The capsid-associated UL25 protein of the alphaherpesvirus pseudorabies virus is nonessential for cleavage and encapsidation of genomic DNA but is required for nuclear egress of capsids

Abstract

Homologs of the UL25 gene product of herpes simplex virus (HSV) have been identified in all three subfamilies of the Herpesviridae. However, their exact function during viral replication is not yet known. Whereas earlier studies indicated that the UL25 protein of HSV-1 is not required for cleavage of newly replicated viral DNA but is necessary for stable encapsidation (A. R. McNab, P. Desai, S. Person, L. Roof, D. R. Thompson, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060-1070, 1998), viral DNA packaging has recently been demonstrated to occur in the absence of UL25, although at significantly decreased levels compared to wild-type HSV-1 (N. Stow, J. Virol. 75:10755-10765 2001). To clarify the functional role of UL25 we analyzed the homologous protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL25 was found to be essential for viral replication, as a mutant virus lacking the UL25 protein required UL25-expressing cells for productive propagation. In the absence of the UL25 protein, newly replicated PrV DNA was cleaved and DNA-containing C-type capsids were detected in infected cell nuclei. However, although capsids were frequently found in close association with the inner nuclear membrane, nuclear egress was not observed. Consequently, no capsids were found in the cytoplasm, resulting in an inhibition of virion morphogenesis. In contrast, the formation of capsidless enveloped tegument structures (L particles) in the cytoplasm was readily observed. Thus, our data demonstrate that the PrV UL25 protein is not essential for cleavage and encapsidation of viral genomes, although both processes occur more efficiently in the presence of the protein. However, the presence of the PrV UL25 protein is a prerequisite for nuclear egress. By immunoelectron microscopy, we detected UL25-specific label on DNA-containing C capsids but not on other intranuclear immature or defective capsid forms. Thus, the PrV UL25 protein may represent the hitherto missing trigger that allows primary envelopment preferably of DNA-filled C capsids.

FIG. 1.

Construction of PrV-ΔUL25F. (A) Map of the PrV genome with the unique long (U_L), unique short (U_S), and inverted repeat (IR, TR) sequences. BamHI restriction sites are indicated, and fragments are numbered according to size. (B) Enlargement of BamHI fragment 9. The UL24, UL25, UL26, and UL26.5 genes (shown as pointed rectangles) are transcribed into 3′-coterminal mRNAs with a common poly(A) addition signal (shown as an arrow pointing up). Relevant restriction sites for construction of the UL25 deletion mutant, PrV-ΔUL25F, are indicated, as is the extent of the introduced deletion. (C) A 0.9-kb SalI subfragment of BamHI fragment 9 was used for prokaryotic expression of GST-UL25.

FIG. 2.

One-step growth kinetics of PrV-ΔUL25F. RK13 or RK13-UL25 cells were infected with PrV-Ka or PrV-ΔUL25F at an MOI of 10. Cells and supernatant were harvested at the indicated time points, and infectious titers were determined on RK13-UL25 cells and plotted. Mean titers of three independent experiments and the corresponding standard deviations are shown.

FIG. 3.

Plaque formation of PrV-ΔUL25F. RK13 and RK13-UL25 cells were infected with PrV-Ka and PrV-ΔUL25F under plaque assay conditions and fixed 2 days p.i. Infected cells were visualized by indirect immunofluorescence with a glycoprotein C-specific monoclonal antibody.

FIG. 4.

Expression kinetics of mutant PrV-ΔUL25F. Western blot analysis of rabbit kidney cells infected with PrV-Ka or PrV-ΔUL25F at an MOI of 10 is shown. Cells were harvested at the indicated time points after infection, and proteins were separated on SDS-10% polyacrylamide gels. After electrotransfer onto nylon membranes, parallel blots were incubated with the indicated antisera. Molecular masses of marker proteins are indicated on the left.

FIG. 5.

Electron microscopy of PrV-ΔUL25F-infected RK13 and RK13-UL25 cells. RK13 cells (A to D) or RK13-UL25 cells (E) were infected with PrV-ΔUL25F at an MOI of 1 and processed for electron microscopy at 14 h p.i. Bars represent 3 μm in panel A (300 nm in inset); 0.5 μm in panels B, D, and E; and 2 μm in panel C.

FIG. 6.

Impairment of cleavage of concatemeric DNA in the absence of PrV UL25. RK13 or RK13-UL25 cells were infected with PrV-Ka, PrV-ΔUL17, or PrV-ΔUL25F at an MOI of 5 and harvested at 16 h p.i. Whole-cell DNA was isolated, digested with BamHI, separated on a 0.8% agarose gel, and blotted onto a nylon membrane. Parallel blots were probed with labeled genome-end-specific BamHI fragments 13 (A) and 14′ (B). Bound radioactivity was recorded by a phosphorimager and quantified. Values were calculated with respect to bound radioactivity present on BamHI fragment 8′, set as 100% for each lane. Identification of fragments and their sizes are indicated. “Junction” refers to the junction fragment derived from head-to-tail-fused termini.

FIG. 7.

Virus fractionation. Purified virion preparations (V) were separated into capsid (C) and envelope (E) fractions and analyzed by immunoblotting with sera against the major capsid protein UL19, the portal protein UL6, the capsid component UL17, envelope glycoprotein H (gH), or the UL37 tegument protein, as well as with the UL25-specific serum. Molecular mass markers are indicated on the left.

FIG. 8.

Immunolabeling of purified virions. Purified virion preparations and capsids in ultrathin sections were analyzed by immunoelectron microscopy after incubation with UL25 antiserum and gold-conjugated secondary antibodies. (A to C) Results from analysis of negatively stained purified virion preparations. (D to F) Intranuclear A, B, and C capsids in PrV-Ka-infected RK13 cells. (G to I) Different intranuclear capsid forms in PrV-ΔUL25F-infected cells serving as negative controls to demonstrate the specificity of the antiserum. Secondary antibodies tagged with 5-nm gold particles were used for panel C, whereas 10-nm gold-tagged antibodies were used for all other panels. Bars, 100 μm.