Probing the RNA Binding Surface of the HIV-1 Nucleocapsid Protein by Site-Directed Mutagenesis

Biochemistry ◽  
2013 ◽  
Vol 52 (19) ◽  
pp. 3358-3368 ◽  
Author(s):  
Wei Ouyang ◽  
Stephen Okaine ◽  
Mark P. McPike ◽  
Yong Lin ◽  
Philip N. Borer
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binding Surface ◽  
Hiv 1

scholarly journals Defining HIV-1 Vif residues that interact with CBFβ by site-directed mutagenesis

Virology ◽  
2014 ◽  
Vol 449 ◽  
pp. 82-87 ◽  
Author(s):  
Yusuke Matsui ◽  
Keisuke Shindo ◽  
Kayoko Nagata ◽  
Katsuhiro Io ◽  
Kohei Tada ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Hiv 1

Site-directed mutagenesis of two trans-regulatory genes (tat-III,trs) of HIV-1

Science ◽  
1988 ◽  
Vol 239 (4842) ◽  
pp. 910-913 ◽  
Author(s):  
M. Sadaie ◽  
T Benter ◽  
F Wong-Staal
Keyword(s):  
Regulatory Genes ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Hiv 1

scholarly journals RNA and Nucleocapsid Are Dispensable for Mature HIV-1 Capsid Assembly

2015 ◽  
Vol 89 (19) ◽  
pp. 9739-9747 ◽  
Author(s):  
Simone Mattei ◽  
Annica Flemming ◽  
Maria Anders-Össwein ◽  
Hans-Georg Kräusslich ◽  
John A. G. Briggs ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Electron Tomography ◽  
Significant Proportion ◽  
Dimensional Structure ◽  
Capsid Assembly ◽  
Interaction Domain ◽  
Cryo Electron Tomography ◽  
Rna Genome ◽  
Hiv 1

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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