Function of the Nonconserved N-Terminal Domain of Pseudorabies Virus pUL31 in Nuclear Egress

Abstract

Nuclear egress of herpesvirus capsids is mediated by the conserved nuclear egress complex (NEC), composed of the membrane-anchored pUL34 and its nucleoplasmic interaction partner, pUL31. The recently solved crystal structures of the NECs from different herpesviruses show a high structural similarity, with the pUL34 homologs building a platform recruiting pUL31 to the inner nuclear membrane. Both proteins possess a central globular fold, while the conserved N-terminal portion of pUL31 forms an extension reaching around the core of pUL34. However, the extreme N terminus of the pUL31 homologs, which is highly variable in length and amino acid composition, had to be removed for crystallization. Several pUL31 homologs contain a classical nuclear localization signal (NLS) within this part mediating efficient nuclear import. In addition, membrane-binding activity, blocking premature interaction with pUL34, nucleocapsid trafficking, and regulation of NEC assembly and disassembly via phosphorylation were assigned to the extreme pUL31 N terminus. To test the functional importance in the alphaherpesvirus pseudorabies virus (PrV) pUL31, N-terminal truncations and site-specific mutations were generated, and the resulting proteins were tested for intracellular localization, interaction with pUL34, and functional complementation of PrV-ΔUL31. Our data show that neither the bipartite NLS nor the predicted phosphorylation sites are essential for pUL31 function during nuclear egress. Moreover, nearly the complete variable N-terminal part was dispensable for function as long as a stretch of basic amino acids was retained. Phosphorylation of this domain controls efficient nucleocapsid release from the perinuclear space.IMPORTANCE Nuclear egress of herpesvirus capsids is a unique vesicle-mediated nucleocytoplasmic transport. Crystal structures of the heterodimeric NECs from different herpesviruses provided important details of this viral nuclear membrane deformation and scission machinery but excluded the highly variable N terminus of the pUL31 component. We present here a detailed mutagenesis study of this important portion of pUL31 and show that basic amino acid residues within this domain play an essential role for proper targeting, complex formation, and function during nuclear egress, while phosphorylation modulates efficient release from the perinuclear space. Thus, our data complement previous structure-function assignments of the nucleocapsid-interacting component of the NEC.

Keywords: herpesvirus; nuclear egress complex (NEC); nuclear envelope; nuclear export signal (NES); nuclear localization signal (NLS); pseudorabies virus.

FIG 1

Summary of PrV pUL31 mutants. (A) The variable N termini of PrV and HSV-1 pUL31 are aligned. Basic amino acids are marked by triangles, and predicted phosphorylation sites are shown by asterisks. (B) The N-terminal amino acid sequences of the full-length, N-terminally truncated, and site-specifically mutated pUL31 are shown. Basic amino acids as well as predicted phosphorylation sites are highlighted as shown for panel A. The black bar indicates location of the predicted bipartite NLS; amino acids substituted in this study are underlined. A summary of the results is presented in the table on the right, with nuclear localization (Nuc loc) after transfection, colocalization in punctate pattern after cotransfection with pcDNA-UL34, and complementation (Comp) of PrV-ΔUL31 by stably expressing cell lines (++, wild-type; +, slightly reduced; +/−, significantly reduced; −, negative).

FIG 2

Localization of N-terminally truncated pUL31 and colocalization with pUL34. The pUL31 expression constructs were transfected (upper row) or cotransfected with pcDNA-UL34 (lower rows) into RK13 cells. Two days posttransfection, cells were fixed, permeabilized, and stained with the monospecific pUL31 antiserum (green) and a monoclonal anti-pUL34 antibody (red). Fluorescent signals were recorded with a confocal laser-scanning microscope (Leica SP5). Bars, 10 μm.

FIG 3

Western blot analysis of the pUL31-expressing cell lines. Stably pUL31-expressing or nontransgenic RK13 cells were harvested and lysed. Proteins were separated on an SDS-10% polyacrylamide gel. After transfer to nitrocellulose, membranes were incubated with the monospecific anti-pUL31 rabbit serum or murine anti-alpha tubulin as a loading control. Molecular masses of marker proteins are given on the left (in kDa).

FIG 4

In vitro replication. RK13, RK13-UL31, and cells expressing truncated pUL31 were infected with PrV-Ka or PrV-ΔUL31 at an MOI of 5 and harvested 24 h p.i. Shown are mean values from four independent experiments with the corresponding standard deviations. Statistically significant differences between the titers of infection with PrV-Ka and PrV-ΔUL31 of each transgenic cell line are indicated: ***, P < 0.001.

FIG 5

Localization of site-specifically mutated pUL31 and colocalization with pUL34. Transfections, cotransfections with pcDNA-UL34, and processing for confocal microscopy were done as described in the legend to Fig. 2. Bars, 10 μm.

FIG 6

In vitro replication. RK13, RK13-UL31, and cells expressing site-specifically mutated pUL31 as indicated were infected with PrV-Ka or PrV-ΔUL31 at an MOI of 5 (A to C). After 24 h p.i. cells and supernatant were harvested and infectious virus titers were determined on RK13-UL31 cells. Given are mean values from at least three independent experiments with the corresponding standard deviations. Statistically significant differences are indicated: **, P < 0.01; ***, P < 0.001.

FIG 7

Site-specific mutation of predicted phosphorylation sites. The predicted phosphorylation sites at positions 12 and 13 were changed in full-length pUL31, giving rise to pUL31-S12/13A (A), or the threonine residues at position 19 and 21 were replaced by alanine in the N-terminally truncated pUL31-N14 mutant singly or in combination (B). Stably expressing cell lines were infected as described in the legend to Fig. 6. Shown are the mean values for three independent experiments with the corresponding standard deviations (*, P < 0.05; ***, P < 0.001).

FIG 8

Ultrastructural analyses. RK13-UL31-N14 (A), RK13-UL31-N14-T19A (B), RK13-UL31-N14-T21A (C), and RK13-UL31-N14-T19/21A (D) cells were infected with PrV-ΔUL31 at an MOI of 1. Cells were processed for electron microscopy 14 h p.i. Bars indicate 1 μm (A and B) and 500 nM (C and D).