Unexpected features and mechanism of heterodimer formation of a herpesvirus nuclear egress complex

Abstract

Herpesvirus nucleocapsids escape from the nucleus in a process orchestrated by a highly conserved, viral nuclear egress complex. In human cytomegalovirus, the complex consists of two proteins, UL50 and UL53. We solved structures of versions of UL53 and the complex by X-ray crystallography. The UL53 structures, determined at 1.93 and 3.0 Å resolution, contained unexpected features including a Bergerat fold resembling that found in certain nucleotide-binding proteins, and a Cys 3 His zinc finger. Substitutions of zinc-coordinating residues decreased UL50-UL53 co-localization in transfected cells, and, when incorporated into the HCMV genome, ablated viral replication. The structure of the complex, determined at 2.47 Å resolution, revealed a mechanism of heterodimerization in which UL50 clamps onto helices of UL53 like a vise. Substitutions of particular residues on the interaction interface disrupted UL50-UL53 colocalization in transfected cells and abolished virus production. The structures and the identification of contacts can be harnessed toward the rational design of novel and highly specific antiviral drugs and will aid in the detailed understanding of nuclear egress.

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

“…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). Although UL50 appeared slightly less robustly expressed in the p53KO cells at earlier timepoints by Western blot, levels appeared comparable to LOX cells by 96–120 h pi (Figure 3A).…”

“…The viral constituents of the NEC begin to be expressed at early stages of infection. UL50 is a transmembrane protein and when properly localized its C-terminus is anchored into the INM (Camozzi et al, 2008; Lye et al., 2015; 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). Extraction of un- and loosely bound UL50 from p53KO cells prior to fixation and staining revealed only 40% were positive for UL50 in the nucleus.…”

“…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).…”

Human Cytomegalovirus nuclear egress and secondary envelopment are negatively affected in the absence of cellular p53

“…Much of the literature on HSV and PrV (both alpha herpesviruses) indicate that these viruses traffic directly through a remodeled INM without the formation of tubules (Fuchs et al, 2002; Klupp et al, 2000; Reynolds et al, 2001; Reynolds et al, 2002; Roller et al, 2000). In addition, it has been shown that the UL50/UL53 equivalents in these viruses are capable of distinct deformation of membranes, indicating that these components of the NEC can alone affect the primary envelopment process (Bigalke et al, 2014; Hagen et al, 2015; Klupp et al, 2007; Lorenz et al, 2015) and references within (Lye et al, 2015). Despite this possible difference, all the herpesviruses require remodeling of the lamina and utilize components of the NEC, and perhaps other cellular components, to accomplish this task (Camozzi et al, 2008; Dal Monte et al, 2002; Farina et al, 2005; Fuchs et al, 2002; Gonnella et al, 2005; Hamirally et al, 2009; Krosky et al, 2003; Marschall et al, 2005; Milbradt et al, 2007; Milbradt et al, 2009; Milbradt et al, 2014; Milbradt et al, 2010; Miller et al, 2010; Reim et al, 2013; Reynolds et al, 2001; Reynolds et al, 2002; Sam et al, 2009; Sharma et al, 2014).…”

Vesicular Nucleo-Cytoplasmic Transport—Herpesviruses as Pioneers in Cell Biology

“…Two proteins that are conserved in sequence between members of the family Herpesviridae (i.e., the ‘classical’ herpesviruses) within the order Herpesvirales form a heterodimeric nuclear egress complex (NEC). The NEC exhibits a highly conserved structure similar in shape and size in the alphaherpesviruses herpes simplex virus 1 (HSV-1) [11] and pseudorabies virus (PrV) [11,12], and the betaherpesvirus human cytomegalovirus (HCMV) [13,14] (Figure 1). The C-terminally membrane-bound component (designated pUL34 in the alphaherpesvirusesHSV-1 and PrV) exhibits a large groove in the globular head domain into which an N-terminally extended α-helix of the otherwise globular pUL31 integrates.…”