The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein.
HIV-1 Tat activates transcription through binding to human cyclin T1, a regulatory subunit of the TAK/P-TEFb CTD kinase complex. Here we show that the cyclin domain of hCycT1 is necessary and sufficient to interact with Tat and promote cooperative binding to TAR RNA in vitro, as well as mediate Tat transactivation in vivo. A Tat:TAR recognition motif (TRM) was identified at the carboxy-terminal edge of the cyclin domain, and we show that hCycT1 can interact simultaneously with Tat and CDK9 on TAR RNA in vitro. Alanine-scanning mutagenesis of the hCycT1 TRM identified residues that are critical for the interaction with Tat and others that are required specifically for binding of the complex to TAR RNA. Interestingly, we find that the interaction between Tat and hCycT1 requires zinc as well as essential cysteine residues in both proteins. Cloning and characterization of the murine CycT1 protein revealed that it lacks a critical cysteine residue (C261) and forms a weak, zinc-independent complex with HIV-1 Tat that greatly reduces binding to TAR RNA. A point mutation in mCycT1 (Y261C) restores high-affinity, zinc-dependent binding to Tat and TAR in vitro, and rescues Tat transactivation in vivo. Although overexpression of hCycT1 in NIH3T3 cells strongly enhances transcription from an integrated proviral promoter, we find that this fails to overcome all blocks to productive HIV-1 infection in murine cells.
Garber
M E
ME
Regulatory Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037-1099, USA.
Wei
P
P
KewalRamani
V N
VN
Mayall
T P
TP
Herrmann
C H
CH
Rice
A P
AP
Littman
D R
DR
Jones
K A
KA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D056906:Cyclin T; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics; D015696:Gene Products, tat / Q000378:metabolism*; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation / Q000235:genetics; D014764:Viral Proteins / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus
C503684:CCNT1 protein, human; C503685:Ccnt1 protein, mouse; D056906:Cyclin T; D016213:Cyclins; D015696:Gene Products, tat; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014764:Viral Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D018844:Cyclin-Dependent Kinases; D015032:Zinc; D003545:Cysteine