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Nature Aug 2022Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR-Cas systems. In response, one family of...
Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR-Cas systems. In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors. However, the principal composition and structure of this compartment remain unknown. Here we find that the bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.
Topics: Bacteria; Bacteriophages; Cell Compartmentation; Cryoelectron Microscopy; Viral Proteins; Virus Assembly
PubMed: 35922510
DOI: 10.1038/s41586-022-05013-4 -
Viruses Aug 2020Since the emergence of HIV and AIDS in the early 1980s, the development of safe and effective therapies has accompanied a massive increase in our understanding of the... (Review)
Review
Since the emergence of HIV and AIDS in the early 1980s, the development of safe and effective therapies has accompanied a massive increase in our understanding of the fundamental processes that drive HIV biology. As basic HIV research has informed the development of novel therapies, HIV inhibitors have been used as probes for investigating basic mechanisms of HIV-1 replication, transmission, and pathogenesis. This positive feedback cycle has led to the development of highly effective combination antiretroviral therapy (cART), which has helped stall the progression to AIDS, prolong lives, and reduce transmission of the virus. However, to combat the growing rates of virologic failure and toxicity associated with long-term therapy, it is important to diversify our repertoire of HIV-1 treatments by identifying compounds that block additional steps not targeted by current drugs. Most of the available therapeutics disrupt early events in the replication cycle, with the exception of the protease (PR) inhibitors, which act at the virus maturation step. HIV-1 maturation consists of a series of biochemical changes that facilitate the conversion of an immature, noninfectious particle to a mature infectious virion. These changes include proteolytic processing of the Gag polyprotein by the viral protease (PR), structural rearrangement of the capsid (CA) protein, and assembly of individual CA monomers into hexamers and pentamers that ultimately form the capsid. Here, we review the development and therapeutic potential of maturation inhibitors (MIs), an experimental class of anti-HIV-1 compounds with mechanisms of action distinct from those of the PR inhibitors. We emphasize the key insights into HIV-1 biology and structure that the study of MIs has provided. We will focus on three distinct groups of inhibitors that block HIV-1 maturation: (1) compounds that block the processing of the CA-spacer peptide 1 (SP1) cleavage intermediate, the original class of compounds to which the term MI was applied; (2) CA-binding inhibitors that disrupt capsid condensation; and (3) allosteric integrase inhibitors (ALLINIs) that block the packaging of the viral RNA genome into the condensing capsid during maturation. Although these three classes of compounds have distinct structures and mechanisms of action, they share the ability to block the formation of the condensed conical capsid, thereby blocking particle infectivity.
Topics: Anti-HIV Agents; Capsid; Capsid Proteins; Clinical Trials as Topic; Drug Resistance, Viral; HIV Infections; HIV Integrase; HIV Integrase Inhibitors; HIV-1; Humans; Indazoles; Protein Processing, Post-Translational; Pyridines; RNA, Viral; Succinates; Triterpenes; Viral Genome Packaging; Virus Assembly; Virus Replication
PubMed: 32858867
DOI: 10.3390/v12090940 -
Nature Communications Oct 2022Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic...
Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. It is unknown how virions assemble around these newly synthesized genomes and how they are then loaded into autophagic membranes for release through secretory autophagy. Here, we use cryo-electron tomography of infected cells to show that poliovirus assembles directly on replication membranes. Pharmacological untethering of capsids from membranes abrogates RNA encapsidation. Our data directly visualize a membrane-bound half-capsid as a prominent virion assembly intermediate. Assembly progression past this intermediate depends on the class III phosphatidylinositol 3-kinase VPS34, a key host-cell autophagy factor. On the other hand, the canonical autophagy initiator ULK1 is shown to restrict virion production since its inhibition leads to increased accumulation of virions in vast intracellular arrays, followed by an increased vesicular release at later time points. Finally, we identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from other types of autophagosome contents. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle.
Topics: Autophagy; Capsid; Class III Phosphatidylinositol 3-Kinases; Enterovirus Infections; Humans; Poliovirus; RNA; Virion; Virus Assembly
PubMed: 36216808
DOI: 10.1038/s41467-022-33483-7 -
Viruses Jan 2021Within the family of , foamy viruses (FVs) are unique and unconventional with respect to many aspects in their molecular biology, including assembly and release of... (Review)
Review
Within the family of , foamy viruses (FVs) are unique and unconventional with respect to many aspects in their molecular biology, including assembly and release of enveloped viral particles. Both components of the minimal assembly and release machinery, Gag and Env, display significant differences in their molecular structures and functions compared to the other retroviruses. This led to the placement of FVs into a separate subfamily, the . Here, we describe the molecular differences in FV Gag and Env, as well as Pol, which is translated as a separate protein and not in an orthoretroviral manner as a Gag-Pol fusion protein. This feature further complicates FV assembly since a specialized Pol encapsidation strategy via a tripartite Gag-genome-Pol complex is used. We try to relate the different features and specific interaction patterns of the FV Gag, Pol, and Env proteins in order to develop a comprehensive and dynamic picture of particle assembly and release, but also other features that are indirectly affected. Since FVs are at the root of the retrovirus tree, we aim at dissecting the unique/specialized features from those shared among the and Such analyses may shed light on the evolution and characteristics of virus envelopment since related viruses within the , for instance LTR retrotransposons, are characterized by different levels of envelopment, thus affecting the capacity for intercellular transmission.
Topics: Capsid; Genome, Viral; Host-Pathogen Interactions; Humans; Retroviridae Infections; Spumavirus; Viral Proteins; Virus Assembly; Virus Physiological Phenomena; Virus Release; Virus Replication
PubMed: 33451128
DOI: 10.3390/v13010105 -
The Enzymes 2021Although the process of genome encapsidation is highly conserved in tailed bacteriophages and eukaryotic double-stranded DNA viruses, there are two distinct packaging... (Review)
Review
Although the process of genome encapsidation is highly conserved in tailed bacteriophages and eukaryotic double-stranded DNA viruses, there are two distinct packaging pathways that these viruses use to catalyze ATP-driven translocation of the viral genome into a preassembled procapsid shell. One pathway is used by ϕ29-like phages and adenoviruses, which replicate and subsequently package a monomeric, unit-length genome covalently attached to a virus/phage-encoded protein at each 5'-end of the dsDNA genome. In a second, more ubiquitous packaging pathway characterized by phage lambda and the herpesviruses, the viral DNA is replicated as multigenome concatemers linked in a head-to-tail fashion. Genome packaging in these viruses thus requires excision of individual genomes from the concatemer that are then translocated into a preassembled procapsid. Hence, the ATPases that power packaging in these viruses also possess nuclease activities that cut the genome from the concatemer at the beginning and end of packaging. This review focuses on proposed mechanisms of genome packaging in the dsDNA viruses using unit-length ϕ29 and concatemeric λ genome packaging motors as representative model systems.
Topics: Bacteriophage lambda; DNA Packaging; DNA, Viral; Viral Genome Packaging; Virus Assembly
PubMed: 34861943
DOI: 10.1016/bs.enz.2021.09.006 -
MBio Apr 2024A member of the Retroviridae, human immunodeficiency virus type 1 (HIV-1), uses the RNA genome packaged into nascent virions to transfer genetic information to its... (Review)
Review
A member of the Retroviridae, human immunodeficiency virus type 1 (HIV-1), uses the RNA genome packaged into nascent virions to transfer genetic information to its progeny. The genome packaging step is a highly regulated and extremely efficient process as a vast majority of virus particles contain two copies of full-length unspliced HIV-1 RNA that form a dimer. Thus, during virus assembly HIV-1 can identify and selectively encapsidate HIV-1 unspliced RNA from an abundant pool of cellular RNAs and various spliced HIV-1 RNAs. Several "" features facilitate the packaging of a dimeric RNA genome. The viral polyprotein ag orchestrates virus assembly and mediates RNA genome packaging. During this process, Gag preferentially binds unpaired uanosines within the highly structured 5' untranslated region (UTR) of HIV-1 RNA. In addition, the HIV-1 unspliced RNA provides a scaffold that promotes Gag:Gag interactions and virus assembly, thereby ensuring its packaging. Intriguingly, recent studies have shown that the use of different uanosines at the junction of U3 and R as transcription start sites results in HIV-1 unspliced RNA species with 99.9% identical sequences but dramatically distinct 5' UTR conformations. Consequently, one species of unspliced RNA is preferentially packaged over other nearly identical RNAs. These studies reveal how conformations affect the functions of HIV-1 RNA elements and the complex regulation of HIV-1 replication. In this review, we summarize - and -acting elements critical for HIV-1 RNA packaging, locations of Gag:RNA interactions that mediate genome encapsidation, and the effects of transcription start sites on the structure and packaging of HIV-1 RNA.
Topics: Humans; HIV-1; RNA, Viral; Virus Assembly; Genome, Viral
PubMed: 38411060
DOI: 10.1128/mbio.00861-23 -
Viruses Aug 2019RNA viruses represent a large and important group of pathogens that infect a broad range of hosts. Segmented RNA viruses are a subclass of this group that encode their... (Review)
Review
RNA viruses represent a large and important group of pathogens that infect a broad range of hosts. Segmented RNA viruses are a subclass of this group that encode their genomes in two or more molecules and package all of their RNA segments in a single virus particle. These divided genomes come in different forms, including double-stranded RNA, coding-sense single-stranded RNA, and noncoding single-stranded RNA. Genera that possess these genome types include, respectively, (e.g., Bluetongue virus), (e.g., Red clover necrotic mosaic virus) and (e.g., Influenza A virus). Despite their distinct genomic features and diverse host ranges (i.e., animals, plants, and humans, respectively) each of these viruses uses -acting RNA-RNA interactions (tRRIs) to facilitate co-packaging of their segmented genome. The tRRIs occur between different viral genome segments and direct the selective packaging of a complete genome complement. Here we explore the current state of understanding of tRRI-mediated co-packaging in the abovementioned viruses and examine other known and potential functions for this class of RNA-RNA interaction.
Topics: Animals; Gene Expression Regulation, Viral; Humans; RNA Viruses; RNA, Viral; Transcriptional Activation; Virus Assembly; Virus Diseases
PubMed: 31416187
DOI: 10.3390/v11080751 -
Acta Virologica 2021Nora virus is a RNA picorna-like virus that produces a persistent infection in Drosophila melanogaster. The genome is approximately 12,300 bases and is divided into four...
Nora virus is a RNA picorna-like virus that produces a persistent infection in Drosophila melanogaster. The genome is approximately 12,300 bases and is divided into four open reading frames (ORFs). Structurally, there are four important viral proteins: VP3, VP4A, VP4B, and VP4C. Three proteins (VP4A, VP4B, and VP4C) that form the virion's capsid are encoded by ORF 4, which produces a polyprotein that is post-translationally cleaved. The fourth protein (VP3) is encoded by ORF 3 and it is hypothesized to play a role in virion stability. The genes for these proteins were individually cloned into Escherichia coli, expressed, and the proteins were purified. Virus-like particles (VLPs) were assembled in vitro by mixing the proteins together in different combinations and measured via electron microscopy. Assemblies that contained VP4A and/or VP3 created VLPs with similar sizes to purified empty Nora virus capsids, potentially indicating that VP4A and/or VP3 are vital for Nora virus capsid structure, assembly, and/or stability. Not only does this study provide insight into the role of Nora virus proteins, but it may also lead to a deeper understanding of how Nora virus or other picorna-like viruses undergo assembly. Keywords: RNA viruses; Nora virus; picorna-like virus; virus-like particles; capsid assembly.
Topics: Animals; Capsid; Capsid Proteins; Drosophila melanogaster; Persistent Infection; RNA Viruses; Virion; Virus Assembly
PubMed: 34796712
DOI: 10.4149/av_2021_403 -
Methods in Molecular Biology (Clifton,... 2022The four serotypes of dengue virus (DENV), belonging to the genus Flavivirus in the family Flaviviridae, are the leading cause of arboviral diseases in humans. The...
The four serotypes of dengue virus (DENV), belonging to the genus Flavivirus in the family Flaviviridae, are the leading cause of arboviral diseases in humans. The clinical presentations range from dengue fever to dengue hemorrhagic fever and dengue shock syndrome. Despite decades of efforts on developing intervention strategies against DENV, there is no licensed antiviral, and safe and effective vaccines remain challenging. Similar to other flaviviruses, the assembly of DENV particles occurs in the membranes derived from endoplasmic reticulum; immature virions bud into the lumen followed by maturation in the trans-Golgi and transport through the secretary pathway. A unique feature of flavivirus replication is the production of small and slowly sedimenting subviral particles, known as virus-like particles (VLPs). Co-expression of premembrane (prM) and envelope (E) proteins can generate recombinant VLPs, which are biophysically and antigenically similar to infectious virions and have been employed to study the function of prM and E proteins, assembly, serodiagnostic antigens, and vaccine candidates. Previously, we have developed several assays including sucrose cushion ultracentrifugation, sucrose gradient ultracentrifugation, membrane flotation, subcellular fractionation, and glycosidase digestion assay to exploit the interaction between DENV prM and E proteins, membrane association, subcellular localization, glycosylation pattern, and assembly of VLPs and replicon particles. The information derived from these assays have implications to further our understanding of DENV assembly, replication cycle, intervention strategies, and pathogenesis.
Topics: Antibodies, Viral; Dengue Virus; Humans; Membrane Proteins; Sucrose; Viral Matrix Proteins; Virus Assembly
PubMed: 34709636
DOI: 10.1007/978-1-0716-1879-0_6 -
PLoS Pathogens Oct 2020HBV is an enveloped DNA virus that replicates its DNA genome via reverse transcription of a pregenomic (pg) RNA intermediate in hepatocytes. Interestingly, HBV RNA can...
HBV is an enveloped DNA virus that replicates its DNA genome via reverse transcription of a pregenomic (pg) RNA intermediate in hepatocytes. Interestingly, HBV RNA can be detected in virus-like particles in chronic hepatitis B (CHB) patient serum and has been utilized as a biomarker for intrahepatic cccDNA activity in treated patients. However, the biogenesis and molecular characteristics of serum HBV RNA remain to be fully defined. In this study, we found that the encapsidated serum HBV RNA predominately consists of pgRNA, which are detergent- and ribonuclease-resistant. Through blocking HBV DNA replication without affecting pgRNA encapsidation by using the priming-defective HBV mutant Y63D or 3TC treatment, we demonstrated that the cell culture supernatant contains a large amount of pgRNA-containing nonenveloped capsids and a minor population of pgRNA-containing virions. The formation of pgRNA-virion requires both capsid assembly and viral envelope proteins, which can be inhibited by capsid assembly modulators and an envelope-knockout mutant, respectively. Furthermore, the pgRNA-virion utilizes the multivesicular body pathway for egress, in a similar way as DNA-virion morphogenesis. Northern blotting, RT-PCR, and 3' RACE assays revealed that serum/supernatant HBV pgRNA are mainly spliced and devoid of the 3'-terminal sequences. Furthermore, pgRNA-virion collected from cells treated with a reversible HBV priming inhibitor L-FMAU was unable to establish infection in HepG2-NTCP cells. In summary, serum HBV RNA is secreted in noninfectious virion-like particle as spliced and poly(A)-free pgRNA. Our study will shed light on the molecular biology of serum HBV RNA in HBV life cycle, and aid the development of serum HBV RNA as a novel biomarker for CHB diagnosis and treatment prognosis.
Topics: Capsid; Capsid Proteins; DNA, Viral; Hepatitis B virus; Hepatocytes; Humans; RNA, Viral; Reverse Transcription; Virus Assembly; Virus Replication
PubMed: 33079954
DOI: 10.1371/journal.ppat.1008945