Identification, subviral localization, and functional characterization of the pseudorabies virus UL17 protein

Abstract

Homologs of the UL17 gene of the alphaherpesvirus herpes simplex virus 1 (HSV-1) are conserved in all three subfamilies of herpesviruses. However, only the HSV-1 protein has so far been characterized in any detail. To analyze UL17 of pseudorabies virus (PrV) the complete 597-amino-acid protein was expressed in Escherichia coli and used for rabbit immunization. The antiserum recognized a 64-kDa protein in PrV-infected cell lysates and purified virions, identifying PrV UL17 as a structural virion component. In indirect immunofluorescence analyses of PrV-infected cells the protein was predominantly found in the nucleus. In electron microscopic studies after immunogold labeling of negatively stained purified virion preparations, UL17-specific label was detected on single, mostly damaged capsids, whereas complete virions and the majority of capsids were free of label. In ultrathin sections of infected cells, label was primarily found dispersed around scaffold-containing B-capsids, whereas on DNA-filled C-capsids it was located in the center. Empty intranuclear A-capsids were free of label, as were extracellular capsid-less L-particles. Functional characterization of PrV-DeltaUL17F, a deletion mutant lacking codons 23 to 444, demonstrated that cleavage of viral DNA into unit-length genomes was inhibited in the absence of UL17. In electron microscopic analyses of PrV-DeltaUL17F-infected RK13 cells, DNA-containing capsids were not detected, while numerous capsidless L-particles were observed. In summary, our data indicate that the PrV UL17 protein is an internal nucleocapsid protein necessary for DNA cleavage and packaging but suggest that the protein is not a prominent part of the tegument.

FIG. 1.

Construction of PrV-ΔUL17F. (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 the PrV UL15-UL17-UL16 gene region. The UL17 and UL16 genes are located within the intron of the spliced UL15 gene (dotted line), which is bracketed by exons I and II of the UL15 gene. UL17 and UL16 are transcribed into 3′ coterminal mRNAs, which share a common polyadenylation signal (arrow pointing up at right), whereas the polyadenylation signal for the UL15 mRNA is located downstream of UL15 exon II (arrow pointing up at left). Relevant restriction sites are indicated. (C) The BamHI-NcoI fragment of BamHI fragment 3 was used for cloning into pro- and eukaryotic expression vectors and for generation of the UL17 deletion mutant. In mutant PrV-ΔUL17F, sequences between the BmgBI sites were deleted and replaced by one copy of a flp recognition target site.

FIG. 2.

Identification of the PrV UL17 protein and characterization of PrV-ΔUL17F. Purified PrV-Ka virions (PrV-Ka V) or lysates of cells infected with PrV-Ka (PrV-Ka L) or PrV-ΔUL17F (PrV-ΔUL17F L) or mock-infected RK13 cells (mock) were separated on sodium dodecyl sulfate-10% polyacrylamide gels and incubated with monospecific antisera against the UL17, gH, UL37, UL16, and UL49 proteins. The locations of molecular mass markers are indicated on the left.

FIG. 3.

Intracellular localization of PrV UL17. RK13 cells were infected with PrV-Ka or PrV-ΔUL17F at an MOI of 10 or mock infected and were fixed with ice-cold acetone 6 h later. Immunofluorescence analysis was performed by laser scanning microscopy using the UL17-specific antiserum and Alexa 488-conjugated secondary antibodies (green). Chromatin was counterstained with propidium iodide (red).

FIG. 4.

Immunoelectron microscopy of purified PrV-Ka virions. Purified PrV-Ka virions (A) were incubated with monospecific antisera against the UL17 (B to E), UL19 (F), UL36 (G), UL48 (H), and UL11 (I) proteins or gB (J). Reactivity of the antisera was visualized after incubation with 10 nm-diameter-gold-tagged secondary anti-rabbit antibodies. Bars represent 100 nm.

FIG. 5.

Immunoelectron microscopy of PrV-Ka-infected cells. RK13 cells were infected with PrV-Ka as described above, and ultrathin sections were labeled with anti-UL17 serum (A to G) or anti-UL48 serum (H). Anti-rabbit sera conjugated with gold (10 nm [B, D, and E to H] or 5 nm [A and C]) were used. For better visualization of the smaller gold particles, counterstaining was omitted in the assays using 5-nm gold. Arrows in panels G and H point to L-particles. Bars represent 100 nm in panels A to D and 150 nm in panels E to H.

FIG. 6.

In vitro growth properties of PrV-ΔUL17F. (A) RK13 or RK13-UL17 cells were infected with PrV-Ka or PrV-ΔUL17F at an MOI of 10, harvested at the indicated times after infection, and titrated on RK13-UL17 cells. Average titers (PFU/ml) and standard deviations for three independent experiments are shown. (B) RK13 and RK13-UL17 cells were infected under plaque assay conditions with PrV-Ka and PrV-ΔUL17F and fixed 2 days postinfection. Infected cells were visualized by indirect immunofluorescence with a gC-specific monoclonal antibody.

FIG. 7.

Defect in cleavage of concatemeric viral DNA in the absence of PrV UL17. RK13 (A) or RK13-UL17 (B) cells were infected with PrV-Ka, PrV-ΔUL17F, or PrV-ΔUL28 at an MOI of 1. Cells were harvested at approximately 12 h p.i., and DNA was isolated, digested with BamHI, and separated on a 0.8% agarose gel. After transfer to nylon membranes, DNA was probed with labeled genome end-specific BamHI fragment 13. BamHI-13 hybridized to fragments BamHI-8′ and BamHI-13, which share homologous sequences since they are both derived from the inverted repeat regions (see Fig. 1), and the junction fragment composed of BamHI-13 and BamHI-14′, which is derived from head-to-tail concatemeric DNA.

FIG.8.

Electron microscopy of PrV-ΔUL17F-infected cells. RK13 (A to D) and RK13-UL17 (E to G) cells were infected with PrV-ΔUL17F at an MOI of 1 and processed for electron microscopy 14 h p.i. (A) Overview of an infected RK13 cell. (B) Pseudocrystalline aggregations of B-capsids in the nucleus. (C and D) L-particles in the cytoplasm (C) and on the cell surface (D). (E) Unimpaired virus morphogenesis on UL17-expressing cells, including production of C-capsids in the nucleus (F) and mature virions on the cell surface (G). Bars represent 2 μm in panel A, 500 nm in panels B and D, 250 nm in panels C, F, and G, and 1 μm in panel E.