Identification of specific molecular structures of human immunodeficiency virus type 1 Tat relevant for its biological effects on vascular endothelial cells.
Human immunodeficiency virus type 1 (HIV-1) Tat transactivates viral genes and is released by infected cells, acting as a soluble mediator. In endothelial cells (EC), it activates a proangiogenic program by activating vascular endothelial growth factor receptor type 2 (VEGFR-2) and integrins. A structure-activity relationship study was performed by functional analysis of Tat substitution and deletion variants to define the Tat determinants necessary for EC activation. Variants were made (i) in the basic and (ii) in the cysteine-rich domains and (iii) in the C-terminal region containing the RGD sequence required for integrin recognition. Our results led to the following conclusions. (i) Besides a high-affinity binding site corresponding to VEGFR-2, EC express low-affinity binding sites. (ii) The basic and the cysteine-rich variants bind only to the low-affinity binding sites and do not promote tyrosine phosphorylation of VEGFR-2. Furthermore, they have a reduced ability to activate EC in vitro, and they lack angiogenic activity. (iii) Mutants with mutations in the C-terminal region are partially defective for in vitro biological activities and in vivo angiogenesis, but they activate VEGFR-2 as Tat wild type. In conclusion, regions encoded by the first exon of tat are necessary and sufficient for activation of VEGFR-2. However, the C-terminal region, most probably through RGD-mediated integrin engagement, is indispensable for full activation of an in vitro and in vivo angiogenic program.
Mitola
S
S
Institute for Cancer Research and Treatment, 10060 Candiolo, Italy.
Soldi
R
R
Zanon
I
I
Barra
L
L
Gutierrez
M I
MI
Berkhout
B
B
Giacca
M
M
Bussolino
F
F
D001483:Base Sequence; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000166:cytology / Q000821:virology*; D015696:Gene Products, tat / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism / Q000502:physiology*; D017978:Receptors, Growth Factor / Q000378:metabolism / Q000502:physiology*; D040262:Receptors, Vascular Endothelial Growth Factor; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D054322:tat Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D015696:Gene Products, tat; D017978:Receptors, Growth Factor; D054322:tat Gene Products, Human Immunodeficiency Virus; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor