Nucleolin Associates with the Human Cytomegalovirus DNA Polymerase Accessory Subunit UL44 and Is Necessary for Efficient Viral Replication

Abstract

In the eukaryotic cell, DNA replication entails the interaction of multiple proteins with the DNA polymerase processivity factor PCNA. As the structure of the presumptive human cytomegalovirus (HCMV) DNA polymerase processivity factor UL44 is highly homologous to that of PCNA, we hypothesized that UL44 also interacts with numerous proteins. To investigate this possibility, recombinant HCMV expressing FLAG-tagged UL44 was generated and used to immunoprecipitate UL44 and associated proteins from infected cell lysates. Unexpectedly, nucleolin, a major protein component of the nucleolus, was identified among these proteins by mass spectrometry and Western blotting. The association of nucleolin and UL44 in infected cell lysate was confirmed by reciprocal coimmunoprecipitation in the presence and absence of nuclease. Western blotting and immunofluorescence assays demonstrated that the level of nucleolin increases during infection and that nucleolin becomes distributed throughout the nucleus. Furthermore, the colocalization of nucleolin and UL44 in infected cell nuclei was observed by immunofluorescence assays. Assays of HCMV-infected cells treated with small interfering RNA (siRNA) targeting nucleolin mRNA indicated that nucleolin was required for efficient virus production, viral DNA synthesis, and the expression of a late viral protein, with a correlation between the efficacy of knockdown and the effect on virus replication. In contrast, the level of neither global protein synthesis nor the replication of an unrelated virus (reovirus) was reduced in siRNA-treated cells. Taken together, our results indicate an association of nucleolin and UL44 in HCMV-infected cells and a role for nucleolin in viral DNA synthesis.DNA polymerase is essential for the replication of DNA. Most replicative DNA polymerases include a catalytic subunit, required for DNA polymerization, and a processivity subunit that holds the catalytic subunit of the polymerase on DNA to permit continuous DNA synthesis and, in some cases, to interact with other proteins required for DNA synthesis as the need arises. For example, proliferating cell nuclear antigen (PCNA), the processivity factor of eukaryotic DNA polymerases ␦ and ε, is capable of numerous interactions with proteins that aid and abet DNA synthesis (33,35). Human cytomegalovirus (HCMV) encodes a dimeric DNA polymerase, which includes the catalytic subunit UL54 and the presumptive processivity factor UL44. Previous studies of UL44 have revealed that UL44 forms a head-to-head homodimer (2) that has structural homology to PCNA (2, 3) and, like PCNA, can wrap around DNA (25). These results give rise to the hypothesis that UL44 can interact with multiple proteins involved in DNA synthesis.Other than UL54 (12), to date, three viral proteins have been reported to associate with UL44 in the infected cell: the viral kinase UL97 (26, 34), the uracil DNA glycosylase UL114 (39,40), and the DNA replication factor UL84 (14, 47). To investigate whether other viral and cellular proteins associate with UL44, ...

The Human Cytomegalovirus Gene Products Essential for Late Viral Gene Expression Assemble into Prereplication Complexes before Viral DNA Replication

“…The UL44 protein is structurally homologous to the eukaryotic proliferating cell nuclear antigen (PCNA), which interacts with multiple proteins (31). Recent proteomic analyses demonstrated that the UL44 protein interacts with a variety of proteins, including transcription factors other than viral replication-related proteins (13,(45)(46)(47). Therefore, it is conceivable that pre-RCs including UL44 protein act to optimize the timing of late gene transcription.…”

Viral and cellular subnuclear structures in human cytomegalovirus-infected cells

“…Thus, nucleolin is required for both proper nuclear and subnuclear localization of HCMV replication compartment proteins. Furthermore, it is possible that, in infected cells, nucleolin, UL44, UL84 and either IRS1 or TRS1 interact within a large multiprotein complex (Strang et al, 2010a, b). Therefore, nucleolin may have an effect on the localization of both IRS1 and TRS1 in the infected cell.…”

“…UL44 could play a similar role and bind multiple proteins in replication compartments. Proteomic studies have indicated that UL44 associates with viral and cellular proteins found in replication compartments, including UL84 (Strang et al, 2010a). The binding of proteins to UL44 is increasingly well characterized.…”

“…Unexpectedly, the multifunctional cellular nucleolar protein nucleolin (Ginisty et al, 1999) is involved in maintaining replication compartment architecture. UL44 and nucleolin associate via the N terminus of UL44 (Strang et al, 2010a) and in infected cells nucleolin colocalizes with UL44, but not viral DNA, on the exterior of replication compartments (Strang et al, 2010a(Strang et al, , 2012a. 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).…”

“…However, the full repertoire of viral and cellular proteins acting in concert during DNA synthesis has yet to be described. These proteins probably include those viral and cellular proteins that associate with UL44 and/or UL84 (Gao et al, 2008;Strang et al, 2009Strang et al, , 2010a, several proteins that have illdefined roles in viral DNA synthesis (for example, UL36, IRS1 and IE2; Sarisky & Hayward, 1996), plus viral and cellular proteins involved in DNA damage responses and viral DNA repair O'Dowd et al, 2012;. How these and other proteins are organized within HCMV replication compartments is unclear but may involve UL44.…”