Protein-
protein and
protein-
nucleic acid interactions within subcellular compartments are required for viral genome replication. To understand the localization of the human cytomegalovirus viral replication factor UL84 relative to other
proteins involved in
viral DNA synthesis and to replicating
viral DNA in infected cells, we created a recombinant virus expressing a FLAG-tagged version of UL84 (UL84FLAG) and used this virus in immunofluorescence assays. UL84FLAG localization differed at early and late times of
infection, transitioning from diffuse distribution throughout the nucleus to exclusion from the interior of replication compartments, with some concentration at the periphery of replication compartments with newly labeled
DNA and the
viral DNA polymerase subunit UL44. Early in
infection, UL84FLAG colocalized with the viral
single-stranded DNA binding protein UL57, but colocalization became less prominent as
infection progressed. A portion of UL84FLAG also colocalized with the host
nucleolar protein nucleolin at the peripheries of both replication compartments and nucleoli.
Small interfering RNA (
siRNA)-mediated knockdown of
nucleolin resulted in a dramatic elimination of UL84FLAG from replication compartments and other parts of the nucleus and its accumulation in the cytoplasm. Reciprocal coimmunoprecipitation of
viral proteins from infected cell lysates revealed association of UL84, UL44, and
nucleolin. These results indicate that UL84 localization during
infection is dynamic, which is likely relevant to its functions, and suggest that its nuclear and subnuclear localization is highly dependent on direct or indirect interactions with
nucleolin. Importance: The
protein-
protein interactions among viral and cellular
proteins required for replication of the human cytomegalovirus (HCMV)
DNA genome are poorly understood. We sought to understand how an enigmatic HCMV
protein critical for virus replication, UL84, localizes relative to other viral and cellular
proteins required for HCMV genome replication and replicating
viral DNA. We found that UL84 localizes with
viral proteins,
viral DNA, and the cellular
nucleolar protein nucleolin in the subnuclear replication compartments in which
viral DNA replication occurs. Unexpectedly, we also found localization of UL84 with
nucleolin in nucleoli and showed that the presence of
nucleolin is involved in localization of UL84 to the nucleus. These results add to previous work showing the importance of
nucleolin in replication compartment architecture and
viral DNA synthesis and are relevant to understanding UL84 function.