Functional analysis of the pseudorabies virus UL51 protein

Abstract

Homologs of the UL51 protein of herpes simplex virus have been identified in all herpesvirus subfamilies, but until now, no function has been assigned to any of them. To investigate function of the UL51 gene product of the alphaherpesvirus pseudorabies virus (PrV), we isolated and analyzed a mutant lacking the major part of the open reading frame, PrV-DeltaUL51F, and a rescuant. One-step growth analysis of PrV-DeltaUL51F revealed only slightly reduced titers, but plaque size was notably diminished and reached only approximately 30% the plaque size of wild-type PrV. Ultrastructurally, intracytoplasmic capsids were found in large numbers either without envelope or in different stages of envelopment, indicating that secondary envelopment in the cytoplasm was less efficient. However, neuroinvasion in the mouse trigeminal pathway after intranasal infection was only slightly delayed. A PrV UL11 mutant also showed a defect in secondary envelopment (M. Kopp, H. Granzow, W. Fuchs, B. G. Klupp, E. Mundt, A. Karger, and T. C. Mettenleiter, J. Virol. 77:5339-5351, 2003). Since both proteins are part of the viral tegument and are predicted to be membrane associated, they may serve similar, possibly redundant functions during viral morphogenesis. Therefore, we also isolated a mutant simultaneously lacking UL51 and UL11. This mutant exhibited further reduced plaque size compared to the single-deletion mutants, but viral titers were comparable to those for the UL11 mutant. In electron microscopic analyses, the observed defect in secondary envelopment was similar to that found in the UL11 single-deletion mutant. In conclusion, both conserved tegument proteins, either singly or in combination, are involved in virion morphogenesis in the cytoplasm but are not essential for viral replication in vitro and in vivo.

FIG. 1.

Construction of virus mutants. (A) A schematic map of the PrV genome showing the unique long (U_L) and unique short (U_S) regions, the inverted repeat sequences (IR, TR), and the positions of the BamHI restriction fragments which are numbered according to their size. (B) Enlargement of the UL51 gene region. The UL51 gene is transcribed into a unique mRNA antiparallel to the neighboring genes UL50, coding for dUTPase, and UL52, encoding the putative primase subunit of the primase/helicase complex (6). A region of reiterated sequences located between ORF-1 and UL54 is shown by a hatched box. Relevant restriction sites are indicated. (C) Construction of PrV-ΔUL51F. Deletion of UL51-specific sequences between the StuI sites is indicated. Locations of the polyadenylation signals are shown by hollow arrows (pointing downward for the UL50 gene and pointing upward for the UL51 gene). (D) Amino-terminal (UL51N) and carboxy-terminal (UL51C) regions used for the generation of specific antisera.

FIG. 2.

Subcellular localization of PrV UL51. (A) RK13 cells were infected with PrV-Ka or PrV-ΔUL51F, fixed 16 h p.i., and incubated with UL51-specific antisera (α-UL51N, α-UL51C) or monoclonal antibodies against gB (A20-c26) or gC (B16-c8). Fluorescence of Alexa 488-conjugated anti-rabbit or anti-mouse antibodies (green) and of propidium iodide-stained chromatin (red) was analyzed in a confocal laser scan microscope. (B and C) RK13 cells were infected with PrV-Ka or transfected with plasmid pcDNA-UL51 and then incubated with the anti-UL51 serum (green fluorescence) and a monoclonal antibody against γ-Adaptin (panel B, red fluorescence) or an unspecified Golgi marker (panel C, red fluorescence) (16). Columns labeled “merge” depict the merger of the green and red fluorescence.

FIG. 3.

Immunoblots of mutant viruses. Purified virions (left panels) or lysates of RK13 cells (right panels) infected with PrV-Ka, PrV-ΔUL51F, PrV-ΔUL51FR, or PrV-ΔUL51F/11G were separated by electrophoresis in sodium dodecyl sulfate gels containing 10% (for detection of gH, UL37, and UL49 proteins) or 15% (for detection of the UL51 and UL11 proteins) polyacrylamide. After transfer to nitrocellulose filters, blots were probed with monospecific sera against UL51N, UL51C, UL11, UL37, UL49, and gH. Locations of molecular mass markers are indicated to the left of each panel.

FIG. 4.

Plaque size of mutant viruses on noncomplementing and complementing cells. RK13, RK15-UL51, or RK13-UL11 cells were infected with the indicated viruses under plaque assay conditions. Plaques were microscopically measured 2 days p.i. Relative plaque sizes were calculated and compared to those of PrV-Ka which were set as 100%. Average values and standard deviations from at least two independent experiments are shown.

FIG. 5.

One-step growth analysis. RK13, RK13-UL51, or RK13-UL11 cells were infected with PrV-Ka or the respective mutants, harvested at the indicated times after infection, and titrated. Average values and standard deviations from three independent experiments are shown.

FIG. 6.

Transmission electron microscopy of infected cells. RK13 (A through C), RK13-UL11 (D), or RK13-UL51 (E) cells were infected with PrV-Ka (A), PrV-ΔUL51F (B), or PrV-ΔUL51F/11G (C through E) at an MOI of 1 and fixed 14 h p.i. Arrows point to intracytoplasmic nucleocapsids, closed triangles point to nucleocapsids undergoing secondary envelopment, and open triangles point to enveloped virions. Open arrows indicate L-particles. In PrV-ΔUL51F/11G-infected RK13 or RK13-UL51 cells, intracytoplasmic nucleocapsids were sometimes observed in close contact with tegument and distorted intracytoplasmic membranes (panels C and E, double-shafted arrows; also panel C, inset) as previously described for a PrV UL11 deletion mutant (23, 24), whereas infected RK13-UL11 cells exhibited an increased number of nucleocapsids in the process of secondary envelopment as observed in RK13 cells infected by PrV-ΔUL51F. Bars, 500 nm (A, B, C inset, D, and E) and 2 μm (C).