The Experts below are selected from a list of 9888 Experts worldwide ranked by ideXlab platform
Carlos Enrique Catalano - One of the best experts on this subject based on the ideXlab platform.
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Thermodynamic Interrogation of the Assembly of a Viral Genome Packaging Motor Complex
Biophysical journal, 2015Co-Authors: Teng Chieh Yang, Gabriel C Lander, David Ortiz, Lyn'al Nosaka, Carlos Enrique CatalanoAbstract:Viral terminase enzymes serve as Genome Packaging motors in many complex double-stranded DNA viruses. The functional motors are multiprotein complexes that translocate viral DNA into a capsid shell, powered by a Packaging ATPase, and are among the most powerful molecular motors in nature. Given their essential role in virus development, the structure and function of these biological motors is of considerable interest. Bacteriophage λ-terminase, which serves as a prototypical Genome Packaging motor, is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. This protomer assembles into a functional higher-order complex that excises a unit length Genome from a concatemeric DNA precursor (Genome maturation) and concomitantly translocates the duplex into a preformed procapsid shell (Genome Packaging). While the enzymology of λ-terminase has been well described, the nature of the catalytically competent nucleoprotein intermediates, and the mechanism describing their assembly and activation, is less clear. Here we utilize analytical ultracentrifugation to determine the thermodynamic parameters describing motor assembly and define a minimal thermodynamic linkage model that describes the effects of salt on protomer assembly into a tetrameric complex. Negative stain electron microscopy images reveal a symmetric ring-like complex with a compact stem and four extended arms that exhibit a range of conformational states. Finally, kinetic studies demonstrate that assembly of the ring tetramer is directly linked to activation of the Packaging ATPase activity of the motor, thus providing a direct link between structure and function. The implications of these results with respect to the assembly and activation of the functional Packaging motor during a productive viral infection are discussed.
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Energy-Independent Helicase Activity of a Viral Genome Packaging Motor
Biochemistry, 2011Co-Authors: Jenny R. Chang, Benjamin T. Andrews, Carlos Enrique CatalanoAbstract:The assembly of complex double-stranded DNA viruses includes a Genome Packaging step where viral DNA is translocated into the confines of a preformed procapsid shell. In most cases, the preferred Packaging substrate is a linear concatemer of viral Genomes linked head-to-tail. Viral terminase enzymes are responsible for both excision of an individual Genome from the concatemer (DNA maturation) and translocation of the duplex into the capsid (DNA Packaging). Bacteriophage λ terminase site-specifically nicks viral DNA at the cos site in a concatemer and then physically separates the nicked, annealed strands to mature the Genome in preparation for Packaging. Here we present biochemical studies on the so-called helicase activity of λ terminase. Previous studies reported that ATP is required for strand separation, and it has been presumed that ATP hydrolysis is required to drive the reaction. We show that ADP and nonhydrolyzable ATP analogues also support strand separation at low (micromolar) concentrations. In addition, the Escherichia coli integration host factor protein (IHF) strongly stimulates the reaction in a nucleotide-independent manner. Finally, we show that elevated concentrations of nucleotide inhibit both ATP- and IHF-stimulated strand separation by λ terminase. We present a model where nucleotide and IHF interact with the large terminase subunit and viral DNA, respectively, to engender a site-specifically bound, catalytically competent Genome maturation complex. In contrast, binding of nucleotide to the low-affinity ATP binding site in the small terminase subunit mediates a conformational switch that down-regulates maturation activities and activates the DNA Packaging activity of the enzyme. This affords a motor complex that binds tightly, but nonspecifically, to DNA as it translocates the duplex into the capsid shell. These studies have yielded mechanistic insight into the assembly of the maturation complex on viral DNA and its transition to a mobile Packaging motor that may be common to all of the complex double-stranded DNA viruses.
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Bacteriophage lambda gpNu1 and Escherichia coli IHF proteins cooperatively bind and bend viral DNA: implications for the assembly of a Genome-Packaging motor.
Biochemistry, 2006Co-Authors: Marcos E. Ortega, Carlos Enrique CatalanoAbstract:Terminase enzymes are common to both prokaryotic and eukaryotic double-stranded DNA viruses and are responsible for Packaging viral DNA into the confines of an empty procapsid shell. In all known cases, the holoenzymes are heteroligomers composed of a large subunit that possesses the catalytic activities required for Genome Packaging and a small subunit that is responsible for specific recognition of viral DNA. In bacteriophage lambda, the DNA recognition protein is gpNu1. The gpNu1 subunit interacts with multiple recognition elements within cos, the Packaging initiation site in viral DNA, to site-specifically assemble the Packaging machinery. Motor assembly is modulated by the Escherichia coli integration host factor protein (IHF), which binds to a consensus sequence also located within cos. On the basis of a variety of biochemical data and the recently solved NMR structure of the DNA binding domain of gpNu1, we proposed a novel DNA binding mode that predicts significant bending of duplex DNA by gpNu1 (d...
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viral Genome Packaging machines genetics structure and mechanism
2005Co-Authors: Carlos Enrique CatalanoAbstract:Viral Genome Packaging Machines.- Bacteriophage Lambda Terminase and the Mechanism of Viral DNA Packaging.- DNA Packaging in Bacteriophage T4.- T3/T7 DNA Packaging.- DNA Packaging by Bacteriophage P22.- Bacteriophage SPP1 DNA Packaging.- The o29 DNA Packaging Motor.- Encapsidation of the Segmented Double-Stranded RNA Genome of Bacteriophage ?6.- Cleavage and Packaging of Herpes Simplex Virus 1 DNA.
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Biochemical characterization of bacteriophage lambda Genome Packaging in vitro.
Virology, 2003Co-Authors: Qin Yang, Carlos Enrique CatalanoAbstract:Abstract Bacteriophage λ has been extensively studied, and the abundance of genetic and biochemical information available makes this an ideal model system to study virus DNA Packaging at the molecular level. Limited in vitro Packaging efficiency has hampered progress toward this end, however. It has been suggested that limited Packaging efficiency is related to poor activity of purified procapsids. We describe the construction of a vector that expresses λ procapsids with a yield that is 40-fold greater than existing systems. Consistent with previous studies, Packaging of a mature λ Genome is very inefficient in vitro, with only 4% of the input procapsids utilized. Concatemeric DNA is the preferred Packaging substrate in vivo, and procapsids interact with a nucleoprotein complex known as complex I to initiate Genome Packaging. When complex I is used as a Packaging substrate in vitro, capsid utilization is extremely efficient, and 40% of the input DNA is packaged. Finally, we provide evidence for a Packaging-stimulated ATPase activity, and kinetically characterize this reaction quantifying the energetic cost of DNA Packaging in bacteriophage λ.
Vinay K. Pathak - One of the best experts on this subject based on the ideXlab platform.
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Specific Guanosines in the HIV-2 Leader RNA are Essential for Efficient Viral Genome Packaging
Journal of molecular biology, 2020Co-Authors: Chijioke N. Umunnakwe, Olga A. Nikolaitchik, Alice Duchon, Abdul Rahman, Yang Liu, Jianbo Chen, S.-h. Sheldon Tai, Vinay K. PathakAbstract:HIV-2, a human pathogen that causes acquired immunodeficiency syndrome, is distinct from the more prevalent HIV-1 in several features including its evolutionary history and certain aspects of viral replication. Like other retroviruses, HIV-2 packages two copies of full-length viral RNA during virus assembly and efficient Genome encapsidation is mediated by the viral protein Gag. We sought to define cis-acting elements in the HIV-2 Genome that are important for the encapsidation of full-length RNA into viral particles. Based on previous studies of murine leukemia virus and HIV-1, we hypothesized that unpaired guanosines in the 5' untranslated region (UTR) play an important role in Gag:RNA interactions leading to Genome Packaging. To test our hypothesis, we targeted 18 guanosines located in 9 sites within the HIV-2 5' UTR and performed substitution analyses. We found that mutating as few as three guanosines significantly reduce RNA Packaging efficiency. However, not all guanosines examined have the same effect; instead, a hierarchical order exists wherein a primary site, a secondary site, and three tertiary sites are identified. Additionally, there are functional overlaps in these sites and mutations of more than one site can act synergistically to cause Genome Packaging defects. These studies demonstrate the importance of specific guanosines in HIV-2 5'UTR in mediating Genome Packaging. Our results also demonstrate an interchangeable and hierarchical nature of guanosine-containing sites, which was not previously established, thereby revealing key insights into the replication mechanisms of HIV-2.
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Unpaired Guanosines in the 5′ Untranslated Region of HIV-1 RNA Act Synergistically to Mediate Genome Packaging
Journal of virology, 2020Co-Authors: Olga A. Nikolaitchik, Xayathed Somoulay, Jonathan M O Rawson, Jennifer A Yoo, Vinay K. PathakAbstract:The viral protein Gag selects full-length HIV-1 RNA from a large pool of mRNAs as virion Genome during virus assembly. Currently, the precise mechanism that mediates the Genome selection is not understood. Previous studies have identified several sites in the 5' untranslated region (5' UTR) of HIV-1 RNA that are bound by nucleocapsid (NC) protein, which is derived from Gag during virus maturation. However, whether these NC binding sites direct HIV-1 RNA Genome Packaging has not been fully investigated. In this report, we examined the roles of single-stranded, exposed guanosines at NC binding sites in RNA Genome Packaging using stable cell lines expressing competing wild-type and mutant HIV-1 RNAs. Mutant RNA Packaging efficiencies were determined by comparing their prevalence in cytoplasmic RNA and in virion RNA. We observed that multiple NC binding sites affected RNA Packaging; of the sites tested, those located within stem-loop 1 of the 5' UTR had the most significant effects. These sites were previously reported as the primary NC binding sites using a chemical probe reverse-footprinting assay and as the major Gag binding sites using an in vitro assay. Of the mutants tested in this report, substituting 3 to 4 guanosines resulted in less than two-fold defects in Packaging. However, when mutations at different NC binding sites were combined, severe defects were observed. Furthermore, combining the mutations resulted in synergistic defects in RNA Packaging, suggesting redundancy in Gag:RNA interactions and a requirement for multiple Gag binding on viral RNA during HIV-1 Genome encapsidation.IMPORTANCE HIV-1 must package its RNA Genome during virus assembly to generate infectious viruses. To better understand how HIV-1 packages its RNA Genome, we examined the roles of RNA elements identified as binding sites for NC, a Gag-derived RNA-binding protein. Our results demonstrate that binding sites within stem-loop 1 of the 5' untranslated region play important roles in Genome Packaging. Although mutating one or two NC-binding sites caused only mild defects in Packaging, mutating multiple sites resulted in severe defects in Genome encapsidation, indicating that unpaired guanosines act synergistically to promote Packaging. Our results suggest that Gag:RNA interactions occur at multiple RNA sites during Genome Packaging; furthermore, there are functionally redundant binding sites in viral RNA.
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unpaired guanosines in the 5 untranslated region of hiv 1 rna act synergistically to mediate Genome Packaging
Journal of Virology, 2020Co-Authors: Olga A. Nikolaitchik, Xayathed Somoulay, Jonathan M O Rawson, Jennifer A Yoo, Vinay K. PathakAbstract:The viral protein Gag selects full-length HIV-1 RNA from a large pool of mRNAs as virion Genome during virus assembly. Currently, the precise mechanism that mediates the Genome selection is not understood. Previous studies have identified several sites in the 5' untranslated region (5' UTR) of HIV-1 RNA that are bound by nucleocapsid (NC) protein, which is derived from Gag during virus maturation. However, whether these NC binding sites direct HIV-1 RNA Genome Packaging has not been fully investigated. In this report, we examined the roles of single-stranded exposed guanosines at NC binding sites in RNA Genome Packaging using stable cell lines expressing competing wild-type and mutant HIV-1 RNAs. Mutant RNA Packaging efficiencies were determined by comparing their prevalences in cytoplasmic RNA and in virion RNA. We observed that multiple NC binding sites affected RNA Packaging; of the sites tested, those located within stem-loop 1 of the 5' UTR had the most significant effects. These sites were previously reported as the primary NC binding sites by using a chemical probe reverse-footprinting assay and as the major Gag binding sites by using an in vitro assay. Of the mutants tested in this report, substituting 3 to 4 guanosines resulted in <2-fold defects in Packaging. However, when mutations at different NC binding sites were combined, severe defects were observed. Furthermore, combining the mutations resulted in synergistic defects in RNA Packaging, suggesting redundancy in Gag-RNA interactions and a requirement for multiple Gag binding on viral RNA during HIV-1 Genome encapsidation.IMPORTANCE HIV-1 must package its RNA Genome during virus assembly to generate infectious viruses. To better understand how HIV-1 packages its RNA Genome, we examined the roles of RNA elements identified as binding sites for NC, a Gag-derived RNA-binding protein. Our results demonstrate that binding sites within stem-loop 1 of the 5' untranslated region play important roles in Genome Packaging. Although mutating one or two NC-binding sites caused only mild defects in Packaging, mutating multiple sites resulted in severe defects in Genome encapsidation, indicating that unpaired guanosines act synergistically to promote Packaging. Our results suggest that Gag-RNA interactions occur at multiple RNA sites during Genome Packaging; furthermore, there are functionally redundant binding sites in viral RNA.
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Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly.
Journal of virology, 2017Co-Authors: Kari A. Dilley, Olga A. Nikolaitchik, Andrea Galli, Ryan C. Burdick, Louis Levine, Alan Rein, Vinay K. PathakAbstract:Most HIV-1 virions contain two copies of full-length viral RNA, indicating that Genome Packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of Genome Packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient Genome Packaging during virus assembly.IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA Genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA Genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly. Understanding the mechanism used by HIV-1 to ensure Genome Packaging provides significant insights into viral assembly and replication.
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Dimeric RNA recognition regulates HIV-1 Genome Packaging.
PLoS pathogens, 2013Co-Authors: Olga A. Nikolaitchik, S.-h. Sheldon Tai, Kari A. Dilley, Robert J. Gorelick, Ferri Soheilian, Roger G. Ptak, Kunio Nagashima, Vinay K. PathakAbstract:How retroviruses regulate the amount of RNA Genome packaged into each virion has remained a long-standing question. Our previous study showed that most HIV-1 particles contain two copies of viral RNA, indicating that the number of Genomes packaged is tightly regulated. In this report, we examine the mechanism that controls the number of RNA Genomes encapsidated into HIV-1 particles. We hypothesize that HIV-1 regulates Genome Packaging by either the mass or copy number of the viral RNA. These two distinct mechanisms predict different outcomes when the Genome size deviates significantly from that of wild type. Regulation by RNA mass would result in multiple copies of a small Genome or one copy of a large Genome being packaged, whereas regulation by copy number would result in two copies of a Genome being packaged independent of size. To distinguish between these two hypotheses, we examined the Packaging of viral RNA that was larger (≈17 kb) or smaller (≈3 kb) than that of wild-type HIV-1 (≈9 kb) and found that most particles packaged two copies of the viral Genome regardless of whether they were 17 kb or 3 kb. Therefore, HIV-1 regulates RNA Genome encapsidation not by the mass of RNA but by Packaging two copies of RNA. To further explore the mechanism that governs this regulation, we examined the Packaging of viral RNAs containing two Packaging signals that can form intermolecular dimers or intramolecular dimers (self-dimers) and found that one self-dimer is packaged. Therefore, HIV-1 recognizes one dimeric RNA instead of two copies of RNA. Our findings reveal that dimeric RNA recognition is the key mechanism that regulates HIV-1 Genome encapsidation and provide insights into a critical step in the generation of infectious viruses.
Michael F. Summers - One of the best experts on this subject based on the ideXlab platform.
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Structural Determinants and Mechanism of HIV-1 Genome Packaging
Journal of molecular biology, 2011Co-Authors: Xiao Heng, Michael F. SummersAbstract:Like all retroviruses, the human immunodeficiency virus selectively packages two copies of its unspliced RNA Genome, both of which are utilized for strand-transfer-mediated recombination during reverse transcription—a process that enables rapid evolution under environmental and chemotherapeutic pressures. The viral RNA appears to be selected for Packaging as a dimer, and there is evidence that dimerization and Packaging are mechanistically coupled. Both processes are mediated by interactions between the nucleocapsid domains of a small number of assembling viral Gag polyproteins and RNA elements within the 5′-untranslated region of the Genome. A number of secondary structures have been predicted for regions of the Genome that are responsible for Packaging, and high-resolution structures have been determined for a few small RNA fragments and protein–RNA complexes. However, major questions regarding the RNA structures (and potentially the structural changes) that are responsible for dimeric Genome selection remain unanswered. Here, we review efforts that have been made to identify the molecular determinants and mechanism of human immunodeficiency virus type 1 Genome Packaging.
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an rna structural switch regulates diploid Genome Packaging by moloney murine leukemia virus
Journal of Molecular Biology, 2010Co-Authors: Yasuyuki Miyazaki, Eric L. Garcia, Steven R King, Kilali Iyalla, Kelsey Loeliger, Patrice Starck, Sameera Syed, Alice Telesnitsky, Michael F. SummersAbstract:Retroviruses selectively package two copies of their RNA Genomes by mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukaemia virus (MoMuLV) Genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5'-untranslated region (5'-UTR) that contains all residues necessary for efficient RNA Packaging (Ψ WT , residues 147–623) also exhibits dimerization-dependent affinity for NC, with the native dimer ([Ψ WT ]2) binding 12 ± 2 NC molecules with high affinity (Kd = 17 ± 7 nM) and the monomer, stabilized by substitution of dimer-promoting loop residues by hairpin-stabilizing sequences (Ψ M ), binding 1–2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit Genome Packaging in vivo (~100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism, and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric Packaging signal.
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An RNA Structural Switch Regulates Diploid Genome Packaging by Moloney Murine Leukemia Virus
Journal of molecular biology, 2009Co-Authors: Yasuyuki Miyazaki, Eric L. Garcia, Steven R King, Kilali Iyalla, Kelsey Loeliger, Patrice Starck, Sameera Syed, Alice Telesnitsky, Michael F. SummersAbstract:Retroviruses selectively package two copies of their RNA Genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) Genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5' untranslated region that contains all residues necessary for efficient RNA Packaging (Psi(WT); residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Psi(WT)](2)) binding 12+/-2 NC molecules with high affinity (K(d)=17+/-7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Psi(M)), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit Genome Packaging in vivo (approximately 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric Packaging signal.
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Insights Into The Mechanism Of Retroviral Genome Packaging And Assembly
Biophysical Journal, 2009Co-Authors: Michael F. SummersAbstract:In HIV-1 infected cells, newly synthesized retroviral Gag polyproteins are directed to specific cellular membranes where they assemble and bud to form immature virions. Membrane binding is mediated by Gag's matrix (MA) domain, a 132-residue polypeptide containing an N-terminal myristyl group that can adopt sequestered and exposed conformations. Membane specificity was recently shown to be regulated by phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2), a cellular factor abundant in the inner leaflet of the plasma membrane (PM). We now show that phosphoinositides, including soluble analogs of PI(4,5)P2 with truncated lipids, bind HIV-1 MA and trigger myristate exposure. The phosphoinositol moiety and one of the fatty acid tails binds to a cleft on the surface of the protein. The other fatty acid chain of PI(4,5)P2 and the exposed myristyl group of MA bracket a conserved basic surface patch implicated in membrane binding. Our findings indicate that PI(4,5)P2 acts as both a trigger of the myristyl switch and as a membrane anchor, and suggest a structure-based mechanism for the specific targeting HIV-1 Gag to PI(4,5)P2-enriched membranes. Retroviral Genomes contain elements within their 5′-untranslated regions (UTRs) that regulate multiple essential functions, including splicing, nuclear export, translational activation, Genome Packaging, and reverse transcription, among others. A number of studies suggest that these processes may be differentially regulated by RNA conformational changes. To gain insights into the structural basis for these processes, we have initiated NMR studies of intact retroviral Packaging elements, including the native, dimeric 200 nucleotide core encapsidation signal (ΨCES) of the Moloney murine leukaemia virus (MLV) and the intact, dimeric 748 nucleotide 5′-UTR of the human immunodeficiency virus Type-1 (HIV-1). Progress toward the implementation of these data as restraints for structure refinement of the dimeric MLV ΨCES will be presented.
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how retroviruses select their Genomes
Nature Reviews Microbiology, 2005Co-Authors: Victoria M Dsouza, Michael F. SummersAbstract:As retroviruses assemble in infected cells, two copies of their full-length, unspliced RNA Genomes are selected for Packaging from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Understanding the molecular details of Genome Packaging is important for the development of new antiviral strategies and to enhance the efficacy of retroviral vectors used in human gene therapy. Recent studies of viral RNA structure in vitro and in vivo and high-resolution studies of RNA fragments and protein–RNA complexes are helping to unravel the mechanism of Genome Packaging and providing the first glimpses of the initial stages of retrovirus assembly.
Olga A. Nikolaitchik - One of the best experts on this subject based on the ideXlab platform.
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Specific Guanosines in the HIV-2 Leader RNA are Essential for Efficient Viral Genome Packaging
Journal of molecular biology, 2020Co-Authors: Chijioke N. Umunnakwe, Olga A. Nikolaitchik, Alice Duchon, Abdul Rahman, Yang Liu, Jianbo Chen, S.-h. Sheldon Tai, Vinay K. PathakAbstract:HIV-2, a human pathogen that causes acquired immunodeficiency syndrome, is distinct from the more prevalent HIV-1 in several features including its evolutionary history and certain aspects of viral replication. Like other retroviruses, HIV-2 packages two copies of full-length viral RNA during virus assembly and efficient Genome encapsidation is mediated by the viral protein Gag. We sought to define cis-acting elements in the HIV-2 Genome that are important for the encapsidation of full-length RNA into viral particles. Based on previous studies of murine leukemia virus and HIV-1, we hypothesized that unpaired guanosines in the 5' untranslated region (UTR) play an important role in Gag:RNA interactions leading to Genome Packaging. To test our hypothesis, we targeted 18 guanosines located in 9 sites within the HIV-2 5' UTR and performed substitution analyses. We found that mutating as few as three guanosines significantly reduce RNA Packaging efficiency. However, not all guanosines examined have the same effect; instead, a hierarchical order exists wherein a primary site, a secondary site, and three tertiary sites are identified. Additionally, there are functional overlaps in these sites and mutations of more than one site can act synergistically to cause Genome Packaging defects. These studies demonstrate the importance of specific guanosines in HIV-2 5'UTR in mediating Genome Packaging. Our results also demonstrate an interchangeable and hierarchical nature of guanosine-containing sites, which was not previously established, thereby revealing key insights into the replication mechanisms of HIV-2.
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Unpaired Guanosines in the 5′ Untranslated Region of HIV-1 RNA Act Synergistically to Mediate Genome Packaging
Journal of virology, 2020Co-Authors: Olga A. Nikolaitchik, Xayathed Somoulay, Jonathan M O Rawson, Jennifer A Yoo, Vinay K. PathakAbstract:The viral protein Gag selects full-length HIV-1 RNA from a large pool of mRNAs as virion Genome during virus assembly. Currently, the precise mechanism that mediates the Genome selection is not understood. Previous studies have identified several sites in the 5' untranslated region (5' UTR) of HIV-1 RNA that are bound by nucleocapsid (NC) protein, which is derived from Gag during virus maturation. However, whether these NC binding sites direct HIV-1 RNA Genome Packaging has not been fully investigated. In this report, we examined the roles of single-stranded, exposed guanosines at NC binding sites in RNA Genome Packaging using stable cell lines expressing competing wild-type and mutant HIV-1 RNAs. Mutant RNA Packaging efficiencies were determined by comparing their prevalence in cytoplasmic RNA and in virion RNA. We observed that multiple NC binding sites affected RNA Packaging; of the sites tested, those located within stem-loop 1 of the 5' UTR had the most significant effects. These sites were previously reported as the primary NC binding sites using a chemical probe reverse-footprinting assay and as the major Gag binding sites using an in vitro assay. Of the mutants tested in this report, substituting 3 to 4 guanosines resulted in less than two-fold defects in Packaging. However, when mutations at different NC binding sites were combined, severe defects were observed. Furthermore, combining the mutations resulted in synergistic defects in RNA Packaging, suggesting redundancy in Gag:RNA interactions and a requirement for multiple Gag binding on viral RNA during HIV-1 Genome encapsidation.IMPORTANCE HIV-1 must package its RNA Genome during virus assembly to generate infectious viruses. To better understand how HIV-1 packages its RNA Genome, we examined the roles of RNA elements identified as binding sites for NC, a Gag-derived RNA-binding protein. Our results demonstrate that binding sites within stem-loop 1 of the 5' untranslated region play important roles in Genome Packaging. Although mutating one or two NC-binding sites caused only mild defects in Packaging, mutating multiple sites resulted in severe defects in Genome encapsidation, indicating that unpaired guanosines act synergistically to promote Packaging. Our results suggest that Gag:RNA interactions occur at multiple RNA sites during Genome Packaging; furthermore, there are functionally redundant binding sites in viral RNA.
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unpaired guanosines in the 5 untranslated region of hiv 1 rna act synergistically to mediate Genome Packaging
Journal of Virology, 2020Co-Authors: Olga A. Nikolaitchik, Xayathed Somoulay, Jonathan M O Rawson, Jennifer A Yoo, Vinay K. PathakAbstract:The viral protein Gag selects full-length HIV-1 RNA from a large pool of mRNAs as virion Genome during virus assembly. Currently, the precise mechanism that mediates the Genome selection is not understood. Previous studies have identified several sites in the 5' untranslated region (5' UTR) of HIV-1 RNA that are bound by nucleocapsid (NC) protein, which is derived from Gag during virus maturation. However, whether these NC binding sites direct HIV-1 RNA Genome Packaging has not been fully investigated. In this report, we examined the roles of single-stranded exposed guanosines at NC binding sites in RNA Genome Packaging using stable cell lines expressing competing wild-type and mutant HIV-1 RNAs. Mutant RNA Packaging efficiencies were determined by comparing their prevalences in cytoplasmic RNA and in virion RNA. We observed that multiple NC binding sites affected RNA Packaging; of the sites tested, those located within stem-loop 1 of the 5' UTR had the most significant effects. These sites were previously reported as the primary NC binding sites by using a chemical probe reverse-footprinting assay and as the major Gag binding sites by using an in vitro assay. Of the mutants tested in this report, substituting 3 to 4 guanosines resulted in <2-fold defects in Packaging. However, when mutations at different NC binding sites were combined, severe defects were observed. Furthermore, combining the mutations resulted in synergistic defects in RNA Packaging, suggesting redundancy in Gag-RNA interactions and a requirement for multiple Gag binding on viral RNA during HIV-1 Genome encapsidation.IMPORTANCE HIV-1 must package its RNA Genome during virus assembly to generate infectious viruses. To better understand how HIV-1 packages its RNA Genome, we examined the roles of RNA elements identified as binding sites for NC, a Gag-derived RNA-binding protein. Our results demonstrate that binding sites within stem-loop 1 of the 5' untranslated region play important roles in Genome Packaging. Although mutating one or two NC-binding sites caused only mild defects in Packaging, mutating multiple sites resulted in severe defects in Genome encapsidation, indicating that unpaired guanosines act synergistically to promote Packaging. Our results suggest that Gag-RNA interactions occur at multiple RNA sites during Genome Packaging; furthermore, there are functionally redundant binding sites in viral RNA.
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Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly.
Journal of virology, 2017Co-Authors: Kari A. Dilley, Olga A. Nikolaitchik, Andrea Galli, Ryan C. Burdick, Louis Levine, Alan Rein, Vinay K. PathakAbstract:Most HIV-1 virions contain two copies of full-length viral RNA, indicating that Genome Packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of Genome Packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient Genome Packaging during virus assembly.IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA Genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA Genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly. Understanding the mechanism used by HIV-1 to ensure Genome Packaging provides significant insights into viral assembly and replication.
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Deciphering the Role of the Gag-Pol Ribosomal Frameshift Signal in HIV-1 RNA Genome Packaging
Journal of virology, 2014Co-Authors: Olga A. NikolaitchikAbstract:A key step of retroviral replication is Packaging of the viral RNA Genome during virus assembly. Specific Packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA Genome. In HIV-1, similar to most retroviruses, the Packaging signal is located within the 5′ untranslated region and extends into the gag-coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA Packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10- to 50-fold) in viral RNA Packaging and virus titer. We examined here the role of the ribosomal frameshift signal in HIV-1 RNA Packaging by studying the RNA Packaging and virus titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently, and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation. IMPORTANCE To generate infectious viruses, HIV-1 must package viral RNA Genome during virus assembly. The specific HIV-1 Genome Packaging is mediated by interactions between the structural protein Gag and elements near the 5′ end of the viral RNA known as Packaging signal. In this study, we examined whether the Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA Packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans, none of the tested ribosomal frameshift signal mutants has defects in RNA Packaging or virus titer. These studies provide important information on how HIV-1 regulates its Genome Packaging and generate infectious viruses necessary for transmission to new hosts.
Gino Cingolani - One of the best experts on this subject based on the ideXlab platform.
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dna conformational changes play a force generating role during bacteriophage Genome Packaging
Biophysical Journal, 2019Co-Authors: Kim A Sharp, Gino Cingolani, Stephen C HarveyAbstract:Abstract Motors that move DNA, or that move along DNA, play essential roles in DNA replication, transcription, recombination, and chromosome segregation. The mechanisms by which these DNA translocases operate remain largely unknown. Some double-stranded DNA (dsDNA) viruses use an ATP-dependent motor to drive DNA into preformed capsids. These include several human pathogens as well as dsDNA bacteriophages—viruses that infect bacteria. We previously proposed that DNA is not a passive substrate of bacteriophage Packaging motors but is instead an active component of the machinery. We carried out computational studies on dsDNA in the channels of viral portal proteins, and they reveal DNA conformational changes consistent with that hypothesis. dsDNA becomes longer (“stretched”) in regions of high negative electrostatic potential and shorter (“scrunched”) in regions of high positive potential. These results suggest a mechanism that electrostatically couples the energy released by ATP hydrolysis to DNA translocation: The chemical cycle of ATP binding, hydrolysis, and product release drives a cycle of protein conformational changes. This produces changes in the electrostatic potential in the channel through the portal, and these drive cyclic changes in the length of dsDNA as the phosphate groups respond to the protein’s electrostatic potential. The DNA motions are captured by a coordinated protein-DNA grip-and-release cycle to produce DNA translocation. In short, the ATPase, portal, and dsDNA work synergistically to promote Genome Packaging.
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simulations and electrostatic analysis suggest an active role for dna conformational changes during Genome Packaging by bacteriophages
bioRxiv, 2018Co-Authors: Kim A Sharp, Gino Cingolani, Stephen C HarveyAbstract:Motors that move DNA, or that move along DNA, play essential roles in DNA replication, transcription, recombination, and chromosome segregation. The mechanisms by which these DNA translocases operate remain largely unknown. Some double-stranded DNA (dsDNA) viruses use an ATP-dependent motor to drive DNA into preformed capsids. These include several human pathogens, as well as dsDNA bacteriophages (viruses that infect bacteria). We previously proposed that DNA is not a passive substrate of bacteriophage Packaging motors but is, instead, an active component of the machinery. Computational studies on dsDNA in the channel of viral portal proteins reported here reveal DNA conformational changes consistent with that hypothesis. dsDNA becomes longer ("stretched") in regions of high negative electrostatic potential, and shorter ("scrunched") in regions of high positive potential. These results suggest a mechanism that couples the energy released by ATP hydrolysis to DNA translocation: The chemical cycle of ATP binding, hydrolysis and product release drives a cycle of protein conformational changes. This produces changes in the electrostatic potential in the channel through the portal, and these drive cyclic changes in the length of dsDNA. The DNA motions are captured by a coordinated protein-DNA grip-and-release cycle to produce DNA translocation. In short, the ATPase, portal and dsDNA work synergistically to promote Genome Packaging.
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portal protein functions akin to a dna sensor that couples Genome Packaging to icosahedral capsid maturation
Nature Communications, 2017Co-Authors: Ravi K Lokareddy, Ankoor Roy, Gino Cingolani, Rajeshwer S Sankhala, Pavel V Afonine, Tina Motwani, Carolyn M Teschke, Kristin N ParentAbstract:Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or 'procapsid') built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome Packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase Packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the Packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of 'Headful Packaging' is a DNA-dependent symmetrization of portal protein.
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small terminase couples viral dna binding to Genome Packaging atpase activity
Structure, 2012Co-Authors: Ankoor Roy, Anshul Bhardwaj, Pinaki Datta, Gabriel C Lander, Gino CingolaniAbstract:Packaging of viral Genomes into empty procapsids is powered by a large DNA-Packaging motor. In most viruses, this machine is composed of a large (L) and a small (S) terminase subunit complexed with a dodecamer of portal protein. Here we describe the 1.75 A crystal structure of the bacteriophage P22 S-terminase in a nonameric conformation. The structure presents a central channel ∼23 A in diameter, sufficiently large to accommodate hydrated B-DNA. The last 23 residues of S-terminase are essential for binding to DNA and assembly to L-terminase. Upon binding to its own DNA, S-terminase functions as a specific activator of L-terminase ATPase activity. The DNA-dependent stimulation of ATPase activity thus rationalizes the exclusive specificity of Genome-Packaging motors for viral DNA in the crowd of host DNA, ensuring fidelity of Packaging and avoiding wasteful ATP hydrolysis. This posits a model for DNA-dependent activation of Genome-Packaging motors of general interest in virology.