RESUMO
The Gag polyprotein of feline immunodeficiency virus (FIV) assembles at the plasma membrane of the infected cells. We aimed to identify the FIV Gag domains that interact and promote Gag multimerization. To do this we generated a series of Gag subdomains and tested their ability to associate with full-length Gag and be recruited into extracellular virus-like particles (VLPs). Removal of 37 residues from the C-terminus of FIV Gag and deletion of the N-terminal and central regions of the nucleocapsid (NC) domain attenuated but did not abrogate association with wild-type Gag, whereas a Gag mutant protein encompassing the matrix (MA) and capsid (CA) domains interacted poorly with full-length Gag. Association with wild-type Gag was abolished by deleting most of the NC together with the N-terminal 40 residues of the MA, which most likely reflects the inability of this Gag mutant to bind RNA. Notably, the CA-NC Gag subdomain both associated with wild-type Gag and was recruited into particles in a proportion close to 50â% of the total Gag-related protein mass of VLPs. Moreover, both a Gag protein lacking the C-terminal p2 peptide and a nonmyristoylated version of the polyprotein exhibited a transdominant-negative effect on the assembly of wild-type Gag. Analysis of Gag mutants carrying internal deletions within the CA revealed that the N-terminal and the C-terminal domains of the CA are necessary for Gag assembly. Our results demonstrate that the FIV CA-NC region constitutes the principal self-interaction domain of Gag and that the RNA-binding capacity of Gag is necessary for its multimerization.
Assuntos
Produtos do Gene gag/genética , Vírus da Imunodeficiência Felina/genética , Multimerização Proteica/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Células COS , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Linhagem Celular , Membrana Celular/virologia , Chlorocebus aethiops , Produtos do Gene gag/biossíntese , Produtos do Gene gag/metabolismo , Vírus da Imunodeficiência Felina/patogenicidade , Dados de Sequência Molecular , Nucleocapsídeo/genética , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Proteínas de Ligação a RNA/genética , Ratos , Alinhamento de Sequência , Vaccinia virus/genética , Vaccinia virus/patogenicidade , Proteínas da Matriz Viral/genética , Montagem de Vírus/genéticaRESUMO
The matrix domain (MA) of the simian immunodeficiency virus (SIV) is encoded by the amino-terminal region of the Gag polyprotein precursor and is the component of the viral capsid that lines the inner surface of the virus envelope. To define domains of the SIV MA protein that are involved in virus morphogenesis, deletion and substitution mutations were introduced in this protein in the context of a gag-protease construct and expressed in the vaccinia virus vector system. The MA mutants were characterized with respect to synthesis and processing of the Gag precursor, assembly and release of virus-like particles, and incorporation of the envelope (Env) glycoprotein into particles. We have identified two regions of the SIV MA which are critical for particle formation. Both domains are located in a central hydrophobic alpha-helix of the SIV MA, according to data on the structure of this protein. In addition, we have characterized a domain whose mutation impairs the incorporation of SIV Env glycoproteins with long transmembrane cytoplasmic tails into particles. Interestingly, these mutant particles retained the ability to associate with SIV Env proteins with short cytoplasmic tails.
Assuntos
Produtos do Gene env/biossíntese , Produtos do Gene gag/biossíntese , Vírus da Imunodeficiência Símia/metabolismo , Proteínas da Matriz Viral/metabolismo , Sequência de Aminoácidos , Animais , Capsídeo/biossíntese , Linhagem Celular , Chlorocebus aethiops , Produtos do Gene gag/genética , Genes gag , HIV-1/metabolismo , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas da Matriz Viral/químicaRESUMO
The matrix protein (MA) of human and simian immunodeficiency viruses (HIV and SIV) is encoded by the amino-terminal region of the Gag precursor and has been suggested to be involved in different processes during the early and late stages of the virus life cycle. The MA protein of SIV contains three cysteine residues at positions 57, 83, and 87, which are also highly conserved among HIV-2 isolates. In order to study the functional significance of these residues in virus morphogenesis, a series of mutations affecting the cysteines of SIV MA were introduced into a gag-protease construct and expressed in the vaccinia vector system. The MA mutants were assayed for their ability to synthesize and process the Gag polyprotein precursor as well as to release particles into the culture medium. In addition, the incorporation of the envelope glycoprotein (Env) into the Gag-made particles was investigated. Substitution of alanine for cysteine 87 had little effect on particle release and Env glycoprotein association. By contrast, the individual replacement of cysteines 57 or 83 by alanine, as well as the simultaneous mutation of cysteines 83 and 87, significantly reduced the ability of Gag polypeptides to produce extracellular particles. Assembly into particles appeared to be also affected, albeit to a lesser extent, when both cysteines 57 and 83 were replaced by alanine. Furthermore, substitution of cysteine 83 in the SIV MA domain was found to be detrimental to Gag polyprotein processing. Analysis of the Env glycoprotein association with recombinant particles revealed that this process was moderately affected in the case of the double mutants lacking cysteines 57 and 83, or cysteines 57 and 87, and the cysteine-minus triple mutant. Our results suggest that the conserved cysteines 57 and 83 in the MA domain are important for efficient SIV Gag particle production.