV-1 uncoating is a pivotal question of long standing. Using atomic force microscopy to analyze individual HIV-1 cores during reverse transcription, we observe a reproducible pattern of stiffness spikes. These spikes were shown to be associated with distinct stages of the reverse transcription reaction. Our findings suggest that these reverse-transcription-induced alterations gradually prepared the core for uncoating at the right time and location in target cells.

Imaging Viral Infection by Fluorescence Microscopy: Focus on HIV-1 Early Stage

During the last two decades, progresses in bioimaging and the development of various strategies to fluorescently label the viral components opened a wide range of possibilities to visualize the early phase of Human Immunodeficiency Virus 1 (HIV-1) life cycle directly in infected cells. After fusion of the viral envelope with the cell membrane, the viral core is released into the cytoplasm and the viral RNA (vRNA) is retro-transcribed into DNA by the reverse transcriptase. During this process, the RNA-based viral complex transforms into a pre-integration complex (PIC), composed of the viral genomic DNA (vDNA) coated with viral and host cellular proteins. The protective capsid shell disassembles during a process called uncoating. The viral genome is transported into the cell nucleus and integrates into the host cell chromatin. Unlike biochemical approaches that provide global data about the whole population of viral particles, imaging techniques enable following individual viruses on a single particle level. In this context, quantitative microscopy has brought original data shedding light on the dynamics of the viral entry into the host cell, the cytoplasmic transport, the nuclear import, and the selection of the integration site. In parallel, multi-color imaging studies have elucidated the mechanism of action of host cell factors implicated in HIV-1 viral cycle progression. In this review, we describe the labeling strategies used for HIV-1 fluorescence imaging and report on the main advancements that imaging studies have brought in the understanding of the infection mechanisms from the viral entry into the host cell until the provirus integration step.