Proteomic Analysis of the Multimeric Nuclear Egress Complex of Human Cytomegalovirus

Abstract

Herpesviral capsids are assembled in the host cell nucleus before being translocated into the cytoplasm for further maturation. The crossing of the nuclear envelope represents a major event that requires the formation of the nuclear egress complex (NEC). Previous studies demonstrated that human cytomegalovirus (HCMV) proteins pUL50 and pUL53, as well as their homologs in all members of Herpesviridae, interact with each other at the nuclear envelope and form the heterodimeric core of the NEC. In order to characterize further the viral and cellular protein content of the multimeric NEC, the native complex was isolated from HCMV-infected human primary fibroblasts at various time points and analyzed using quantitative proteomics. Previously postulated components of the HCMVspecific NEC, as well as novel potential NEC-associated proteins such as emerin, were identified. In this regard, interaction and colocalization between emerin and pUL50 were confirmed by coimmunoprecipitation and confocal microscopy analyses, respectively. A functional validation of viral and cellular NEC constituents was achieved through siRNA-mediated knockdown experiments. The important role of emerin in NEC functionality was demonstrated by a reduction of viral replication when emerin expression was down-regulated. Moreover, under such conditions, reduced production of viral proteins and deregulation of viral late cytoplasmic maturation were observed. Combined, these data prove the functional importance of emerin as an NEC component, associated with pUL50, pUL53, pUL97, p32/ gC1qR, and further regulatory proteins. Summarized, our findings provide the first proteomics-based characterization and functional validation of the HCMV-specific multimeric NEC. Molecular & Cellular

Crystal Structure of the Human Cytomegalovirus pUL50-pUL53 Core Nuclear Egress Complex Provides Insight into a Unique Assembly Scaffold for Virus-Host Protein Interactions

“…Although intracellular membrane trafficking of pUL50 is determined by residues and sequential domains other than Glu 56 /Tyr 57 (19), the two residues were both found to be essential not only for pUL50-pUL53 complex formation but also for the recruitment of pUL53 to the nuclear rim promoting viral replication efficiency (14,15). As depicted in Fig.…”

“…Specific residues within the interaction surfaces of the core NEC proteins (in particular Glu 56 and Tyr 57 in pUL50) seem to be essential not only for heterodimerization but also for subsequent scaffolding steps during multimeric NEC formation (9,15). Although intracellular membrane trafficking of pUL50 is determined by residues and sequential domains other than Glu 56 /Tyr 57 (19), the two residues were both found to be essential not only for pUL50-pUL53 complex formation but also for the recruitment of pUL53 to the nuclear rim promoting viral replication efficiency (14,15).…”

“…As an essential step in HCMV replication, pUL50 and pUL53 heterodimerize and form the core of the NEC that serves as a scaffold for the recruitment of a group of viral and cellular NEC-associated proteins. The composition of the multimeric NEC of human and murine CMVs has recently been defined by proteomic analyses (15,18).…”

Therapeutics to prevent congenital cytomegalovirus during pregnancy: what is available now and in the future?

“…Nuclear egress is a multi-step regulatory process that involves a phosphorylationtriggered distortion of the nuclear lamina [42][43][44][45] . Pivotal for nuclear egress is the role of the two viral nuclear egress proteins pUL50 and pUL53 that heterodimerise and form a core for the multimeric viralcellular NEC 46 …”

Viral and cellular subnuclear structures in human cytomegalovirus-infected cells

“…This should facilitate capsid movement through the lamina to the nuclear membrane. The requirement for other viral (including c-ORF29 and UL45) and cellular (including emerin, p32 and protein kinase C) proteins implicated in nuclear egress in this model remains unclear (Milbradt et al, 2014;Miller et al, 2010). It is unknown what mechanism selects only DNA-containing capsids for nuclear egress in HCMV-infected cells.…”