Mutational analysis of functional domains in the HIV-1 Rev trans-regulatory protein.
HIV-1 replication depends on the expression of trans-regulatory genes (tat, rev) encoded in the 3' part of the retroviral genome. HIV-1 Rev trans-activator protein allows the cytoplasmic translocation of incompletely spliced retroviral mRNA which is required for the translational switch from regulatory (Tat, Rev, Nef) to structural proteins (Gag, Pol, Env). The HIV-1 Rev regulatory protein comprises an activation domain (RAD) and a RNA binding domain (RBD). Both functional domains are not well defined and the RBD appears to overlap with the nuclear localization signal (NLS). Our mutational analysis localized the Rev protein domain important for RRE (nucleotide 7781 to 8000) binding in vitro to amino acid residues 31 to 50. Mutations in this domain always resulted in exclusion from the nucleoli. Furthermore, these mutants did not support Rev-dependent p24 Gag production in vivo. Sequences immediately upstream of this domain (RevM4, RevM19) were attenuated in their in vivo activity possibly indicating a role in Rev protein oligomerization. The observed tight correlation between subcellular localization and RNA binding in vitro indicates that this short stretch of amino acids supports two essential functions required for HIV-1 replication.
Berger
J
J
SANDOZ Research Institute, Vienna, Austria.
Aepinus
C
C
Dobrovnik
M
M
Fleckenstein
B
B
Hauber
J
J
Böhnlein
E
E
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D015694:Gene Products, rev / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D012367:RNA, Viral / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014779:Virus Replication / Q000235:genetics; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D012367:RNA, Viral; D015534:Trans-Activators; D054321:rev Gene Products, Human Immunodeficiency Virus