Antiviral chemokines: Intracellular life of recombinant C-C chemokine RANTES

Objective: Select C-C and C-X-C chemokines can suppress HN infection. This is because their receptors are the gateways for HIV-1 entry, determinants o f viral tropism and sensitivity. C-C chemokines are most effective against macrophage-tropic viruses, and C-X-C chemokines are most effective against T-tropic viruses. The epitopes on the chemokine molecule responsible for vi rus inhibition and for chemokines' specificities are not known. The objecti ve of this study was to map the functional domains of prototypic antiviral chemokine, namely, RANTES (regulated-on-activation normal T-expressed and s ecreted). Study Design: Optimal folding of the chemokine molecule is thought to be im portant for its biologic activity. Anticipating that it will provide a nati ve milieu for folding, we expressed recombinant RANTES molecules in an HIV- 2-derived lentivirus mammalian expression system. We focused on the structu ral landmarks of RANTES to determine their role in its life and function. Results: We found that the flexible amino-terminal region of RANTES was not important for its structural integrity or antiviral activity, either posit ively or negatively. It was also not important for binding to the CCR5 rece ptor. Modification of all other domains was detrimental, implying a functio nal role. However, a more careful analysis revealed that these domains were crucial for controlling stability, transport, and secretion of the molecul e. Although all recombinant clones contained signal sequence and were trans criptionally active, they presented three different phenotypes: normal synt hesis and secretion, normal synthesis but blocked secretion, and presumed n ormal synthesis but rapid degradation. Structural considerations and prelim inary experiments showing a lack of effect of proteasome inhibitors suggest ed that the signal recognition particle pathway of translocation and protea somal pathway of destruction may not be the major determinant of the life o f the chemokine. Conclusions: The amino-terminal domain of RANTES was not essential for its antiviral activity of for its binding to the CCR5 receptor. Although the I- domain of the core and carboxyterminal domain may contribute to the antivir al activity of RANTES, they were more important for its intracellular life.