Quantification of the HIV‐1 virus‐like particle production process by super‐resolution imaging: From VLP budding to nanoparticle analysis

2020 ◽  
Vol 117 (7) ◽  
pp. 1929-1945 ◽  
Author(s):  
Irene González‐Domínguez ◽  
Eduard Puente‐Massaguer ◽  
Laura Cervera ◽  
Francesc Gòdia
Keyword(s):  
Production Process ◽  
Particle Production ◽  
Super Resolution ◽  
Virus Like Particle ◽  
Resolution Imaging ◽  
Hiv 1

scholarly journals Late Assembly Domain Function Can Exhibit Context Dependence and Involves Ubiquitin Residues Implicated in Endocytosis

2002 ◽  
Vol 76 (11) ◽  
pp. 5472-5479 ◽  
Author(s):  
Bettina Strack ◽  
Arianna Calistri ◽  
Heinrich G. Göttlinger
Keyword(s):  
Particle Production ◽  
Ebola Virus ◽  
Point Mutations ◽  
Core Motif ◽  
Virus Particles ◽  
Virus Like Particle ◽  
Immunodeficiency Virus ◽  
Late Assembly Domain ◽  
Hiv 1

ABSTRACT Retroviral Gag polyproteins contain regions that promote the separation of virus particles from the plasma membrane and from each other. These Gag regions are often referred to as late assembly (L) domains. The L domain of human immunodeficiency virus type 1 (HIV-1) is in the C-terminal p6 gag domain and harbors an essential P(T/S)APP motif, whereas the L domains of oncoretroviruses are in the N-terminal half of the Gag precursor and have a PPXY core motif. We recently observed that L domains induce the ubiquitination of a minimal HIV-1 Gag construct and that point mutations which abolish L domain activity prevent Gag ubiquitination. In that study, a peptide from the Ebola virus L domain with overlapping P(T/S)APP and PPXY motifs showed exceptional activity in promoting Gag ubiquitination and the release of virus-like particles. We now show that a substitution which disrupts the PPXY motif but leaves the P(T/S)APP motif intact abolishes L domain activity in the minimal Gag context, but not in the context of a near full-length HIV-1 Gag precursor. Our results reveal that the P(T/S)APP motif does not function autonomously and indicate that the HIV-1 nucleocapsid-p1 region, which is proximal to p6 gag , can cooperate with the conserved L domain core motif. We have also examined the effects of ubiquitin mutants on virus-like particle production, and the results indicate that residues required for the endocytosis function of ubiquitin are also involved in virus budding.

scholarly journals Super-Resolution Imaging of ESCRT-Proteins at HIV-1 Assembly Sites

2015 ◽  
Vol 11 (2) ◽  
pp. e1004677 ◽  
Author(s):  
Jens Prescher ◽  
Viola Baumgärtel ◽  
Sergey Ivanchenko ◽  
Adriano A. Torrano ◽  
Christoph Bräuchle ◽  
...  
Keyword(s):  
Super Resolution ◽  
Resolution Imaging ◽  
Hiv 1

scholarly journals Single-molecule super-resolution imaging of T-cell plasma membrane CD4 redistribution upon HIV-1 binding

2021 ◽  
Author(s):  
Yue Yuan ◽  
Caron Jacobs ◽  
Isabel Llorente Garcia ◽  
Pedro M. Pereira ◽  
Scott P. Lawrence ◽  
...  
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
Single Molecule ◽  
Super Resolution ◽  
Fold Increase ◽  
Membrane Receptors ◽  
Host Cells ◽  
Virus Binding ◽  
Resolution Imaging ◽  
Hiv 1

The first step of cellular entry for the human immunodeficiency virus type-1 (HIV-1) occurs through the binding of its envelope protein (Env) with the plasma membrane receptor CD4 and co-receptor CCR5 or CXCR4 on susceptible cells, primarily CD4+ T cells and macrophages. Although there is considerable knowledge of the molecular interactions between Env and host cell receptors that lead to successful fusion, the precise way in which HIV-1 receptors redistribute to sites of virus binding at the nanoscale remains unknown. Here, we quantitatively examine changes in the nanoscale organisation of CD4 on the surface of CD4+ T cells following HIV-1 binding. Using single-molecule super-resolution imaging, we show that CD4 molecules are distributed mostly as either individual molecules or small clusters of up to 4 molecules. Following virus binding, we observe a local 3-to-10-fold increase in cluster diameter and molecule number for virus-associated CD4 clusters. Moreover, a similar but smaller magnitude reorganisation of CD4 was also observed with recombinant gp120. For the first time, our results quantify the nanoscale CD4 reorganisation triggered by HIV-1 on host cells. Our quantitative approach provides a robust methodology for characterising the nanoscale organisation of plasma membrane receptors in general with the potential to link spatial organisation to function.

scholarly journals Single-Molecule Super-Resolution Imaging of T-Cell Plasma Membrane CD4 Redistribution upon HIV-1 Binding

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 142
Author(s):  
Yue Yuan ◽  
Caron A. Jacobs ◽  
Isabel Llorente Garcia ◽  
Pedro M. Pereira ◽  
Scott P. Lawrence ◽  
...  
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
Single Molecule ◽  
Cd4 T Cells ◽  
Super Resolution ◽  
Fold Increase ◽  
Membrane Receptors ◽  
Virus Binding ◽  
Resolution Imaging ◽  
Hiv 1

The first step of cellular entry for the human immunodeficiency virus type-1 (HIV-1) occurs through the binding of its envelope protein (Env) with the plasma membrane receptor CD4 and co-receptor CCR5 or CXCR4 on susceptible cells, primarily CD4+ T cells and macrophages. Although there is considerable knowledge of the molecular interactions between Env and host cell receptors that lead to successful fusion, the precise way in which HIV-1 receptors redistribute to sites of virus binding at the nanoscale remains unknown. Here, we quantitatively examine changes in the nanoscale organisation of CD4 on the surface of CD4+ T cells following HIV-1 binding. Using single-molecule super-resolution imaging, we show that CD4 molecules are distributed mostly as either individual molecules or small clusters of up to 4 molecules. Following virus binding, we observe a local 3-to-10-fold increase in cluster diameter and molecule number for virus-associated CD4 clusters. Moreover, a similar but smaller magnitude reorganisation of CD4 was also observed with recombinant gp120. For one of the first times, our results quantify the nanoscale CD4 reorganisation triggered by HIV-1 on host CD4+ T cells. Our quantitative approach provides a robust methodology for characterising the nanoscale organisation of plasma membrane receptors in general with the potential to link spatial organisation to function.

scholarly journals Divergent Bro1 Domains Share the Capacity To Bind Human Immunodeficiency Virus Type 1 Nucleocapsid and To Enhance Virus-Like Particle Production

2009 ◽  
Vol 83 (14) ◽  
pp. 7185-7193 ◽  
Author(s):  
Sergei Popov ◽  
Elena Popova ◽  
Michio Inoue ◽  
Heinrich G. Göttlinger
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Particle Production ◽  
Intrinsic Property ◽  
Significant Activity ◽  
Virus Like Particle ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT To promote the release of infectious virions, human immunodeficiency virus type 1 (HIV-1) exploits the endosomal sorting complex required for transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in p6 Gag. An LYPxnL motif in p6 serves as docking site for the central V domain of ALIX and is required for its ability to stimulate HIV-1 budding. Additionally, the nucleocapsid (NC) domain of Gag binds to the N-terminal Bro1 domain of ALIX, which connects ALIX to the membrane-deforming ESCRT-III complex via its CHMP4 subunits. Since the isolated Bro1 domain of ALIX is sufficient to markedly stimulate virus-like particle (VLP) production in a minimal Gag rescue assay, we examined whether the Bro1 domains of other human proteins possess a similar activity. We now show that the Bro1 domain-only protein Brox and the isolated Bro1 domains of HD-PTP and rhophilin all bind to HIV-1 NC. Furthermore, all shared the capacity to stimulate VLP production by a minimal HIV-1 Gag molecule, and Brox in particular was as potent as the Bro1 domain of ALIX in this assay. Unexpectedly, Brox retained significant activity even if its CHMP4 binding site was disrupted. Thus, the ability to assist in VLP production may be an intrinsic property of the boomerang-shaped Bro1 domain.

Stable Sf9 cell pools as a system for rapid HIV ‐1 virus‐like particle production

2021 ◽  
Author(s):  
Eduard Puente‐Massaguer ◽  
Paula Grau‐Garcia ◽  
Florian Strobl ◽  
Reingard Grabherr ◽  
Gerald Striedner ◽  
...  
Keyword(s):  
Particle Production ◽  
Virus Like Particle ◽  
Hiv 1
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