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Titolo:
PERTURBATION OF THE CARBOXY-TERMINUS OF HIV-1 REV AFFECTS MULTIMERIZATION ON THE REV RESPONSIVE ELEMENT
Autore:
DALY TJ; RENNERT P; LYNCH P; BARRY JK; DUNDAS M; RUSCHE JR; DOTEN RC; AUER M; FARRINGTON GK;
Indirizzi:
REPLIGEN CORP,1 KENDALL SQ,BLDG 700 CAMBRIDGE MA 02139 SANDOZ GMBH A-1235 VIENNA AUSTRIA
Titolo Testata:
Biochemistry
fascicolo: 34, volume: 32, anno: 1993,
pagine: 8945 - 8954
SICI:
0006-2960(1993)32:34<8945:POTCOH>2.0.ZU;2-N
Fonte:
ISI
Lingua:
ENG
Soggetto:
IMMUNODEFICIENCY-VIRUS TYPE-1; GENE-EXPRESSION REQUIRES; RNA-BINDING; ACTIVATION DOMAIN; TRANS-ACTIVATOR; PROTEIN; RECOGNITION; SEQUENCE; REGION; PHOSPHORYLATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
27
Recensione:
Indirizzi per estratti:
Citazione:
T.J. Daly et al., "PERTURBATION OF THE CARBOXY-TERMINUS OF HIV-1 REV AFFECTS MULTIMERIZATION ON THE REV RESPONSIVE ELEMENT", Biochemistry, 32(34), 1993, pp. 8945-8954

Abstract

Perturbations within the transactivation and carboxy-terminal domainsof HIV-1 Rev were examined for effects on Rev responsive element (RRE) binding activities in vitro and biological activity in vivo. Bindingaffinities, specificities, and multimerization of the transactivationmutants M10 and Rev/Rex M10-16 on the RRE were equivalent to wild-type Rev. Substitution of the Rex transactivation domain within Rev resulted in the incorporation of an internal methionine residue which, whencleaved with CNBr and subsequently purified, produced a protein species (CNBr-Rev) unable to fully multimerize on the RRE. Instead, two discrete protein-dependent species were generated in the gel shift assay. Furthermore, CNBr-Rev was observed to bind to the RRE with high specificity and an equilibrium binding constant of 6 x 10(-10) M. A C-terminal Rev deletion mutant (Rev M9DELTA14) lacking amino acids 68-112 displayed identical RRE binding characteristics to the CNBr-Rev protein. This protein, which lacks both the activation and the C-terminal domains, was biologically inactive but maintained the ability to discriminate the RRE from nonspecific RNA. Deletion of amino acids 92-112 resulted in a Rev mutant (Rev M11DELTA14) which bound to the RRE with wild-type affinity and high specificity. This purified mutant was observed to be aberrant in multimerization activity on the RRE with reduced multimerization apparent in the gel shift assay. However, Rev M11DELTA14 possessed biological activity equivalent to wild-type Rev in a cell-based p24 ELISA assay. These results suggest that polymerization on the RRE is dispensable for Rev activity and that two monomeric Rev proteinsbound to the RRE are sufficient for biological activity. Furthermore,in vivo experiments using the Rev/Rex chimeric mutant and the M10 transdominant mutant as well as in vitro dissociation rate studies with Rev M11DELTA14 and Rev M9DELTA14 suggest that the M9 through M11 domainof the protein may be involved in RRE-dependent specific Rev dimerization.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 28/09/20 alle ore 21:53:24