Cytomegalovirus pUL50 is the multi-interacting determinant of the core nuclear egress complex (NEC) that recruits cellular accessory NEC components

Abstract

Nuclear egress of herpesvirus capsids through the nuclear envelope is mediated by the multimeric nuclear egress complex (NEC). The human cytomegalovirus (HCMV) core NEC is defined by an interaction between the membrane-anchored pUL50 and its nuclear co-factor pUL53, tightly associated through heterodimeric corecruitment to the nuclear envelope. Cellular proteins, such as p32/gC1qR, emerin and protein kinase C (PKC), are recruited by direct interaction with pUL50 for the multimeric extension of the NEC. As a functionally important event, the recruitment of both viral and cellular protein kinases leads to site-specific lamin phosphorylation and nuclear lamina disassembly. In this study, interaction domains within pUL50 for its binding partners were defined by co-immunoprecipitation. The interaction domain for pUL53 is located within the pUL50 N-terminus (residues 10-169), interaction domains for p32/gC1qR (100-358) and PKC (100-280) overlap in the central part of pUL50, and the interaction domain for emerin is located in the C-terminus (265-397). Moreover, expression and formation of core NEC proteins at the nuclear rim were consistently detected in cells permissive for productive HCMV replication, including two trophoblast-cell lines. Importantly, regular nuclear-rim formation of the core NEC was blocked by inhibition of cyclin-dependent kinase (CDK) activity. In relation to the recently published crystal structure of the HCMV core NEC, our findings result in a refined view of NEC assembly. In particular, we suggest that CDKs may play an important regulatory role in NEC formation during HCMV replication.

The absence of p53 during Human Cytomegalovirus infection leads to decreased UL53 expression, disrupting UL50 localization to the inner nuclear membrane, and thereby inhibiting capsid nuclear egress

“…A functioning viral protein UL97 must be present for normal capsid egress from the nucleus to occur (Azzeh et al, 2006; Hamirally et al, 2009; Krosky et al, 2003; Milbradt et al, 2010; Prichard et al, 2005; Reim et al, 2013; Sharma et al, 2015; Sonntag et al, 2016). UL97 KO viruses fail to traffic capsids efficiently from the nucleus and produce dramatically lower titers (Azzeh et al, 2006; Prichard et al, 1999).…”

“…Several studies report that UL50 is crucial for efficient capsid nuclear egress during HCMV infection (Camozzi et al, 2008; Dal Monte et al, 2002; Leigh et al, 2015; Milbradt et al, 2007; Muranyi et al, 2002; Sharma et al, 2014; Sonntag et al, 2016). UL50 is a key component of the NEC, which directly recruits UL53, and indirectly recruits other NEC components, to the nuclear membrane (Camozzi et al, 2008; Lye et al, 2015; Marschall et al, 2005; Milbradt et al, 2009; Milbradt et al, 2010; Miller et al, 2010; Sam et al, 2009; Sharma et al, 2015; Sharma et al, 2014; Sonntag et al, 2016).…”

“…UL50 lacks its own NLS and it has been suggested that translocation of this protein is accomplished via membrane bound diffusion along the pore membrane (Milbradt et al, 2012; Schmeiser et al, 2013). UL50 directly recruits UL53, and indirectly recruits other potential NEC components, to the nuclear membrane (Camozzi et al, 2008; Lye et al, 2015; Marschall et al, 2005; Milbradt et al, 2009; Milbradt et al, 2010; Miller et al, 2010; Sam et al, 2009; Sharma et al, 2015; Sharma et al, 2014; Sonntag et al, 2016). The C-terminus of UL50 is embedded in the inner nuclear membrane (INM) and secures the NEC to the INM (Camozzi et al., 2008; Milbradt et al, 2007; Milbradt et al, 2012; Milbradt et al, 2009; Milbradt et al, 2014; Miller et al, 2010; Muranyi et al, 2002; Sam et al, 2009; Schmeiser et al, 2013; Sharma et al, 2014; Sonntag et al, 2016).…”

“…UL53 localizes to the nucleus beginning at 48 h pi, presumably via its nuclear localization signal (NLS) (Milbradt et al, 2012; Schmeiser et al, 2013). From the nuclear rim, UL50 recruits UL53 to that location (Camozzi et al, 2008; Milbradt et al, 2012; Milbradt et al, 2014; Sam et al, 2009; Schmeiser et al, 2013; Sonntag et al, 2016). Several cellular proteins have been identified as potential components of the NEC, including the lamin B receptor (LBR), protein kinase C (PKC), p32, peptidyl-prolyl cis-trans isomerase (pin1), and emerin (Camozzi et al, 2008; Hamirally et al, 2009; Krosky et al, 2003; Marschall et al, 2005; Milbradt et al, 2009; Milbradt et al, 2014; Milbradt et al, 2010; Miller et al, 2010; Sam et al, 2009; Sharma et al., 2014).…”

Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins

“…On the one hand, cyclin-dependent kinases (CDKs), which play functionally relevant roles during HCMV infection, are promising candidates. CDK inhibitors, such as roscovitine (targeting CDK1, 2, 5, 9), R25 (alsterpaullone; CDK1, 2, 5), R22 (CDK9) and LDC4297 (CDK7), have shown strong antiviral activities in cell culture models including both laboratory and clinically relevant virus strains [12,13,14,15,16]. On the other hand, herpesviral protein kinases, such as HCMV pUL97, may also represent attractive targets.…”