Special Issue "The 11th International Retroviral Nucleocapsid and Assembly Symposium"

A special issue of Viruses (ISSN 1999-4915). This special issue belongs to the section "Animal Viruses".

Deadline for manuscript submissions: closed (31 May 2020).

Special Issue Editors

Dr. Mark Williams
Website
Guest Editor
Department of Physics, Northeastern University, Boston, Massachusetts, 02115, USA
Interests: single molecule biophysics, nucleic acid-protein interactions, retrovirus and retrotransposon replication, retroviral restriction factors, DNA replication and packaging
Dr. Akira Ono
Website
Guest Editor
Department of Microbiology and Immunology, University of Michigan, Ann Arbor , Michigan 48109, USA
Interests: assembly of enveloped viruses including retroviruses, lipid membrane-protein interactions, RNA-protein interactions, cell contact-mediated virus spread, virion incorporation of host proteins
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Special Issue Information

Dear Colleagues,

The 11th International Retroviral Nucleocapsid and Assembly Symposium took place in Boston, MA, USA, on the campus of Northeastern University, from August 15-17, 2019. The goal of the meeting was to discuss retroviral replication and assembly, focusing on the roles of nucleocapsid and Gag proteins in these processes, as well as interactions with cellular factors. These interactions were addressed with a wide range of interdisciplinary methods, and the conference facilitated interactions between experts in a variety of methods, furthering research through networking and collaboration.

In this Special Issue, we welcome research papers and review articles related to all aspects of retroviral replication and assembly. Topics of interest include biophysical and structural studies, retroviral Gag and NC interactions with RNA and/or membranes, interactions with cellular factors, immature virus assembly and budding, and therapeutic strategies.

Prof. Dr. Mark Williams
Prof. Dr. Akira Ono
Guest Editors

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Viruses is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2000 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Gag
  • nucleocapsid protein
  • retroviral
  • retroviral assembly
  • retroviral budding
  • nucleic acid chaperone
  • membrane domains
  • Env incorpoation
  • phospholipids
  • cellular membrane proteins

Published Papers (8 papers)

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Research

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Open AccessArticle
Abrogating ALIX Interactions Results in Stuttering of the ESCRT Machinery
Viruses 2020, 12(9), 1032; https://doi.org/10.3390/v12091032 - 16 Sep 2020
Abstract
Endosomal sorting complexes required for transport (ESCRT) proteins assemble on budding cellular membranes and catalyze their fission. Using live imaging of HIV virions budding from cells, we followed recruitment of ESCRT proteins ALIX, CHMP4B and VPS4. We report that the ESCRT proteins transiently [...] Read more.
Endosomal sorting complexes required for transport (ESCRT) proteins assemble on budding cellular membranes and catalyze their fission. Using live imaging of HIV virions budding from cells, we followed recruitment of ESCRT proteins ALIX, CHMP4B and VPS4. We report that the ESCRT proteins transiently co-localize with virions after completion of virion assembly for durations of 45 ± 30 s. We show that mutagenizing the YP domain of Gag which is the primary ALIX binding site or depleting ALIX from cells results in multiple recruitments of the full ESCRT machinery on the same virion (referred to as stuttering where the number of recruitments to the same virion >3). The stuttering recruitments are approximately 4 ± 3 min apart and have the same stoichiometry of ESCRTs and same residence time (45 ± 30 s) as the single recruitments in wild type interactions. Our observations suggest a role for ALIX during fission and question the linear model of ESCRT recruitment, suggesting instead a more complex co-assembly model. Full article
(This article belongs to the Special Issue The 11th International Retroviral Nucleocapsid and Assembly Symposium)
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Open AccessArticle
Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity
Viruses 2020, 12(5), 568; https://doi.org/10.3390/v12050568 - 22 May 2020
Abstract
Retroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a “Ψ” packaging signal located in the gRNA 5′-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs [...] Read more.
Retroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a “Ψ” packaging signal located in the gRNA 5′-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs efficiently. The simplicity of RSV Ψ makes it an informative model to examine the mechanism of retroviral gRNA packaging, which is incompletely understood. Little is known about the structure of dimerization initiation sites or specific Gag interaction sites of RSV gRNA. Using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), we probed the secondary structure of the entire RSV 5′-leader RNA for the first time. We identified a putative bipartite dimerization initiation signal (DIS), and mutation of both sites was required to significantly reduce dimerization in vitro. These mutations failed to reduce viral replication, suggesting that in vitro dimerization results do not strictly correlate with in vivo infectivity, possibly due to additional RNA interactions that maintain the dimers in cells. UV crosslinking-coupled SHAPE (XL-SHAPE) was next used to determine Gag-induced RNA conformational changes, revealing G218 as a critical Gag contact site. Overall, our results suggest that disruption of either of the DIS sequences does not reduce virus replication and reveal specific sites of Gag–RNA interactions. Full article
(This article belongs to the Special Issue The 11th International Retroviral Nucleocapsid and Assembly Symposium)
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Open AccessArticle
Significant Differences in RNA Structure Destabilization by HIV-1 Gag∆p6 and NCp7 Proteins
Viruses 2020, 12(5), 484; https://doi.org/10.3390/v12050484 - 25 Apr 2020
Abstract
Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play distinct roles in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structures, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed [...] Read more.
Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play distinct roles in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structures, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a complementary RNA hairpin. In contrast, during viral assembly, NC, as a domain of the group-specific antigen (Gag) polyprotein, binds the genomic RNA and facilitates packaging into new virions. It is not clear how the same protein, alone or as part of Gag, performs such different RNA binding functions in the viral life cycle. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium stability and unfolding barrier for TAR RNA. Comparing measured results with a model of discrete protein binding allows us to localize affected binding sites, in addition to quantifying hairpin stability. We find that, while both NCp7 and Gag∆p6 destabilize the TAR hairpin, Gag∆p6 binding is localized to two sites in the stem, while NCp7 targets sites near the top loop. Unlike Gag∆p6, NCp7 destabilizes this loop, shifting the location of the reaction barrier toward the folded state and increasing the natural rate of hairpin opening by ~104. Thus, our results explain why Gag cleavage and NC release is an essential prerequisite for reverse transcription within the virion. Full article
(This article belongs to the Special Issue The 11th International Retroviral Nucleocapsid and Assembly Symposium)
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Open AccessArticle
RNA Binding Suppresses Tsg101 Recognition of Ub-Modified Gag and Facilitates Recruitment to the Plasma Membrane
Viruses 2020, 12(4), 447; https://doi.org/10.3390/v12040447 - 15 Apr 2020
Cited by 1
Abstract
The ESCRT-I factor Tsg101 is essential for sorting endocytic cargo and is exploited by viral pathogens to facilitate egress from cells. Both the nucleocapsid (NC) domain and p6 domain in HIV-1 Gag contribute to recruitment of the protein. However, the role of NC [...] Read more.
The ESCRT-I factor Tsg101 is essential for sorting endocytic cargo and is exploited by viral pathogens to facilitate egress from cells. Both the nucleocapsid (NC) domain and p6 domain in HIV-1 Gag contribute to recruitment of the protein. However, the role of NC is unclear when the P(S/T)AP motif in p6 is intact, as the motif recruits Tsg101 directly. The zinc fingers in NC bind RNA and membrane and are critical for budding. Tsg101 can substitute for the distal ZnF (ZnF2) and rescue budding of a mutant made defective by deletion of this element. Here, we report that the ubiquitin (Ub) E2 variant (UEV) domain in Tsg101 binds tRNA in vitro. We confirmed that Tsg101 can substitute for ZnF2 when provided at the viral assembly site as a chimeric Gag-Tsg101 protein (Gag-ΔZnF2-Tsg101) and rescue budding. The UEV was not required in this context; however, mutation of the RNA binding determinants in UEV prevented Tsg101 recruitment from the cell interior when Gag and Tsg101 were co-expressed. The same Tsg101 mutations increased recognition of Gag-Ub, suggesting that tRNA and Ub compete for binding sites. This study identifies a novel Tsg101 binding partner that may contribute to its function in recognition of Ub-modified cargo. Full article
(This article belongs to the Special Issue The 11th International Retroviral Nucleocapsid and Assembly Symposium)
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Open AccessArticle
Distinct Contributions of Different Domains within the HIV-1 Gag Polyprotein to Specific and Nonspecific Interactions with RNA
Viruses 2020, 12(4), 394; https://doi.org/10.3390/v12040394 - 02 Apr 2020
Cited by 1
Abstract
Viral genomic RNA is packaged into virions with high specificity and selectivity. However, in vitro the Gag specificity towards viral RNA is obscured when measured in buffers containing physiological salt. Interestingly, when the binding is challenged by increased salt concentration, the addition of [...] Read more.
Viral genomic RNA is packaged into virions with high specificity and selectivity. However, in vitro the Gag specificity towards viral RNA is obscured when measured in buffers containing physiological salt. Interestingly, when the binding is challenged by increased salt concentration, the addition of competing RNAs, or introducing mutations to Gag protein, the specificity towards viral RNA becomes detectable. The objective of this work was to examine the contributions of the individual HIV-1 Gag polyprotein domains to nonspecific and specific RNA binding and stability of the initial protein-RNA complexes. Using a panel of Gag proteins with mutations disabling different Gag-Gag or Gag-RNA interfaces, we investigated the distinct contributions of individual domains which distinguish the binding to viral and nonviral RNA by measuring the binding of the proteins to RNAs. We measured the binding affinity in near-physiological salt concentration, and then challenged the binding by increasing the ionic strength to suppress the electrostatic interactions and reveal the contribution of specific Gag–RNA and Gag–Gag interactions. Surprisingly, we observed that Gag dimerization and the highly basic region in the matrix domain contribute significantly to the specificity of viral RNA binding. Full article
(This article belongs to the Special Issue The 11th International Retroviral Nucleocapsid and Assembly Symposium)
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Open AccessArticle
RNA-Binding Domains of Heterologous Viral Proteins Substituted for Basic Residues in the RSV Gag NC Domain Restore Specific Packaging of Genomic RNA
Viruses 2020, 12(4), 370; https://doi.org/10.3390/v12040370 - 27 Mar 2020
Abstract
The Rous sarcoma virus Gag polyprotein transiently traffics through the nucleus, which is required for efficient incorporation of the viral genomic RNA (gRNA) into virus particles. Packaging of gRNA is mediated by two zinc knuckles and basic residues located in the nucleocapsid (NC) [...] Read more.
The Rous sarcoma virus Gag polyprotein transiently traffics through the nucleus, which is required for efficient incorporation of the viral genomic RNA (gRNA) into virus particles. Packaging of gRNA is mediated by two zinc knuckles and basic residues located in the nucleocapsid (NC) domain in Gag. To further examine the role of basic residues located downstream of the zinc knuckles in gRNA encapsidation, we used a gain-of-function approach. We replaced a basic residue cluster essential for gRNA packaging with heterologous basic residue motif (BR) with RNA-binding activity from either the HIV-1 Rev protein (Rev BR) or the HSV ICP27 protein (ICP27 BR). Compared to wild-type Gag, the mutant ICP27 BR and Rev BR Gag proteins were much more strongly localized to the nucleus and released significantly lower levels of virus particles. Surprisingly, both the ICP27 BR and Rev BR mutants packaged normal levels of gRNA per virus particle when examined in the context of a proviral vector, yet both mutants were noninfectious. These results support the hypothesis that basic residues located in the C-terminal region of NC are required for selective gRNA packaging, potentially by binding non-specifically to RNA via electrostatic interactions. Full article
(This article belongs to the Special Issue The 11th International Retroviral Nucleocapsid and Assembly Symposium)
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Review

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Open AccessReview
Rendezvous at Plasma Membrane: Cellular Lipids and tRNA Set up Sites of HIV-1 Particle Assembly and Incorporation of Host Transmembrane Proteins
Viruses 2020, 12(8), 842; https://doi.org/10.3390/v12080842 - 31 Jul 2020
Abstract
The HIV-1 structural polyprotein Gag drives the virus particle assembly specifically at the plasma membrane (PM). During this process, the nascent virion incorporates specific subsets of cellular lipids and host membrane proteins, in addition to viral glycoproteins and viral genomic RNA. Gag binding [...] Read more.
The HIV-1 structural polyprotein Gag drives the virus particle assembly specifically at the plasma membrane (PM). During this process, the nascent virion incorporates specific subsets of cellular lipids and host membrane proteins, in addition to viral glycoproteins and viral genomic RNA. Gag binding to the PM is regulated by cellular factors, including PM-specific phospholipid PI(4,5)P2 and tRNAs, both of which bind the highly basic region in the matrix domain of Gag. In this article, we review our current understanding of the roles played by cellular lipids and tRNAs in specific localization of HIV-1 Gag to the PM. Furthermore, we examine the effects of PM-bound Gag on the organization of the PM bilayer and discuss how the reorganization of the PM at the virus assembly site potentially contributes to the enrichment of host transmembrane proteins in the HIV-1 particle. Since some of these host transmembrane proteins alter release, attachment, or infectivity of the nascent virions, the mechanism of Gag targeting to the PM and the nature of virus assembly sites have major implications in virus spread. Full article
(This article belongs to the Special Issue The 11th International Retroviral Nucleocapsid and Assembly Symposium)
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Open AccessReview
Revisiting Membrane Microdomains and Phase Separation: A Viral Perspective
Viruses 2020, 12(7), 745; https://doi.org/10.3390/v12070745 - 10 Jul 2020
Abstract
Retroviruses selectively incorporate a specific subset of host cell proteins and lipids into their outer membrane when they bud out from the host plasma membrane. This specialized viral membrane composition is critical for both viral survivability and infectivity. Here, we review recent findings [...] Read more.
Retroviruses selectively incorporate a specific subset of host cell proteins and lipids into their outer membrane when they bud out from the host plasma membrane. This specialized viral membrane composition is critical for both viral survivability and infectivity. Here, we review recent findings from live cell imaging of single virus assembly demonstrating that proteins and lipids sort into the HIV retroviral membrane by a mechanism of lipid-based phase partitioning. The findings showed that multimerizing HIV Gag at the assembly site creates a liquid-ordered lipid phase enriched in cholesterol and sphingolipids. Proteins with affinity for this specialized lipid environment partition into it, resulting in the selective incorporation of proteins into the nascent viral membrane. Building on this and other work in the field, we propose a model describing how HIV Gag induces phase separation of the viral assembly site through a mechanism involving transbilayer coupling of lipid acyl chains and membrane curvature changes. Similar phase-partitioning pathways in response to multimerizing structural proteins likely help sort proteins into the membranes of other budding structures within cells. Full article
(This article belongs to the Special Issue The 11th International Retroviral Nucleocapsid and Assembly Symposium)
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