Kaposi's sarcoma-associated herpesvirus encodes a functional cyclin

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus 8) is consistently found in Kaposi's sarcoma lesions and in body-cavity-based lymphomas. A 17-kb KSHV lambda clone was obtained directly from a Kaposi's sarcoma lesion. DNA sequence analysis of this clone identified an open reading frame which has 32% amino acid identity and 53% similarity to the virus-encoded cyclin (v-cyclin) of herpesvirus saimiri (HVS) and 31% identity and 53% similarity to human cellular cyclin D2. This KSHV open reading frame was shown to encode a 29-to 30-kDa protein with the properties of a v-cyclin. KSHV v-cyclin protein was found to associate predominantly with cdk6, a cellular cyclin-dependent kinase known to interact with cellular type D cyclins and HVS v-cyclin. The KSHV v-cyclin was also found to associate weakly with cdk4. KSHV v-cyclin-cdk6 complexes strongly phosphorylated glutathione S-transferase-Rb fusion protein and histone H1 as substrates in vitro. Thus, KSHV v-cyclin resembles the v-cyclin of the T-lymphocyte-transforming HVS in its specificity for association with cdk6 and in its ability to strongly activate cdk6 protein kinase activity.Cell culture and transfection. COS-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Sf9 cells were maintained at 27ЊC in Grace's medium containing 10% fetal calf serum, yeastolate, and lactalbumin hydrolysate. A DEAE-dextran transfection procedure was used for transient expression in COS-1 cells (7).Cloning of KSHV from KS. A single KS biopsy specimen from a male AIDS patient was digested in melting buffer and extracted with phenol-chloroform twice. DNA was partially digested with restriction endonuclease Sau3A and ligated with BamHI-digested arms of the bacteriophage-lambda vector DASH2 (Stratagene Inc., La Jolla, Calif.). The ligation reaction product was in vitro packaged and amplified once. A DNA corresponding to the ORF75 homolog of KSHV (5) was synthesized by PCR with primers H8-75-1 and H8-75-2. With this PCR product as a probe for plaque hybridization, the KSHV-specific phage lambda clone SY3-2 with an insert of about 17 kb was identified.Shotgun cloning and DNA sequencing. The complete DNA sequence of the 17-kb insert of phage lambda clone SY3-2 was determined by a shotgun approach. Briefly, purified insert DNA of SY3-2 was sonicated (maximum output, 70% cycle, 60 s), and ends were filled in with Klenow and T4 DNA polymerases (New England Biolabs). DNA fragments ranging from 1 to 4 kb were prepared from agarose gels and ligated into the SmaI-digested vector BluescriptKSIIminus (Stratagene Inc.). DNA from the shotgun plasmids was sequenced on an ABI377 automated DNA sequencer by using the dye-terminator cycle sequencing chemistry according to the instructions of the manufacturer (Perkin-Elmer Inc., Foster City, Calif.). Sequence assembly and analysis of the contiguous sequence were performed with the suite of sequence analysis tools from the Genetics Computer Group, Madison, Wis.Expression and purification o...

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