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Journal of Virology, February 2001, p. 1487-1506, Vol. 75, No. 3
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.3.1487-1506.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Origin-Independent Assembly of Kaposi's
Sarcoma-Associated Herpesvirus DNA Replication Compartments in
Transient Cotransfection Assays and Association with the ORF-K8 Protein
and Cellular PML
Frederick Y.
Wu,1,2
Jin-Hyun
Ahn,2
Donald J.
Alcendor,1
Won-Jong
Jang,1
Jinsong
Xiao,1
S. Diane
Hayward,1,2 and
Gary S.
Hayward1,2,*
Molecular Virology Laboratories, Department
of Pharmacology and Molecular Sciences,1 and
Viral Oncology Program, Department of
Oncology,2 Johns Hopkins University School of
Medicine, Baltimore, Maryland 21231-1000
Received 22 September 2000/Accepted 7 November 2000
Six predicted Kaposi's sarcoma virus herpesvirus (KSHV) proteins
have homology with other well-characterized herpesvirus core DNA
replication proteins and are expected to be essential for viral DNA
synthesis. Intact Flag-tagged protein products from all six were
produced from genomic expression vectors, although the ORF40/41
transcript encoding a primase-helicase component proved to be spliced
with a 127-bp intron. The intracellular localization of these six KSHV
replication proteins and the mechanism of their nuclear translocation
were investigated. SSB (single-stranded DNA binding protein, ORF6) and
PPF (polymerase processivity factor, ORF59) were found to be intrinsic
nuclear proteins, whereas POL (polymerase, ORF9), which localized in
the cytoplasm on its own, was translocated to the nucleus when
cotransfected with PPF. PAF (primase-associated factor, ORF40/41), a
component of the primase-helicase tripartite subcomplex together with
PRI (primase, ORF56) and HEL (helicase, ORF44), required the presence
of all five other replication proteins for efficient nuclear
translocation. Surprisingly, even in the absence of a lytic cycle
replication origin (ori-Lyt) and any known initiator or origin binding
protein, the protein products of all six KSHV core replication genes
cooperated in a transient cotransfection assay to form large globular
shaped pseudo-replication compartments (pseudo-RC), which excluded
cellular DNA. These pseudo-RC structures were confirmed to include POL,
SSB, PRI, and PAF but did not contain any newly synthesized DNA.
Similar to the human cytomegalovirus system, the peripheries of these
KSHV pre-RC were also found to be surrounded by punctate PML oncogenic
domains (PODs). Furthermore, by transient cotransfection, the six KSHV core replication machinery proteins successfully replicated a plasmid
containing EBV ori-Lyt in the presence of the Epstein-Barr virus-encoded DNA binding initiator protein, ZTA. The KSHV-encoded K8
(ORF-K8) protein, which is a distant evolutionary homologue to ZTA, was
incorporated into pseudo-RC structures formed by transient cotransfection with the six core KSHV replication genes. However, unlike ZTA, K8 displayed a punctate nuclear pattern both in transfected cells and at early stages of lytic infection and colocalized with the
cellular PML proteins in PODs. Finally, K8 was also found to accumulate
in functional viral RC, detected by incorporation of pulse-labeled
bromodeoxyuridine into newly synthesized DNA in both tetradecanoyl
phorbol acetate-induced JSC-1 primary effusion lymphoblasts and in KSHV
lytically infected endothelial cells.
*
Corresponding author. Mailing address: CRB-3M08, 1650 Orleans St., Baltimore, MD 21231-1000. Phone: (410) 955-8684. Fax:
(410) 955-8685. E-mail: ghayward{at}jhmi.edu.
Journal of Virology, February 2001, p. 1487-1506, Vol. 75, No. 3
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.3.1487-1506.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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