Functional Characterization of Nuclear Trafficking Signals in Pseudorabies Virus pUL31

Abstract

The herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in exclusively cytosolic localization, indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutant constructs lacking a functional NLS resulted in diffuse nuclear and cytosolic localization, indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31 deletion mutant. Our data demonstrate that the N terminus of pUL31, encompassing the NLS, is required for efficient nuclear targeting but not for pUL34 interaction, while the C terminus, containing the NES but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-⌬NLS displayed a dominant negative effect on wild-type PrV replication, probably by diverting pUL34 to cytoplasmic membranes. IMPORTANCEThe molecular details of nuclear egress of herpesvirus capsids are still enigmatic. Although the key players, homologs of herpes simplex virus pUL34 and pUL31, which interact and form the heterodimeric nuclear egress complex, are well known, the molecular basis of this interaction and the successive budding, vesicle formation, and scission from the INM, as well as capsid release into the cytoplasm, remain largely obscure. Here we show that classical cellular targeting signals for nuclear import and export are important for proper localization and function of the NEC, thus regulating herpesvirus nuclear egress.

Characterization of the nuclear import and export signals of pseudorabies virus UL31

“…To further test the function of the predicted NES, a plasmid expressing an EYFP dimer (dEYFP) fused to the C-terminus of aa246-254 was constructed, and aa246-254-dEYFP also showed similar subcellular localization to aa231-254-EYFP, confirming that the predicted NES of pUL31 is nonfunctional and that the functional NES of UL31 may depend on its spatial conformation. These results are conflict with those of Paßvogel and colleagues [13]. This can probably be explained by subtle differences in experimental design.…”

“…However, it is still an endemic problem in many countries, although efforts to eradicate PRV in many countries have made great progress [14]. The UL31 protein (pUL31), a PRV-encoded late protein, is composed of 271 amino acid (aa) residues [3], is among the first and best characterized pUL31 homologues [3,8,13]. Herpes simplex virus 1 (HSV-1) UL31 [1,3,7,10,11,[15][16][17][18][19] and Epstein-Barr virus (EBV) BFLF2 [4,6,9], the homologues of PRV UL31, have also been extensively studied.…”

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

“…This behavior appears to be a beta herpesvirus phenomenon, although there is some indication of similar behavior in psuedorabies virus (Klupp et al, 2007). There are reports that transiently co-expressed UL50 and UL53 (and their homologues) are capable of deforming the INM (Gonnella et al, 2005; Hagen et al, 2015; Klupp et al, 2007; Lorenz et al, 2015; Milbradt et al, 2012; Passvogel et al, 2015; Reynolds et al, 2001; Sharma et al, 2014; Zeev-Ben-Mordehai et al, 2015), which has begun to be explained through structural analysis of these proteins (as reviewed in (Bigalke and Heldwein, 2015)). Our results (see Figure 4F) confirmed this behavior in the absence of p53 during transient expression.…”

“…In support of our immunofluorescent localization, Dalmonte et al used immuno-EM at late times pi to determine that UL53 was located within large invaginations (IINMs) within the nucleus (Dal Monte et al, 2002). In addition, transient expression of the equivalent PrV proteins (UL31/UL34) produces "speckles", which have been determined to be "vesicle formation in the perinuclear space" (Hagen et al, 2015; Passvogel et al, 2015; Zeev-Ben-Mordehai et al, 2015). We found that the majority of LOX cells formed abundant puncta at the late stages of infection.…”

Multiple Roles of the Cytoplasmic Domain of Herpes Simplex Virus 1 Envelope Glycoprotein D in Infected Cells

“…In particular, we focused on the arginine cluster in the gD cytoplasmic domain, since positively charged amino acid clusters in proteins have been reported to be involved in various protein functions, including binding to other proteins, membrane lipids, RNA, and DNA and regulation of subcellular localization (24)(25)(26)(27). We report here that the arginine cluster in the gD cytoplasmic domain is required for the efficient formation of microvilluslike projections at the plasma membrane of HSV-1-infected cells and for viral secondary envelopment in HSV-1-infected cells.…”