Dynamic and Nucleolin-Dependent Localization of Human Cytomegalovirus UL84 to the Periphery of Viral Replication Compartments and Nucleoli

Abstract

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 FLAGtagged 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. IMPORTANCEThe 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.

Viral and cellular subnuclear structures in human cytomegalovirus-infected cells

“…Several viral proteins can localize to nucleoli. It has been reported that HCMV pp65 and UL84 proteins are a nucleolar antigens (Arcangeletti et al, 2009;Bender et al, 2014). Also, transfection experiments have indicated that several epitopetagged versions of HCMV proteins (TLR5, TLR7, US33, UL29, UL31 and UL76) localize to the nucleolus in uninfected cells (Salsman et al, 2008).…”

“…The recruitment of nucleolin to replication compartments may be related to the interaction of nucleolin with viral proteins found in replication compartments. It has been observed that the HCMV protein UL84 co-localizes with nucleolin in nucleoli and at the periphery of replication compartments (Bender et al, 2014). Therefore, it is possible that UL84 recruits nucleolin to replication compartments from nucleoli, which results in changes to nucleolar composition and architecture.…”

“…The absence of nucleolin in the infected cell causes mislocalization of UL44 at the periphery of replication compartments and compromises efficient viral DNA synthesis (Strang et al, 2012a). Nucleolin is also required for UL84 localization with UL44 at the periphery of replication compartments, although in the absence of nucleolin, UL84 does not redistribute throughout the nucleus but relocates to the cytoplasm (Bender et al, 2014). Thus, nucleolin is required for both proper nuclear and subnuclear localization of HCMV replication compartment proteins.…”

“…Within these structures, unphosphorylated and phosphorylated forms of RNA polymerase II localize with the viral protein UL69 (Feichtinger et al, 2011), which is known to be required for viral RNA processing and export to the cytoplasm. The localization of RNA polymerase II in relation to UL44 has yet to be described; however, viral proteins involved in transcription and RNA metabolism, for example IE1, IE2 and UL84 (Bender et al, 2014;Silva et al, 2010), are found throughout the nucleus and co-localize with UL44 in viral replication compartments. Therefore, it is likely that viral transcription occurs throughout the interior of replication compartments and, unlike viral DNA synthesis, is not restricted to specific areas of replication compartments such as the layer of UL44 that accumulates at the periphery of replication compartments.…”

Viral noncoding RNAs: more surprises

“…This observation may be related to the interaction of EBER2 with nucleolin (V Fok and J Steitz, unpubl. ), as during the replication of human cytomegalovirus (HCMV), a β-herpesvirus, the viral homolog of the host DNA polymerase processivity factor (UL44) associates with nucleolin (Bender et al 2014). Because EBV lytic replication has been implicated in promoting tumor formation (Katsumura et al 2011), uncovering a connection to DNA replication may relate to the tumorigenic potential of EBER2 (Komano et al 1999).…”