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JHEP Reports : Innovation in Hepatology Jan 2024Hepatitis D virus (HDV) is the causative agent of chronic hepatitis delta, the most severe form of viral hepatitis. HDV encodes one protein, hepatitis delta antigen...
BACKGROUND & AIMS
Hepatitis D virus (HDV) is the causative agent of chronic hepatitis delta, the most severe form of viral hepatitis. HDV encodes one protein, hepatitis delta antigen (HDAg), in two isoforms: S- and L-HDAg. They are identical in sequence except that L-HDAg contains an additional 19-20 amino acids at its C-terminus, which confer regulatory roles that are distinct from those of S-HDAg. Notably, these residues are divergent between different genotypes. We aimed to elucidate the molecular determinants within the C-termini that are essential for the regulatory role of L-HDAg in HDV replication and assembly.
METHODS
Northern blot, reverse-transcription quantitative PCR, and a newly established HDV trans-complementary system were used in this study.
RESULTS
C-termini of L-HDAg, albeit with high sequence variation among different genotypes, are interchangeable with respect to the trans-inhibitory function of L-HDAg and HDV assembly. The C-terminus of L-HDAg features a conserved prenylation CXXQ motif and is enriched with proline and hydrophobic residues. Abolishment of the CXXQ motif attenuated the inhibitory effect of L-HDAg on HDV replication. In contrast, the enrichment of proline and hydrophobic residues does not modify the trans-inhibitory function of L-HDAg. Nevertheless, these residues are essential for HDV assembly. Mechanistically, prolines and hydrophobic residues contribute to HDV assembly via a mode of action independent of the prenylated CXXQ motif.
CONCLUSIONS
Within the C-terminus of L-HDAg, the CXXQ motif and the enrichment of proline and hydrophobic residues are all essential determinants of L-HDAg's regulatory roles in HDV replication and assembly. This intrinsic viral regulatory mechanism we elucidated deepens our understanding of the unique life cycle of HDV.
IMPACT AND IMPLICATIONS
Hepatitis D virus (HDV) encodes one protein, hepatitis delta antigen (HDAg), in two isoforms: S- and L-HDAg. They are identical in sequence except that L-HDAg contains an additional 19-20 amino acids at its C-terminus. This C-terminal extension in L-HDAg confers regulatory roles in the HDV life cycle that are distinct from those of S-HDAg. Herein, we found that C-termini of L-HDAg, although with high sequence variation, are interchangeable among different HDV genotypes. Within the C-terminus of L-HDAg, the prenylation motif, and the enrichment of proline and hydrophobic residues are all essential determinants of L-HDAg's regulatory roles in HDV replication and assembly.
PubMed: 38192534
DOI: 10.1016/j.jhepr.2023.100961 -
Journal of Molecular Biology Aug 2023Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated....
Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that the pan-retroviral nucleocapsid (NC) and HIV-1 pr55 (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yields self-assembling BMCs that have HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs, and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4 T cell nuclear lysates led to the formation of larger BMCs compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggest that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.
Topics: Biomolecular Condensates; gag Gene Products, Human Immunodeficiency Virus; HIV-1; RNA, Viral; Virus Assembly; Genome, Viral; Host-Pathogen Interactions; Humans
PubMed: 37385580
DOI: 10.1016/j.jmb.2023.168190 -
FEBS Letters May 2024Intracellular pathogens rely on host metabolic networks for multiplication. Enveloped viruses need lipids for formation of the viral envelope and positive sense RNA... (Review)
Review
Intracellular pathogens rely on host metabolic networks for multiplication. Enveloped viruses need lipids for formation of the viral envelope and positive sense RNA viruses that replicate in membranous inclusions require lipids for formation of the replication compartments. In addition, all intracellular pathogens need energy for their replicative cycle. As triglycerides in lipid droplets are the main energy storage unit of cells and major source of membrane lipids, it is not surprising that viruses have evolved various strategies to exploit different aspects of lipid droplet biology.
Topics: Virus Replication; Lipid Droplets; Humans; Animals; Viral Envelope; RNA Viruses; Lipid Metabolism; Triglycerides
PubMed: 38348563
DOI: 10.1002/1873-3468.14819 -
Veterinary Research May 2024Pseudorabies virus (PRV) is recognized as the aetiological agent responsible for Aujeszky's disease, or pseudorabies, in swine populations. Rab6, a member of the small...
Pseudorabies virus (PRV) is recognized as the aetiological agent responsible for Aujeszky's disease, or pseudorabies, in swine populations. Rab6, a member of the small GTPase family, is implicated in various membrane trafficking processes, particularly exocytosis regulation. Its involvement in PRV infection, however, has not been documented previously. In our study, we observed a significant increase in the Rab6 mRNA and protein levels in both PK-15 porcine kidney epithelial cells and porcine alveolar macrophages, as well as in the lungs and spleens of mice infected with PRV. The overexpression of wild-type Rab6 and its GTP-bound mutant facilitated PRV proliferation, whereas the GDP-bound mutant form of Rab6 had no effect on viral propagation. These findings indicated that the GTPase activity of Rab6 was crucial for the successful spread of PRV. Further investigations revealed that the reduction in Rab6 levels through knockdown significantly hampered PRV proliferation and disrupted virus assembly and egress. At the molecular level, Rab6 was found to interact with the PRV glycoproteins gB and gE, both of which are essential for viral assembly and egress. Our results collectively suggest that PRV exploits Rab6 to expedite its assembly and egress and identify Rab6 as a promising novel target for therapeutic treatment for PRV infection.
Topics: Animals; Herpesvirus 1, Suid; Swine; rab GTP-Binding Proteins; Mice; Pseudorabies; Virus Release; Virus Assembly; Swine Diseases; Cell Line
PubMed: 38807225
DOI: 10.1186/s13567-024-01328-4 -
Journal of Viral Hepatitis Nov 2023Over 250 million people are living with chronic infection caused by the hepatitis B virus (HBV). HBV has three surface proteins, namely small (SHBs), medium (MHBs) and... (Review)
Review
Over 250 million people are living with chronic infection caused by the hepatitis B virus (HBV). HBV has three surface proteins, namely small (SHBs), medium (MHBs) and large (LHBs), and they play different roles in the virus life cycle. The approved hepatitis B vaccine only contains the SHBs protein and many studies have focused on characterising the functional domains in SHBs. Although the LHBs protein is less studied, recent studies have shown that it plays important roles in mediating viral entry, replication and assembly. Over the years, there have been major advancements in monoclonal antibody (mAb) discovery tools and multiple mAbs have been developed to specifically target the preS1 domain in LHBs. We summarise the HBV infection systems and antibody discovery strategies that have been utilised by various research groups to assess the potential use of anti-preS1 mAbs as therapeutic antibodies against HBV or in the development of new diagnostic assays.
Topics: Humans; Hepatitis B virus; Antibodies, Monoclonal; Hepatitis B Surface Antigens; Membrane Proteins; Hepatitis B; Hepatitis B Antibodies
PubMed: 37525419
DOI: 10.1111/jvh.13880 -
Methods in Molecular Biology (Clifton,... 2024Plant viruses such as brome mosaic virus and cowpea chlorotic mottle virus are effectively purified through PEG precipitation and sucrose cushion ultracentrifugation....
Plant viruses such as brome mosaic virus and cowpea chlorotic mottle virus are effectively purified through PEG precipitation and sucrose cushion ultracentrifugation. Increasing ionic strength and an alkaline pH cause the viruses to swell and disassemble into coat protein subunits. The coat proteins can be reassembled into stable virus-like particles (VLPs) that carry anionic molecules at low ionic strength and through two-step dialysis from neutral pH to acidic buffer. VLPs have been extensively studied due to their ability to protect and deliver cargo, particularly RNA, while avoiding degradation under physiological conditions. Furthermore, chemical functionalization of the surface of VLPs allows for the targeted drug delivery. VLPs derived from plants have demonstrated great potential in nanomedicine by offering a versatile platform for drug delivery, imaging, and therapeutic applications.
Topics: Plant Viruses; Capsid Proteins; Virion; Bromovirus; RNA; Hydrogen-Ion Concentration; RNA, Viral
PubMed: 38907930
DOI: 10.1007/978-1-0716-3918-4_24 -
MBio Feb 2024Vaccinia virus assembly in the cytoplasm of infected cells involves the formation of a biconcave viral core inside the maturing viral particle. The boundary of the core...
Vaccinia virus assembly in the cytoplasm of infected cells involves the formation of a biconcave viral core inside the maturing viral particle. The boundary of the core is defined by a pseudohexagonal palisade layer, composed of trimers projecting from an inner wall. To understand the assembly of this complex core architecture, we obtained a subnanometer structure of the palisade trimer by cryo-electron tomography and subtomogram averaging of purified intact virions. Using AlphaFold2 structure predictions, we determined that the palisade is formed from trimers of the proteolytically processed form of the viral protein A10. In addition, we found that each A10 protomer associates with an α-helix (residues 24-66) of A4. Cellular localization assays outside the context of infection demonstrate that the A4 N-terminus is necessary and sufficient to interact with A10. The interaction between A4 and A10 provides insights into how the palisade layer might become tightly associated with the viral membrane during virion maturation. Reconstruction of the palisade layer reveals that, despite local hexagonal ordering, the A10/A4 trimers are widely spaced, suggesting that additional components organize the lattice. This spacing would, however, allow the adoption of the characteristic biconcave shape of the viral core. Finally, we also found that the palisade incorporates multiple copies of a hexameric portal structure. We suggest that these portals are formed by E6, a viral protein that is essential for virion assembly and required to release viral mRNA from the core early in infection.IMPORTANCEPoxviruses such as variola virus (smallpox) and monkeypox cause diseases in humans. Other poxviruses, including vaccinia and modified vaccinia Ankara, are used as vaccine vectors. Given their importance, a greater structural understanding of poxvirus virions is needed. We now performed cryo-electron tomography of purified intact vaccinia virions to study the structure of the palisade, a protein lattice that defines the viral core boundary. We identified the main viral proteins that form the palisade and their interaction surfaces and provided new insights into the organization of the viral core.
Topics: Humans; Vaccinia virus; Vaccinia; Virus Assembly; Virion; Viral Proteins; Piperidones; Benzeneacetamides
PubMed: 38171004
DOI: 10.1128/mbio.03134-23 -
Biochimie Apr 2024Viral nanoparticles (VNPs) are self-assembling, adaptable delivery systems for vaccines and other therapeutic agents used in a variety of biomedical applications. The... (Review)
Review
Viral nanoparticles (VNPs) are self-assembling, adaptable delivery systems for vaccines and other therapeutic agents used in a variety of biomedical applications. The potential of viruses to invade and infect various hosts and cells renders them suitable as potential nanocarriers, possessing distinct functional characteristics, immunogenic properties, and improved biocompatibility and biodegradability. VNPs are frequently produced through precise genetic or chemical engineering, which involves adding diverse sequences or functional payloads to the capsid protein (CP). Several spherical and helical plant viruses, bacteriophages, and animal viruses are currently being used as VNPs, or non-infectious virus-like particles (VLPs). In addition to their broad use in cancer therapy, vaccine technology, diagnostics, and molecular imaging, VNPs have made important strides in the realms of tissue engineering, biosensing, and antimicrobial prophylaxis. They are also being used in energy storage cells due to their binding and piezoelectric properties. The large-scale production of VNPs for research, preclinical testing, and clinical use is fraught with difficulties, such as those relating to cost-effectiveness, scalability, and purity. Consequently, many plants- and microorganism-based platforms are being developed, and newer viruses are being explored. The goal of the current review is to provide an overview of these advances.
Topics: Animals; Plant Viruses; Nanoparticles; Capsid Proteins; Molecular Imaging; Plants
PubMed: 37573018
DOI: 10.1016/j.biochi.2023.08.006 -
Proceedings of the National Academy of... Mar 2024Carriers for RNA delivery must be dynamic, first stabilizing and protecting therapeutic RNA during delivery to the target tissue and across cellular membrane barriers...
Carriers for RNA delivery must be dynamic, first stabilizing and protecting therapeutic RNA during delivery to the target tissue and across cellular membrane barriers and then releasing the cargo in bioactive form. The chemical space of carriers ranges from small cationic lipids applied in lipoplexes and lipid nanoparticles, over medium-sized sequence-defined xenopeptides, to macromolecular polycations applied in polyplexes and polymer micelles. This perspective highlights the discovery of distinct virus-inspired dynamic processes that capitalize on mutual nanoparticle-host interactions to achieve potent RNA delivery. From the host side, subtle alterations of pH, ion concentration, redox potential, presence of specific proteins, receptors, or enzymes are cues, which must be recognized by the RNA nanocarrier via dynamic chemical designs including cleavable bonds, alterable physicochemical properties, and supramolecular assembly-disassembly processes to respond to changing biological microenvironment during delivery.
Topics: Cell Membrane; Cues; Micelles; Polymers; RNA
PubMed: 38437544
DOI: 10.1073/pnas.2307799120 -
Methods in Molecular Biology (Clifton,... 2024Mycoviruses exist in all major groups of fungi. With the continuous development of science and technology, the methods of studying viruses are constantly updated, and...
Mycoviruses exist in all major groups of fungi. With the continuous development of science and technology, the methods of studying viruses are constantly updated, and progressively mycoviruses have been discovered where most of these viruses are RNA viruses. Therefore, double-stranded RNA has traditionally been used as the hallmark of RNA mycovirus detection. This report describes in detail the method of mycovirus identification using extraction of dsRNA. Besides, extraction of viral dsRNA, and the assembly methods of viral genome and identification of virus type are presented.
Topics: Fungal Viruses; RNA, Double-Stranded; Genome, Viral; Technology
PubMed: 38285397
DOI: 10.1007/978-1-0716-3702-9_15