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Virology Nov 2019We present a novel kinetic Monte Carlo model to simulate the real process time-scale of the assembly of Human Papillomavirus (HPV) virus-like particles (VLPs)...
We present a novel kinetic Monte Carlo model to simulate the real process time-scale of the assembly of Human Papillomavirus (HPV) virus-like particles (VLPs) incorporating the formation of intercapsomeric disulfide bonds. The objective was to develop insights into the underlying mechanisms of HPV VLP assembly and cross-linking during in vitro production of the HPV vaccine. The model integrates actual experimental data and detailed information of VLP geometrical structure in microscopic mechanistic steps. The principal novelty of this model is in the concurrent simulation of VLP assembly and cross-linking including a variable for spatial angular arrangement of capsomeres during their assembly that affects the overall rates of VLP assembly and cross-linking. The cross-linking modeled by using the mechanistic probability rules between involved cysteine residues. The model was utilized to better understand the actual process data and check on the hypothesis related to factors affecting the rates of HPV growth and maturation.
Topics: Humans; Models, Biological; Papillomaviridae; Protein Multimerization; Time Factors; Viral Proteins; Virosomes; Virus Assembly
PubMed: 31450047
DOI: 10.1016/j.virol.2019.08.018 -
Viruses Mar 2022HIV-1 viral particle assembly occurs specifically at the plasma membrane and is driven primarily by the viral polyprotein Gag. Selective association of Gag with the... (Review)
Review
HIV-1 viral particle assembly occurs specifically at the plasma membrane and is driven primarily by the viral polyprotein Gag. Selective association of Gag with the plasma membrane is a key step in the viral assembly pathway, which is traditionally attributed to the MA domain. MA regulates specific plasma membrane binding through two primary mechanisms including: (1) specific interaction of the MA highly basic region (HBR) with the plasma membrane phospholipid phosphatidylinositol (4,5) bisphosphate [PI(4,5)P], and (2) tRNA binding to the MA HBR, which prevents Gag association with non-PI(4,5)P containing membranes. Gag multimerization, driven by both CA-CA inter-protein interactions and NC-RNA binding, also plays an essential role in viral particle assembly, mediating the establishment and growth of the immature Gag lattice on the plasma membrane. In addition to these functions, the multimerization of HIV-1 Gag has also been demonstrated to enhance its membrane binding activity through the MA domain. This review provides an overview of the mechanisms regulating Gag membrane binding through the MA domain and multimerization through the CA and NC domains, and examines how these two functions are intertwined, allowing for multimerization mediated enhancement of Gag membrane binding.
Topics: Cell Membrane; HIV-1; Membranes; Phosphatidylinositol 4,5-Diphosphate; Protein Binding; Virus Assembly; gag Gene Products, Human Immunodeficiency Virus
PubMed: 35337029
DOI: 10.3390/v14030622 -
Nature Communications Mar 2021Adeno-associated viruses (AAVs) are increasingly used as gene therapy vectors. AAVs package their genome in a non-enveloped T = 1 icosahedral capsid of ~3.8...
Adeno-associated viruses (AAVs) are increasingly used as gene therapy vectors. AAVs package their genome in a non-enveloped T = 1 icosahedral capsid of ~3.8 megaDalton, consisting of 60 subunits of 3 distinct viral proteins (VPs), which vary only in their N-terminus. While all three VPs play a role in cell-entry and transduction, their precise stoichiometry and structural organization in the capsid has remained elusive. Here we investigate the composition of several AAV serotypes by high-resolution native mass spectrometry. Our data reveal that the capsids assemble stochastically, leading to a highly heterogeneous population of capsids of variable composition, whereby even the single-most abundant VP stoichiometry represents only a small percentage of the total AAV population. We estimate that virtually every AAV capsid in a particular preparation has a unique composition. The systematic scoring of the simulations against experimental native MS data offers a sensitive new method to characterize these therapeutically important heterogeneous capsids.
Topics: Animals; Capsid; Capsid Proteins; Dependovirus; HEK293 Cells; Humans; Serogroup; Sf9 Cells; Viral Proteins; Virus Assembly
PubMed: 33712599
DOI: 10.1038/s41467-021-21935-5 -
Viruses Oct 2020Viral RNA genomes change shape as virus particles disassemble, form replication complexes, attach to ribosomes for translation, evade host defense mechanisms, and... (Review)
Review
Viral RNA genomes change shape as virus particles disassemble, form replication complexes, attach to ribosomes for translation, evade host defense mechanisms, and assemble new virus particles. These structurally dynamic RNA shapeshifters present a challenging RNA folding problem, because the RNA sequence adopts multiple structures and may sometimes contain regions of partial disorder. Recent advances in high resolution asymmetric cryoelectron microscopy and chemical probing provide new ways to probe the degree of structure and disorder, and have identified more than one conformation in dynamic equilibrium in viral RNA. Chemical probing and the Detection of RNA Folding Ensembles using Expectation Maximization (DREEM) algorithm has been applied to studies of the dynamic equilibrium conformations in HIV RNA in vitro, in virio, and in vivo. This new type of data provides insight into important questions about virus assembly mechanisms and the fundamental physical forces driving virus particle assembly.
Topics: Base Sequence; Cryoelectron Microscopy; Genome, Viral; Nucleic Acid Conformation; RNA Folding; RNA, Viral; Virion; Virus Assembly
PubMed: 33027988
DOI: 10.3390/v12101126 -
Journal of Molecular Biology Mar 2020The portal proteins of tailed bacteriophage and Herpesvirus capsids form dodecameric rings that occupy one capsid vertex and are incorporated during the assembly of...
The portal proteins of tailed bacteriophage and Herpesvirus capsids form dodecameric rings that occupy one capsid vertex and are incorporated during the assembly of capsid precursors called procapsids or proheads. Portals are essential and serve as the pore for DNA transit and the site of tail attachment; however, bacteriophage HK97 capsid proteins assemble efficiently without a portal when expressed from plasmids. Following portal co-expression, portals were incorporated into about half of the proheads that were made. In the absence of active capsid maturation protease, uncleaved proheads formed dimers, trimers, and tetramers of proheads during purification, but only if they had portals. These appeared bound to membrane-like fragments by their portals and could be disaggregated by detergents, supporting a role for membranes in their formation and in capsid assembly. The precursors to prohead oligomers were detected in cell extracts. These were able to bind to Octyl-Sepharose and could be released by detergent, while uncleaved proheads without portal or cleaved proheads with portal did not bind. Our results document a discrete change in the HK97 portal's hydrophobicity induced by cleavage of the procapsid shell in which it is embedded. Additionally, we detected an increase in the rate of expansion induced by the presence of a portal complex in cleaved HK97 proheads. These results suggest that portals and capsids influence each other's conformation during assembly. The formation of prohead oligomers also provides a rapid and sensitive assay for identification and analysis of portal incorporation mutants.
Topics: Bacteriophages; Capsid; Capsid Proteins; Models, Molecular; Molecular Conformation; Viral Proteins; Virus Assembly
PubMed: 32035900
DOI: 10.1016/j.jmb.2020.01.022 -
Viruses Oct 2022The genome packaging motor of bacteriophages and herpesviruses is built by two terminase subunits, known as large (TerL) and small (TerS), both essential for viral... (Review)
Review
The genome packaging motor of bacteriophages and herpesviruses is built by two terminase subunits, known as large (TerL) and small (TerS), both essential for viral genome packaging. TerL structure, composition, and assembly to an empty capsid, as well as the mechanisms of ATP-dependent DNA packaging, have been studied in depth, shedding light on the chemo-mechanical coupling between ATP hydrolysis and DNA translocation. Instead, significantly less is known about the small terminase subunit, TerS, which is dispensable or even inhibitory in vitro, but essential in vivo. By taking advantage of the recent revolution in cryo-electron microscopy (cryo-EM) and building upon a wealth of crystallographic structures of phage TerSs, in this review, we take an inventory of known TerSs studied to date. Our analysis suggests that TerS evolved and diversified into a flexible molecular framework that can conserve biological function with minimal sequence and quaternary structure conservation to fit different packaging strategies and environmental conditions.
Topics: Virus Assembly; Cryoelectron Microscopy; Viral Proteins; DNA, Viral; DNA Packaging; Endodeoxyribonucleases; Bacteriophages; Adenosine Triphosphate
PubMed: 36298770
DOI: 10.3390/v14102215 -
Journal of the American Chemical Society Dec 2023Lassa virus is a negative-strand RNA virus with only four structural proteins that causes periodic outbreaks in West Africa. The nucleoprotein (NP) encapsidates the...
Lassa virus is a negative-strand RNA virus with only four structural proteins that causes periodic outbreaks in West Africa. The nucleoprotein (NP) encapsidates the viral genome, forming ribonucleoprotein complexes (RNPs) together with the viral RNA and the L protein. RNPs must be continuously restructured during viral genome replication and transcription. The Z protein is important for membrane recruitment of RNPs, viral particle assembly, and budding and has also been shown to interact with the L protein. However, the interaction of NP, viral RNA, and Z is poorly understood. Here, we characterize the interactions between Lassa virus NP, Z, and RNA using structural mass spectrometry. We identify the presence of RNA as the driver for the disassembly of ring-like NP trimers, a storage form, into monomers to subsequently form higher order RNA-bound NP assemblies. We locate the interaction site of Z and NP and demonstrate that while NP binds Z independently of the presence of RNA, this interaction is pH-dependent. These data improve our understanding of RNP assembly, recruitment, and release in Lassa virus.
Topics: Lassa virus; Ribonucleoproteins; Nucleoproteins; Virus Assembly; RNA, Viral
PubMed: 38104324
DOI: 10.1021/jacs.3c07325 -
Nature Reviews. Microbiology Apr 2021Traditionally, the viral replication cycle is envisioned as a single, well-defined loop with four major steps: attachment and entry into a target cell, replication of... (Review)
Review
Traditionally, the viral replication cycle is envisioned as a single, well-defined loop with four major steps: attachment and entry into a target cell, replication of the viral genome, maturation of viral proteins and genome packaging into infectious progeny, and egress and dissemination to the next target cell. However, for many viruses, a growing body of evidence points towards extreme heterogeneity in each of these steps. In this Review, we reassess the major steps of the viral replication cycle by highlighting recent advances that show considerable variability during viral infection. First, we discuss heterogeneity in entry receptors, followed by a discussion on error-prone and low-fidelity polymerases and their impact on viral diversity. Next, we cover the implications of heterogeneity in genome packaging and assembly on virion morphology. Last, we explore alternative egress mechanisms, including tunnelling nanotubes and host microvesicles. In summary, we discuss the implications of viral phenotypic, morphological and genetic heterogeneity on pathogenesis and medicine. This Review highlights common themes and unique features that give nuance to the viral replication cycle.
Topics: Animals; Host-Pathogen Interactions; Humans; Receptors, Virus; Viral Genome Packaging; Virus Assembly; Virus Internalization; Virus Replication; Viruses
PubMed: 33024309
DOI: 10.1038/s41579-020-00449-9 -
The FEBS Journal Apr 2023Flaviviruses comprise a number of mosquito- or tick-transmitted human pathogens of global public health importance. Advances in structural biology techniques have... (Review)
Review
Flaviviruses comprise a number of mosquito- or tick-transmitted human pathogens of global public health importance. Advances in structural biology techniques have contributed substantially to our current understanding of the life cycle of these small enveloped RNA viruses and led to deep insights into details of virus assembly, maturation and cell entry. In addition to large-scale conformational changes and oligomeric rearrangements of envelope proteins during these processes, there is increasing evidence that smaller-scale protein dynamics (referred to as virus "breathing") can confer extra flexibility to these viruses for the fine-tuning of their interactions with the immune system and possibly with cellular factors they encounter in their complex ecological cycles in arthropod and vertebrate hosts. In this review, we discuss how work with tick-borne encephalitis virus has extended our view on flavivirus breathing, leading to the identification of a novel mechanism of antibody-mediated infection enhancement and demonstrating breathing intermediates of the envelope protein in the process of membrane fusion. These data are discussed in the context of other flaviviruses and the perspective of a potential role of virus breathing to cope with the requirements of adaptation and replication in evolutionarily very different hosts.
Topics: Animals; Humans; Encephalitis Viruses, Tick-Borne; Virus Assembly
PubMed: 35246954
DOI: 10.1111/febs.16419 -
Viruses Jun 2021The proper assembly and dissemination of progeny virions is a fundamental step in virus replication. As a whole, viruses have evolved a myriad of strategies to exploit... (Review)
Review
The proper assembly and dissemination of progeny virions is a fundamental step in virus replication. As a whole, viruses have evolved a myriad of strategies to exploit cellular compartments and mechanisms to ensure a successful round of infection. For enveloped viruses such as retroviruses and herpesviruses, acquisition and incorporation of cellular membrane is an essential process during the formation of infectious viral particles. To do this, these viruses have evolved to hijack the host Endosomal Sorting Complexes Required for Transport (ESCRT-I, -II, and -III) to coordinate the sculpting of cellular membrane at virus assembly and dissemination sites, in seemingly different, yet fundamentally similar ways. For instance, at the plasma membrane, ESCRT-I recruitment is essential for HIV-1 assembly and budding, while it is dispensable for the release of HSV-1. Further, HSV-1 was shown to recruit ESCRT-III for nuclear particle assembly and egress, a process not used by retroviruses during replication. Although the cooption of ESCRTs occurs in two separate subcellular compartments and at two distinct steps for these viral lifecycles, the role fulfilled by ESCRTs at these sites appears to be conserved. This review discusses recent findings that shed some light on the potential parallels between retroviral budding and nuclear egress and proposes a model where HSV-1 nuclear egress may occur through an ESCRT-dependent mechanism.
Topics: DNA Viruses; Endosomal Sorting Complexes Required for Transport; HIV-1; Host Microbial Interactions; Humans; Protein Transport; Retroviridae; Virion; Virus Assembly; Virus Release; Virus Replication
PubMed: 34199191
DOI: 10.3390/v13061138