Human T-lymphotrophic virus type I nucleocapsid protein NCp15: structural study and stability of the N-terminal zinc-finger

BERTOLA, Françoise; MANIGAND, Claude; PICARD, Philippe; BELGHAZI, Maya; PRECIGOUX, Gilles

doi:10.1042/0264-6021:3520293

Cited by 3 publications

(4 citation statements)

References 46 publications

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“…In vitro studies suggest that the HTLV-1 Gag shell is very likely to be organized differently compared to that of HIV-1 Gag [16-18]. When examining the cryo-TEM images of HTLV-1-like particles, we rarely observed a highly ordered Gag lattice next to the lipid bilayer (Figure 3A), a feature frequently observed in immature HIV-1 particles.…”

Section: Discussionmentioning

confidence: 87%

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Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stoichiometry

et al. 2010

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BackgroundHuman T-lymphotropic virus type 1 (HTLV-1) is an important human retrovirus that is a cause of adult T-cell leukemia/lymphoma. While an important human pathogen, the details regarding virus replication cycle, including the nature of HTLV-1 particles, remain largely unknown due to the difficulties in propagating the virus in tissue culture. In this study, we created a codon-optimized HTLV-1 Gag fused to an EYFP reporter as a model system to quantitatively analyze HTLV-1 particles released from producer cells.ResultsThe codon-optimized Gag led to a dramatic and highly robust level of Gag expression as well as virus-like particle (VLP) production. The robust level of particle production overcomes previous technical difficulties with authentic particles and allowed for detailed analysis of particle architecture using two novel methodologies. We quantitatively measured the diameter and morphology of HTLV-1 VLPs in their native, hydrated state using cryo-transmission electron microscopy (cryo-TEM). Furthermore, we were able to determine HTLV-1 Gag stoichiometry as well as particle size with the novel biophysical technique of fluorescence fluctuation spectroscopy (FFS). The average HTLV-1 particle diameter determined by cryo-TEM and FFS was 71 ± 20 nm and 75 ± 4 nm, respectively. These values are significantly smaller than previous estimates made of HTLV-1 particles by negative staining TEM. Furthermore, cryo-TEM reveals that the majority of HTLV-1 VLPs lacks an ordered structure of the Gag lattice, suggesting that the HTLV-1 Gag shell is very likely to be organized differently compared to that observed with HIV-1 Gag in immature particles. This conclusion is supported by our observation that the average copy number of HTLV-1 Gag per particle is estimated to be 510 based on FFS, which is significantly lower than that found for HIV-1 immature virions.ConclusionsIn summary, our studies represent the first quantitative biophysical analysis of HTLV-1-like particles and reveal novel insights into particle morphology and Gag stochiometry.

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Section: Discussionmentioning

confidence: 87%

“…Despite limited amino acid sequence homology among different retroviruses, the atomic tertiary structures of individual Gag domains exhibit high similarity [16-18]. Therefore, structural and assembly mechanisms of HIV-1 are generally used as a reference model for other retroviruses.…”

Section: Introductionmentioning

confidence: 99%

Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stoichiometry

et al. 2010

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“…However, NMR structures have been reported for several isolated zinc knuckle domains 120–123 , intact NC proteins 96,124–129 , and NC:RNA complexes 66,128,130–137 . The zinc knuckles form highly stable mini-globular domains that are structurally distinct from other CCHC-type modules 131,138,139 .…”

Section: Genome Selection Is Mediated By the Nc Domain Of Gagmentioning

confidence: 99%

Structural Determinants and Mechanism of HIV-1 Genome Packaging

Heng

Summers

2011

Journal of Molecular Biology

226

288

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Like all retroviruses, the Human Immunodeficiency Virus (HIV) 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 (NC) domains of a small number of assembling viral Gag polyproteins and RNA elements within the 5′-untranslated region (5′-UTR) 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 remain open regarding the RNA structures, and potentially the structural changes, that are responsible for dimeric genome selection. Here we review efforts that have been made to identify the molecular determinants and mechanism of HIV-1 genome packaging.

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“…NMR structures were reported for several isolated retroviral zinc knuckle domains [ 92 , 93 , 95 , 96 , 97 , 98 , 99 ], for intact NC proteins [ 82 , 100 , 101 , 102 , 103 , 104 , 105 , 106 ], and NC–RNA complexes [ 66 , 104 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 ] (see sections below for more details). Weak NOEs between aromatic residues of the N- and C-terminal HIV-1 NC zinc knuckles [ 102 ] and NMR relaxation and chemical shift data [ 115 ] were consistent with a model in which the zinc knuckles transiently interact with each other like “beads on a string” [ 82 , 115 ]; Figure 2 B.…”

Section: Protein Components Important For Genome Packagingmentioning

confidence: 99%

NMR Studies of Retroviral Genome Packaging

Boyd

Brown

et al. 2020

Viruses

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Nearly all retroviruses selectively package two copies of their unspliced RNA genomes from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Over the past four decades, combinations of genetic experiments, phylogenetic analyses, nucleotide accessibility mapping, in silico RNA structure predictions, and biophysical experiments were employed to understand how retroviral genomes are selected for packaging. Genetic studies provided early clues regarding the protein and RNA elements required for packaging, and nucleotide accessibility mapping experiments provided insights into the secondary structures of functionally important elements in the genome. Three-dimensional structural determinants of packaging were primarily derived by nuclear magnetic resonance (NMR) spectroscopy. A key advantage of NMR, relative to other methods for determining biomolecular structure (such as X-ray crystallography), is that it is well suited for studies of conformationally dynamic and heterogeneous systems—a hallmark of the retrovirus packaging machinery. Here, we review advances in understanding of the structures, dynamics, and interactions of the proteins and RNA elements involved in retroviral genome selection and packaging that are facilitated by NMR.

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Human T-lymphotrophic virus type I nucleocapsid protein NCp15: structural study and stability of the N-terminal zinc-finger

Cited by 3 publications

References 46 publications

Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stoichiometry

Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stoichiometry

Structural Determinants and Mechanism of HIV-1 Genome Packaging

NMR Studies of Retroviral Genome Packaging

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