Complex formation between the UL16 and UL21 tegument proteins of pseudorabies virus

Abstract

The products of the UL16 and UL21 genes represent tegument proteins which are conserved throughout the mammalian herpesviruses. To identify and functionally characterize the respective proteins in the alphaherpesvirus pseudorabies virus, monospecific antisera against bacterially expressed fusion proteins were generated. In immunoblots the UL16 antiserum detected a ca. 40-kDa protein in infected cells and purified virion preparations, whereas the anti-UL21 serum recognized a protein of approximately 60 kDa. Interestingly, in immunoprecipitations using either antiserum, both proteins were coprecipitated, demonstrating the formation of a physical complex. To investigate protein function, viruses lacking either UL16, UL21, or both were constructed. Mutant viruses could be propagated on noncomplementing cells, indicating that these proteins, either alone or in combination, are not required for viral replication in cell culture. However, plaque sizes and viral titers were reduced. Electron microscopy showed only slight alterations in cytoplasmic virion morphogenesis, whereas intranuclear maturation stages were not affected. Similar results were obtained with a triple mutant simultaneously lacking the three conserved tegument proteins UL11, UL16, and UL21. In summary, our results uncover a novel interaction between conserved herpesvirus tegument proteins that increases the complexity of the intricate network of protein-protein interactions involved in herpesvirus morphogenesis.

FIG. 1.

Construction of virus mutants. Schematic map of the PrV genome shows the unique long (U_L) and unique short (U_S) regions, the inverted repeat regions (IR, TR), and the positions of BamHI restriction fragments. Black bars labeled I and II indicate locations of the relevant genomic regions. (I) Enlargement of the PrV UL15-UL17-UL16 gene region (27). The UL17 and UL16 genes are located within the intron of the spliced UL15 gene (dotted line), which is flanked by exons I (exI) and II (exII) of the UL15 gene. UL17 and UL16 are transcribed into 3′-coterminal mRNAs which share a common polyadenylation signal (arrow pointing up). Relevant restriction sites are indicated. (II) Enlargement of the UL20-UL21 gene region (32). The left part of BamHI fragment 4 is shown. The UL21 open reading frame is located adjacent to an origin of replication (ori L) and is transcribed antiparallel to the UL20 gene. Pointed rectangles show locations of the respective open reading frames and also indicate transcriptional orientation. Relevant restriction site are given, and the location of the UL21 polyadenylation signal is indicated by an arrow pointing up.

FIG. 2.

Identification of the PrV UL16 protein and characterization of mutant viruses. Infected-cell lysates (A) or purified virions (B) of PrV-Ka, PrV-ΔUL11F, PrV-ΔUL16F, PrV-ΔUL21F, PrV-ΔUL16F/11G, PrV-ΔUL16F/21K, PrV-ΔUL16F/21K/11G, or mock-infected RK13 cells were separated on SDS-10 or 15% (for demonstration of UL11) polyacrylamide gels and incubated with monospecific antisera against the UL16, UL21, UL11, UL17, UL19, UL37, gH, or UL49 protein. Locations of molecular mass markers (in kilodaltons) are shown to the left (A) and right (B) of the gels.

FIG. 3.

Intracellular localization of UL16 and UL21 proteins. RK13 cells were either transiently transfected with expression plasmids for UL16 (upper left panel) or UL21 (lower left panel) or infected with PrV-Ka (right panels). Immunofluorescence analysis was performed by confocal laser scanning microscopy using monospecific anti-UL16 or anti-UL21 serum and Alexa 488-conjugated secondary antibodies (green). Chromatin was counterstained with propidium iodide (red).

FIG. 4.

Growth properties of PrV-ΔUL16F. (A) RK13 or RK13-UL16 cells were infected with PrV-Ka or PrV-ΔUL16F at an MOI of 10 and were harvested immediately or after 4, 8, 12, 24, or 36 h p.i. Virus progeny was titrated on RK13 cells. Mean virus titers and standard deviations from three independent experiments are shown. (B) RK13 or RK13-UL16 cells were infected with PrV-Ka or PrV-ΔUL16F under plaque assay conditions, and the diameters of 30 plaques each were measured microscopically after 48 h. The mean value for PrV-Ka was set at 100%, and the average plaque size of PrV-ΔUL16 was calculated accordingly. Means and standard deviations are shown for three independent experiments.

FIG. 5.

Coimmunoprecipitation of UL16 and UL21 proteins. Lysates from metabolically labeled PSEK cells infected with PrV-Ka, PrV-ΔUL16F, or PrV-ΔUL21F were precipitated with the UL16-specific antiserum (α-UL16) or the corresponding preimmune serum (α-UL16NS), the UL21-specific antiserum (α-UL21), or an anti-gH (α-gH) monospecific serum. Precipitates were separated on an SDS-10% polyacrylamide gel and analyzed with a phosphorimager. Labeled marker proteins are shown in the left lane, and their relative molecular masses are indicated.

FIG. 6.

Growth properties of PrV-ΔUL21F. RK13 and RK13-UL21 cells were infected in parallel with PrV-Ka or PrV-ΔUL21F. One-step growth kinetics (A) and plaque sizes (B) were analyzed as described in the legend to Fig. 4.

FIG. 7.

Processing of viral DNA. RK13 cells were infected with PrV-Ka, PrV-ΔUL28, PrV-ΔUL11F, PrV-ΔUL16F, PrV-ΔUL21F, PrV-ΔUL16F/11G, PrV-ΔUL16F/21K, or PrV-ΔUL16F/21K/11G at an MOI of 1, and cells were harvested when the cytopathic effect approached 100%. Whole-cell DNA was digested with BamHI and separated on an 0.8% agarose gel. (A) Ethidium bromide-stained gel. (B) After transfer to a nylon membrane, the filter was hybridized with labeled terminal BamHI fragment 13. Hybridization reveals signals corresponding to fragments 8′ and 13, which share homologous sequences because both originate from the inverted repeat regions (see Fig. 1), as well as the junction fragment composed of BamHI fragments 13 and 14′, which is derived from head-to-tail concatemeric or circular DNA.

FIG. 8.

Growth properties of mutants PrV-ΔUL16F/11G, PrV-ΔUL16F/21K, and PrV-ΔUL16F/21K/11G. (A) One-step growth kinetics. RK13 cells were infected with the indicated viruses at an MOI of 5 and harvested as described in the legend to Fig. 4. Progeny virus titers were determined on RK13 cells. Shown are means and standard deviations from three independent experiments. (B) Plaque size determination. RK13, RK13-UL11, RK13-UL16, or RK13-UL21 cells were infected with PrV-Ka, PrV-ΔUL11F, PrV-ΔUL16F, PrV-ΔUL21F, PrV-ΔUL16F/11G, PrV-ΔUL16F/21K, or PrV-ΔUL16F/21K/11G under plaque assay conditions. Plaque size was determined 2 days p.i. Mean relative values and standard deviations from three different experiments are shown, with the PrV-Ka plaque size set at 100%.

FIG.9.

Electron microscopy of PrV-ΔUL16F/11G, PrV-ΔUL16F/21K, and PrV-ΔUL16F/21K/11G. RK13 cells were infected with PrV-ΔUL16F/11G (A), PrV-ΔUL16F/21K (B), or PrV-ΔUL16F/21K/11G (C) at an MOI of 1 and were processed for electron microscopy at 14 h p.i. Arrows in panel A point to capsids in association with cytoplasmic vesicles, and the arrowhead indicates distorted, tightly connected membranes. Arrows in panel B show capsid accumulations. Arrowheads in panel C again point to distorted membranes, whereas the arrow indicates a large cytoplasmic vesicle. Bars in panels A and B, 1.5 μm; bar in panel C, 500 nm.