Envelope Lipids Regulate the In Vitro Assembly of the HIV-1 Capsid

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) normally assembles into particles of 100 to 120 nm in diameter by budding through the plasma membrane of the cell. The Gag polyprotein is the only viral protein that is required for the formation of these particles. We have used an in vitro assembly system to examine the assembly properties of purified, recombinant HIV-1 Gag protein and of Gag missing the C-terminal p6 domain (Gag Δp6). This system was used previously to show that the CA-NC fragment of HIV-1 Gag assembled into cylindrical particles. We now report that both HIV-1 Gag and Gag Δp6 assemble into small, 25- to 30-nm-diameter spherical particles in vitro. The multimerization of Gag Δp6 into units larger than dimers and the formation of spherical particles required nucleic acid. Removal of the nucleic acid with NaCl or nucleases resulted in the disruption of the multimerized complexes. We conclude from these results that (i) N-terminal extension of HIV-1 CA-NC to include the MA domain results in the formation of spherical, rather than cylindrical, particles; (ii) nucleic acid is required for the assembly and maintenance of HIV-1 Gag Δp6 virus-like particles in vitro and possibly in vivo; (iii) a wide variety of RNAs or even short DNA oligonucleotides will support assembly; (iv) protein-protein interactions within the particle must be relatively weak; and (v) recombinant HIV-1 Gag Δp6 and nucleic acid are not sufficient for the formation of normal-sized particles.

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A Structural Perspective of the Role of IP6 in Immature and Mature Retroviral Assembly

Viruses ◽

10.3390/v13091853 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1853

Author(s):

Martin Obr ◽

Florian K. M. Schur ◽

Robert A. Dick

Keyword(s):

Life Cycle ◽

Binding Site ◽

Core Stability ◽

The Family ◽

In Vitro Assembly ◽

Retroviral Assembly ◽

Immature Virus ◽

Hiv 1

The small cellular molecule inositol hexakisphosphate (IP6) has been known for ~20 years to promote the in vitro assembly of HIV-1 into immature virus-like particles. However, the molecular details underlying this effect have been determined only recently, with the identification of the IP6 binding site in the immature Gag lattice. IP6 also promotes formation of the mature capsid protein (CA) lattice via a second IP6 binding site, and enhances core stability, creating a favorable environment for reverse transcription. IP6 also enhances assembly of other retroviruses, from both the Lentivirus and the Alpharetrovirus genera. These findings suggest that IP6 may have a conserved function throughout the family Retroviridae. Here, we discuss the different steps in the viral life cycle that are influenced by IP6, and describe in detail how IP6 interacts with the immature and mature lattices of different retroviruses.

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Kinetic Studies of the In Vitro Assembly of HIV-1 Capsid Protein

Virus Research ◽

10.1016/s0168-1702(01)00327-6 ◽

2001 ◽

Vol 77 (2) ◽

pp. 109-110

Keyword(s):

Capsid Protein ◽

Kinetic Studies ◽

In Vitro Assembly ◽

Hiv 1

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Distinct Roles for Nucleic Acid in In Vitro Assembly of Purified Mason-Pfizer Monkey Virus CANC Proteins

Journal of Virology ◽

10.1128/jvi.02694-05 ◽

2006 ◽

Vol 80 (14) ◽

pp. 7089-7099 ◽

Cited By ~ 28

Author(s):

Pavel Ulbrich ◽

Sarka Haubova ◽

Milan V. Nermut ◽

Eric Hunter ◽

Michaela Rumlova ◽

...

Keyword(s):

Conformational Changes ◽

Spherical Particles ◽

Genomic Rna ◽

Cylindrical Structures ◽

Immunodeficiency Virus ◽

In Vitro Assembly ◽

Transient Interaction ◽

Hiv 1

ABSTRACT In contrast to other retroviruses, Mason-Pfizer monkey virus (M-PMV) assembles immature capsids in the cytoplasm. We have compared the ability of minimal assembly-competent domains from M-PMV and human immunodeficiency virus type 1 (HIV-1) to assemble in vitro into virus-like particles in the presence and absence of nucleic acids. A fusion protein comprised of the capsid and nucleocapsid domains of Gag (CANC) and its N-terminally modified mutant (ΔProCANC) were used to mimic the assembly of the viral core and immature particles, respectively. In contrast to HIV-1, where CANC assembled efficiently into cylindrical structures, the same domains of M-PMV were assembly incompetent. The addition of RNA or oligonucleotides did not complement this defect. In contrast, the M-PMV ΔProCANC molecule was able to assemble into spherical particles, while that of HIV-1 formed both spheres and cylinders. For M-PMV, the addition of purified RNA increased the efficiency with which ΔProCANC formed spherical particles both in terms of the overall amount and the numbers of completed spheres. The amount of RNA incorporated was determined, and for both rRNA and MS2-RNA, quantities similar to that of genomic RNA were encapsidated. Oligonucleotides also stimulated assembly; however, they were incorporated into ΔProCANC spherical particles in trace amounts that could not serve as a stoichiometric structural component for assembly. Thus, oligonucleotides may, through a transient interaction, induce conformational changes that facilitate assembly, while longer RNAs appear to facilitate the complete assembly of spherical particles.

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In vitro assembly of 2-D crystals from partially purified EA1 protein secreted by Bacillus anthracis strain Delta Sterne-1

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100169110 ◽

1994 ◽

Vol 52 ◽

pp. 276-277

Author(s):

Mary Beth Downs ◽

Wilson Ribot ◽

Joseph W. Farchaus

Keyword(s):

Cell Wall ◽

Molecular Sieves ◽

Cell Envelope ◽

Single Species ◽

Supporting Cell ◽

Surface Layers ◽

Rabbit Igg ◽

In Vitro Assembly ◽

Covalently Linked

Many bacteria possess surface layers (S-layers) that consist of a two-dimensional protein lattice external to the cell envelope. These S-layer arrays are usually composed of a single species of protein or glycoprotein and are not covalently linked to the underlying cell wall. When removed from the cell, S-layer proteins often reassemble into a lattice identical to that found on the cell, even without supporting cell wall fragments. S-layers exist at the interface between the cell and its environment and probably serve as molecular sieves that exclude destructive macromolecules while allowing passage of small nutrients and secreted proteins. Some S-layers are refractory to ingestion by macrophages and, generally, bacteria are more virulent when S-layers are present.When grown in rich medium under aerobic conditions, B. anthracis strain Delta Sterne-1 secretes large amounts of a proteinaceous extractable antigen 1 (EA1) into the growth medium. Immunocytochemistry with rabbit polyclonal anti-EAl antibody made against the secreted protein and gold-conjugated goat anti-rabbit IgG showed that EAI was localized at the cell surface (fig 1), which suggests its role as an S-layer protein.

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Faculty Opinions recommendation of Loop-loop interaction of HIV-1 TAR RNA with N3'-->P5' deoxyphosphoramidate aptamers inhibits in vitro Tat-mediated transcription.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1008473.106958 ◽

2002 ◽

Author(s):

Anthony Czarnik

Keyword(s):

Tar Rna ◽

Hiv 1

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DEVELOPMENT OF APPROACHES FOR DETECTION OF GENOME-INTEGRATED PROVIRAL DNA OF THE HUMAN IMMUNODEFICIENCY VIRUS (HIV-1) IN ULTRA LOW CONCENTRATIONS USING THE CRISPR/CAS SYSTEM

Molecular Diagnostics and Biosafety – 2020. Russian national scientific and practical conference with international participation (October, 6–8, 2020): Conference Proceedings ◽

10.36233/978-5-9900432-9-9-118 ◽

2020 ◽

Author(s):

M.A. Tyumentseva ◽

◽

A.I. Tyumentsev ◽

V.G. Akimkin ◽

◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Health Monitoring ◽

Biological Samples ◽

Proviral Dna ◽

Guide Rnas ◽

Ribonucleoprotein Complexes ◽

Low Concentrations ◽

Immunodeficiency Virus ◽

Hiv 1

For the effective functioning of supervisory and health monitoring services, it is necessary to introduce modern molecular technologies into their practice. Therefore, the task of developing new effective methods for detecting pathogen, for example HIV, based on CRISPR/CAS genome editing systems, remains urgent. In the present work, guide RNAs and specific oligonucleotides were developed for preliminary amplification of highly conserved regions of the HIV-1 genome. The developed guide RNAs make it possible to detect single copies of HIV-1 proviral DNA in vitro as part of CRISPR/CAS ribonucleoprotein complexes in biological samples after preliminary amplification.

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