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15144359416
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note
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Three HHV-8-encoded chemokines have been deposited in GenBank [accession numbers U74585 (vMIP-1A or vMIP-I), U67775 (vMIP-1B or vMIP-II), and U83351 (BCK or vMIP-lll)].
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10
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15144340195
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note
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A biopsy was taken from a Kaposi's sarcoma skin lesion on an HIV-infected patient and a QIAamp blood kit (Qiagen) was used to extract total DNA. Based on the nucleotide sequence deposited in GenBank, the vMIP-II gene was amplified by PCR. The full-length coding sequence was inserted into the eukaryotic expression vector pTEJ8, which uses the ubiquitin UbC promoter (28). Sequence analysis was performed on an AIf Express sequencing system (Pharmacia Biotech). COS-7 cells were transiently transfected by a calcium phosphate precipitate method with addition of chloroquine. Serum-free medium was collected 24 to 48 and at 48 to 72 hours after transfection. COS cells were transfected in parallel with the empty pTEJ8 vector and serum-free medium was collected as a control.
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11
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15144357922
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note
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125I-SDF-Iα was prepared in-house by oxidative iodination with lodogen followed by HPLC purification to remove unlabeled from labeled compound. The chemokine peptides RANTES and IL-8 were expressed in Escherichia coli and purified in-house (Glaxo Biomdical Research Institute, Plan-lesouates, Switzerland); MCP-1 and MIP-1α were obtained from Pepro Tech (London, UK) and SDF-1α was a generous gift from Michael Luther (Glaxo Wellcome, Research Triangle Park, NC).
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12
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15144343350
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note
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2-terminal sequence analysis or mass spectroscopy.
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13
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15144361567
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note
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Conceivably, the carboxypeptidase B-like enzyme, which is found in the secretory pathway of most eukaryotic cells (29), is responsible for the COOH-terminal trimming of the vMIP-II protein by removal of the Arg residue. Nevertheless, the synthetic form of vMIP-ll, which includes the COOH-terminal Arg residue [see (9)], and the recombinantly produced peptide had similar receptor binding and signal transduction profiles.
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-
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14
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15144343104
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note
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vMIP-II including the COOH-terminal Arg, which normally is removed in the secretory pathway, was synthesized with t-butoxycarbonyl chemistry and automated solid-phase methods as described elsewhere (30). The peptide was reconstituted in 1 M guanidinium hydrochloride and tris acetate at pH 8.5 and folded in 10% dimethyl sulfoxide in guanidine hydrochloride at pH 8.5, purified by reversed-phase HPLC, and analyzed by electrospray mass spectroscopy (30).
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15
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15144341602
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note
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2+]) in CHO and HEK-293 cells stably expressing various chemokine receptors were determined by loading with Fura-2 AM (Fluka), and fluorescence was monitored in a Jobin Yvon FluroMax-2 (results expressed as the ratio of 340 nm to 380 nm) or a JASCO PF777 spectrofluorometer. For studies without calcium in the extracellular medium (Krebs-Ringer buffer at pH 7.4) EDTA was added to a final concentration of 10 mM.
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16
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15144356188
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note
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The possible biological consequences associated with the slow, prolonged calcium response to vMIP-II are unclear. However, a secondary, similarly slow and prolonged increase in intracellular calcium after stimulation with RANTES has been shown to be G-protein independent and possibly connected to activation of tyrosine kinases (12). Thus, agonistic effects of vMIP-II on cell differentiation or proliferation possibly related to the Kaposi's sarcoma lesion should be investigated.
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the HHV-8-encoded chemokine receptor ORF74 is deposited in GenBank (accession number U24275)
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Actual binding of vMIP-II to CCR3 was not determined because of the lack of success in establishing a valid binding assay with radioactively labeled eotaxin or MCP-3. However, the observation that pretreatment with vMIP-11 could inhibit the calcium mobilizing effect of MCP-3 in cells expressing CCR3 indicates that vMIP-II does bind to CCR3 (Fig. 3A).
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note
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3 cells per well. On the following day chemokines were added and incubated for 30 min at 37°C before virus was added and incubated an additional 3 hours before washing three times to remove residual virus. The cells were then incubated for 5 days at 37°C before the medium was harvested and p24 concentrations were estimated (31). Initial time-course studies had determined that optimal production of p24 was obtained after 5 days with the virus strains that were used in the U87 cells before confluency was reached.
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15144339620
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note
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Abbreviations for amino acids are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.
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49
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15144361317
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We thank Lisbet Elbak, Helle Iversen, and Tina Jakobsen for technical help and Ulrik Gether for use of the spectrofluorometer. Supported by the Danish Medical Research Council (MRC), the Biotechnology Research Unit for Molecular Recognition, and the Danish AIDSFoundation (to T.W.S.) and by the UK MRC (to G.S. and P.R.C.).
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