Estudio del rol del dominio cápside de la poliproteína Gag en el ensamblado del virus de la inmunodeficiencia de felinos
Lentiviruses assemble at the plasma membrane of infected cells as the result of Gag\npolyprotein multimerization into spherical particles that bud into the extracellular\nmedium. We have previously shown for both feline immunodeficiency virus (FIV)\nand simian immunodeficiency virus (SIV) that the c...
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| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
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Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
2022
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_6744 https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_6744.dir/6744.PDF |
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| Sumario: | Lentiviruses assemble at the plasma membrane of infected cells as the result of Gag\npolyprotein multimerization into spherical particles that bud into the extracellular\nmedium. We have previously shown for both feline immunodeficiency virus (FIV)\nand simian immunodeficiency virus (SIV) that the capsid domain (CA) of the Gag\nprecursor plays two roles during viral replication. As a domain of Gag, it participates\nin the Gag-Gag interactions that drive viral particle assembly and, as a mature\nprotein of infectious virions, it forms the viral core that preserves the integrity of the\ncomplex between the genomic RNA and the nucleocapsid protein (NC). To establish\nthe structural and functional relationship between the CA domains of FIV and SIV\nwe constructed chimeric FIV gag genes in which the CA domain was partially or\ncompletely replaced by that of SIV. We found that FIV Gag proteins carrying the\ncomplete CA together with SP1 and the first eight amino acids of the NC, the aminoterminal\ndomain of the CA (CA-NTD) or the carboxyl region of this protein (CA-CTD)\nare unable to assemble into viral particles. However, all these assembly-deficient\nchimeric Gag proteins are rescued into extracellular particles when co-expressed\nwith the wild-type SIV Gag protein. Importantly, our laboratory has previously\ndemonstrated that SIV Gag proteins carrying the CA-p1-NC(amino acids 1-9) region\nof FIV or the CA-CTD of this feline virus are assembly competent. Taken together,\nour studies demonstrate that although the FIV and SIV CA proteins exhibit a similar\norganization (amino and carboxyl domains with helical structure linked by a flexible\nregion) and 52% similarity at the amino acid level, the functional exchange of CA\ndomains between these two lentiviruses is dependent on the recipient Gag protein.\nOur phenotypic characterization of chimeric Gag proteins derived from FIV and SIV\nprovides relevant information regarding the structural requirements for lentiviral\nassembly.\nImmature lentiviral virions are composed of a lattice of Gag hexamers, which is\nmaintained by inter- and intrahexameric interactions of the CA-NTD and by\ninterhexameric associations of the CA-CTD. Notably, the viral core consists of CA\nprotein hexamers in which the CA-CTD also establishes interhexameric interactions\ninvolving ?-helix 9. In HIV-1, it has been proposed that the W184/M185 motif of the\nCA-CTD helix 9 is important for viral morphogenesis. The FIV CA exhibits at\nequivalent positions the amino acids YL whereas, in another lentivirus, equine\ninfectious anemia virus (EIAV), the motif is FL. We therefore decided to introduce a\nseries of amino acid substitutions in the Y176/L177 motif of the FIV CA to investigate\ntheir effect on Gag assembly, CA oligomerization (which is essential for viral core\nformation), production of mature virions and viral infection. The results obtained\ndemonstrate that the sole substitution of alanine for Y176 or L177 in the FIV CA is\nsufficient to suppress both Gag multimerization into extracellular viral particles and\nin vitro Gag assembly. Furthermore, the recombinant CA protein carrying the Y176A\nmutation is unable to oligomerize in vitro. Furthermore, while the Y176W mutation\ninhibits particle formation by 80%, the amino acid substitutions Y176F, L177M and\nY176W/L177M reduce Gag assembly by 50% with respect to wild-type Gag. The\nY176F and Y176W/L177M mutations were designed to convert the original YL motif\nof the FIV CA into the FL or WM motifs, which are found at equivalent positions in\nthe CA proteins of EIAV and HIV-1, respectively. Notably, the presence in the FIV\nCA of the WM or FL motifs allows the production of mature virions that replicate in\nfeline cells. Our results demonstrate that in FIV the YL motif of the CA ?-helix 9 is\nessential for both Gag multimerization into virions and oligomerization of the mature\nCA protein. Moreover, we demonstrate the existence of functionally equivalent\nmotifs in the CA-CTD of FIV, EIAV and HIV-1. |
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