scholarly journals Structural Characterization of Tethered HIV-1 VLPs by Light Microscopy and Cryo-Electron Tomography

2014 ◽  
Vol 20 (S3) ◽  
pp. 1256-1257 ◽  
Author(s):  
Joshua D. Strauss ◽  
Jason E. Hammonds ◽  
Paul W. Spearman ◽  
Elizabeth R. Wright
2016 ◽  
Vol 22 (S3) ◽  
pp. 80-81
Author(s):  
Bäuerlein FJB ◽  
Saha ◽  
A Mishra ◽  
I Dudanova ◽  
M Hipp ◽  
...  

2007 ◽  
Vol 13 (S03) ◽  
Author(s):  
F Frischknecht ◽  
M Kudryashev ◽  
S Lepper ◽  
S Münter ◽  
S Hegge ◽  
...  

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 114
Author(s):  
R. Elliot Murphy ◽  
Alexandra B. Samal ◽  
Gunnar Eastep ◽  
Ruba H. Ghanam ◽  
Peter E. Prevelige ◽  
...  

During the late phase of the HIV-1 replication cycle, the Gag polyproteins are transported to the plasma membrane (PM) for assembly. Gag targeting and assembly on the PM is dependent on interactions between its matrix (MA) domain and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Subsequent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles. Evidence suggests that the incorporation of the Env protein is mediated by interactions between the MA domain of Gag and the cytoplasmic tail of the gp41 subunit of Env (gp41CT), a mechanism that remains to be elucidated. Trimerization of the MA domain of Gag appears to be an obligatory step for this interaction. The interplay between gp41CT, the MA trimer, and the membrane has yet to be determined. Our lab has pioneered methods and approaches to investigate, at the molecular level, how the retroviral MA domains of Gag interact with membranes, a key requirement for understanding the Gag assembly and Env incorporation. Herein, we devised innovative approaches that will enable the structural characterization of the gp41CT–MA–membrane interactions. We employed structural biology (NMR and cryo-electron microscopy, biophysical methods, and biochemical tools to generate a macromolecular picture of how the MA domain of Gag binds to the membrane and how it interacts with gp41CT. To this end, we: (i) determined the three-dimensional structure of HIV-1 gp41CT and characterized its interaction with the membrane, (ii) engineered trimeric constructs of gp41CT and the MA to recapitulate the native and functional states of the proteins, and (iii) utilized membrane nanodisc technology to anchor the MA and gp41CT proteins. Our studies will allow for a detailed structural characterization of the gp41CT–MA–membrane interactions, which will advance our knowledge of HIV-1 Gag assembly and Env incorporation.


2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Maolin Lu ◽  
Xiaochu Ma ◽  
Nick Reichard ◽  
Daniel S. Terry ◽  
James Arthos ◽  
...  

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer of gp120-gp41 heterodimers mediates virus entry into CD4-positive (CD4+) cells. Single-molecule fluorescence resonance energy transfer (smFRET) has revealed that native Env on the surface of viruses predominantly exists in a pretriggered conformation (state 1) that is preferentially recognized by many broadly neutralizing antibodies (bNAbs). Env is activated by binding receptor CD4, which drives transitions through a default intermediate conformation (state 2) into the three-CD4-bound open conformation (state 3). The application of smFRET to assess the conformational state of existing Env constructs and ligand complexes recently revealed that all current high-resolution structures correspond to downstream states 2 and 3. The structure of state 1, therefore, remains unknown. We sought to identify conditions whereby HIV-1 Env could be stabilized in the pretriggered state 1 for possible structural characterization. Shedding of gp120, known to severely complicate structural studies, can be prevented by using the uncleaved gp160JR-FL precursor with alterations in the protease cleavage site (R508S/R511S) or by introducing a disulfide bridge between gp120 and gp41 designated “SOS” (A501C/T605C). smFRET demonstrated that both shedding-preventing modifications shifted the conformational landscape of Env downstream toward states 2 and 3. However, both membrane-bound Env proteins on the surface of intact viruses remained conformationally dynamic, responsive to state-stabilizing ligands, and able to be stabilized in state 1 by specific ligands such as the Bristol-Myers Squibb (BMS) entry inhibitors. The here-described identification of state 1-stabilizing conditions may enable structural characterization of the state 1 conformation of HIV-1 Env. IMPORTANCE The HIV-1 envelope glycoprotein (Env) opens in response to receptor CD4 binding from a pretriggered (state 1) conformation through a necessary intermediate to the three-CD4-bound conformation. The application of smFRET to test the conformational state of existing Env constructs and ligand complexes used for high-resolution structures recently revealed that they correspond to the downstream conformations. The structure of the pretriggered Env conformation, preferentially recognized by broadly neutralizing antibodies, remains unknown. Here, we identify experimental conditions that stabilize membrane-bound and shedding-resistant virus Env trimers in state 1, potentially facilitating structural characterization of this unknown conformational state.


2010 ◽  
Vol 6 (11) ◽  
pp. e1001173 ◽  
Author(s):  
Lars-Anders Carlson ◽  
Alex de Marco ◽  
Heike Oberwinkler ◽  
Anja Habermann ◽  
John A. G. Briggs ◽  
...  

Retrovirology ◽  
2014 ◽  
Vol 11 (1) ◽  
pp. 42 ◽  
Author(s):  
Philipp Arnold ◽  
Patricia Himmels ◽  
Svenja Weiß ◽  
Tim-Michael Decker ◽  
Jürgen Markl ◽  
...  

2003 ◽  
Vol 278 (44) ◽  
pp. 43188-43201 ◽  
Author(s):  
Karsten Bruns ◽  
Torgils Fossen ◽  
Victor Wray ◽  
Peter Henklein ◽  
Uwe Tessmer ◽  
...  

2015 ◽  
Vol 89 (19) ◽  
pp. 9739-9747 ◽  
Author(s):  
Simone Mattei ◽  
Annica Flemming ◽  
Maria Anders-Össwein ◽  
Hans-Georg Kräusslich ◽  
John A. G. Briggs ◽  
...  

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) is released from infected cells in an immature, noninfectious form in which the structural polyprotein Gag is arranged in a hexameric lattice, forming an incomplete spherical shell. Maturation to the infectious form is mediated by the viral protease, which cleaves Gag at five sites, releasing the CA (capsid) protein, which forms a conical capsid encasing the condensed RNA genome. The pathway of this structural rearrangement is currently not understood, and it is unclear how cone assembly is initiated. RNA represents an integral structural component of retroviruses, and the viral nucleoprotein core has previously been proposed to nucleate mature capsid assembly. We addressed this hypothesis by replacing the RNA-binding NC (nucleocapsid) domain of HIV-1 Gag and the adjacent spacer peptide 2 (SP2) by a leucine zipper (LZ) protein-protein interaction domain [Gag(LZ)] in the viral context. We found that Gag(LZ)-carrying virus [HIV(LZ)] was efficiently released and viral polyproteins were proteolytically processed, though with reduced efficiency. Cryo-electron tomography revealed that the particles lacked a condensed nucleoprotein and contained an increased proportion of aberrant core morphologies caused either by the absence of RNA or by altered Gag processing. Nevertheless, a significant proportion of HIV(LZ) particles contained mature capsids with the wild-type morphology. These results clearly demonstrate that the nucleoprotein complex is dispensable as a nucleator for mature HIV-1 capsid assembly in the viral context.IMPORTANCEFormation of a closed conical capsid encasing the viral RNA genome is essential for HIV-1 infectivity. It is currently unclear what viral components initiate and regulate the formation of the capsid during virus morphogenesis, but it has been proposed that the ribonucleoprotein complex plays a role. To test this, we prepared virus-like particles lacking the viral nucleocapsid protein and RNA and analyzed their three-dimensional structure by cryo-electron tomography. While most virions displayed an abnormal morphology under these conditions, some particles showed a normal mature morphology with closed conical capsids. These data demonstrate that the presence of RNA and the nucleocapsid protein is not required for the formation of a mature, cone-shaped HIV-1 capsid.


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