RESUMO
The mechanism by which lentivirus envelope (Env) glycoproteins are packaged into budding virions is poorly understood. Simian immunodeficiency virus (SIV) contains an Env protein with an unusually long cytoplasmic tail. To investigate the role of this domain in the incorporation of the SIV Env into virions, we generated a series of SIV Env mutants carrying small in-frame deletions within the cytoplasmic domain. The effects of these mutations on Env synthesis, processing, and association with Gag particles were analyzed by means of the vaccinia virus expression system. All of the mutant Env glycoproteins were synthesized and processed in a manner similar to that of the wild-type Env. However, deletions affecting domains C-terminal to residue 832 in the SIV Env protein significantly impaired Env incorporation into particles. Cell surface biotinylation assays showed that this phenotype could not be attributed to inefficient cell surface expression of the Env mutants. Furthermore, when the Env deletion mutants were tested for their ability to mediate virus entry in single-cycle infectivity assays, those mutations that impaired Env incorporation also caused a severe defect in virus infectivity. Our results suggest that domains in the C-terminal third of the SIV Env protein are required for Env incorporation into particles and Env-mediated virus entry.
Assuntos
Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas dos Retroviridae/química , Proteínas dos Retroviridae/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/patogenicidade , Vírion/metabolismo , Animais , Células Cultivadas , Chlorocebus aethiops , Citoplasma/química , Glicoproteínas de Membrana/genética , Estrutura Terciária de Proteína , Ratos , Proteínas dos Retroviridae/genética , Deleção de Sequência , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/metabolismo , Vaccinia virus/genéticaRESUMO
To study the process of feline immunodeficiency virus (FIV) assembly, we examined the suitability of the vaccinia vector system to reproduce FIV particle formation. To this end, we constructed a recombinant vaccinia virus carrying the FIV gag gene. Biochemical and electron microscopy analyses of cells infected with this recombinant virus showed that the FIV Gag polyprotein self-assembled into lentivirus-like particles that were released into the culture medium. As a first step in the identification of molecular determinants in FIV Gag that are involved in virus assembly, we performed a site-directed mutagenesis analysis of the N-terminal matrix (MA) domain of the FIV Gag precursor. To this end, a series of amino acid substitutions and small in-frame deletions were introduced into the FIV MA and the mutated FIV gag gene constructs were expressed by means of the vaccinia system. Characterization of the assembly phenotype of these FIV Gag mutants led to the identification of amino acidic regions within the MA domain that are necessary for efficient transport of the Gag precursor to the plasma membrane and particle assembly. Our results reveal the role that the FIV MA plays in virus morphogenesis and contribute to the understanding of the assembly process in non-primate lentiviruses.
Assuntos
Produtos do Gene gag/metabolismo , Vírus da Imunodeficiência Felina/metabolismo , Vírus da Imunodeficiência Felina/ultraestrutura , Mutação/genética , Proteínas da Matriz Viral/metabolismo , Montagem de Vírus , Sequência de Aminoácidos , Animais , Linhagem Celular , DNA Recombinante/genética , Fibroblastos , Produtos do Gene gag/química , Produtos do Gene gag/genética , Genes gag/genética , Vetores Genéticos/genética , Vírus da Imunodeficiência Felina/genética , Microscopia Eletrônica , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Timidina Quinase/genética , Transfecção , Vaccinia virus/genética , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genéticaRESUMO
Simian immunodeficiency viruses (SIVs) have an envelope (Env) glycoprotein with an unusually long cytoplasmic domain of 164 amino acids. In this article, we have characterized a series of SIV Env truncation mutants in which the cytoplasmic domain was progressively shortened from its carboxyl terminus by 20 amino acids. Expression by means of the vaccinia virus system showed that all of the SIV Env mutants were expressed and processed into the surface and transmembrane (TM) subunits. When the ability of the Env mutants to associate with SIV Gag particles was examined, we found that deletion of 20 to 80 residues from the carboxyl terminus of the SIV TM cytoplasmic tail abrogated the incorporation of the Env glycoprotein into particles. By contrast, further truncation of the SIV TM protein by 100 to 140 amino acids restored the ability of the Env protein to associate with Gag particles. Interestingly, mutants bearing a 44- or 24-amino acid cytoplasmic domain were incorporated at levels significantly higher than those of the wild-type Env. Single-cycle infectivity assays showed that Env mutants bearing cytoplasmic tails of 144 to 64 amino acids were highly inefficient at mediating virus entry. By contrast, truncation of the cytoplasmic domain to 44 or 24 amino acids drastically enhanced virus infectivity with respect to that conferred by the full-length Env protein. Our results demonstrate that small variations in the length of the SIV Env cytoplasmic domain dramatically influence Env-mediated viral functions.