Partial functional complementation of a pseudorabies virus UL25 deletion mutant by herpes simplex virus type 1 pUL25 indicates overlapping functions of alphaherpesvirus pUL25 proteins

Abstract

Homologs of the UL25 gene product of herpes simplex virus 1 (HSV-1) are highly conserved among the Herpesviridae. However, their exact function during viral replication is unknown. Current evidence suggests that in the alphaherpesvirus pseudorabies virus (PrV) the capsid-associated pUL25 plays a role in primary envelopment of DNA-containing mature capsids at the inner nuclear membrane. In the absence of pUL25, capsids were found in close association with the inner nuclear membrane, but nuclear egress was not observed (B. G. Klupp, H. Granzow, G. M. Keil, and T. C. Mettenleiter, J. Virol. 80:6235-6246, 2006). In contrast, HSV-1 pUL25 has been assigned a role in stable packaging of viral genomes (N. Stow, J. Virol. 75:10755-10765, 2001). Despite these apparently divergent functions, we wanted to assess whether the high sequence homology translates into functional homology. Therefore, we first analyzed a newly constructed HSV-1 UL25 deletion mutant in our assay system and observed a similar phenotype as in PrV. In the nuclei of infected cells, numerous electron-dense C capsids were detected, whereas primary envelopment of these capsids did not ensue. In agreement with results from PrV, vesicles were observed in the perinuclear space. Since these data indicated functional homology, we analyzed the ability of pUL25 of HSV-1 to complement a PrV UL25 deletion mutant and vice versa. Whereas a HSV-1 pUL25-expressing cell line partially complemented the pUL25 defect in PrV, reciprocal complementation of a HSV-1 UL25 deletion mutant by PrV pUL25 was not observed. Thus, our data demonstrate overlapping, although not identical functions of these two conserved herpesvirus proteins, and point to a conserved functional role in herpes virion formation.

FIG. 1.

Construction of HSV1-ΔUL25. (A) Map of the HSV-1 genome with the unique long (U_L), unique short (U_S), and inverted repeat (TR_L, TR_S, IR_L, and IR_S) sequences. Arbitrary map units are indicated. (B) Enlargement of the HSV-1 UL25 gene region. The open reading frames for UL24, UL25, UL26, and UL26.5 genes are shown as pointed rectangles. Relevant restriction sites for construction of HSV1-ΔUL25 are indicated, as is the extent of the introduced deletion. (C) The complete UL25 open reading frame was used for prokaryotic expression as GST-UL25(HSV-1).

FIG. 2.

Western blot analyses. (A and B) Lysates of cells infected for 24 h with PrV-Ka (lanes 2), PrV-ΔUL25 (lanes 3), HSV-1 KOS (lanes 4), or HSV1-ΔUL25 (lanes 5) or mock-infected RK13 cells (lanes 1) were separated on SDS-10% polyacrylamide gels. After electrotransfer onto nitrocellulose membranes, parallel blots were incubated with monospecific antisera against pUL25(PrV) (A) or pUL25(HSV-1) (B). (C) Purified virions of HSV-1 KOS (lane 1), HSV1-ΔUL25 propagated on RK13-UL25(HSV-1) cells (lane 3) and PrV-ΔUL25 propagated on RK13-UL25(HSV-1) cells (lane 4) were analyzed by immunoblotting with antiserum to HSV-1 pUL25. As a control, supernatant of HSV1-ΔUL25-infected RK13 cells was processed identically (lane 2). Molecular masses of marker proteins are indicated on the left.

FIG. 3.

One-step growth analysis. RK13, RK13-UL25(PRV), and RK13-UL25(HSV-1) cells were infected at an MOI of 5 with HSV1-KOS or HSV1-ΔUL25 (A) or with PrV-Ka or PrV-ΔUL25 (B), harvested at the indicated times after infection, and titrated on either RK13-UL25(HSV-1) (A) or RK13-UL25(PrV) (B) cells. Average titers (PFU/ml) and standard deviations from three independent experiments are shown.

FIG. 4.

Plaque formation of PrV and HSV UL25 deletion mutants. RK13, RK13-UL25(PrV), and RK13-UL25(HSV-1) cells were infected with PrV-Ka, PrV-ΔUL25, HSV1-KOS, and HSV1-ΔUL25 under plaque assay conditions and fixed 2 days (for PrV) or 4 days (for HSV-1) after infection. Infected cells were visualized by indirect immunofluorescence with a PrV gC-specific monoclonal antibody. (B) Calculation of plaque diameters. Plaques on RK13, RK13-UL25(PrV), or RK13-UL25(HSV-1) cells infected with PrV-Ka or PrV-ΔUL25 were microscopically measured at 2 days postinfection. Relative plaque sizes were calculated and compared to those of PrV-Ka, which were set as 100%. Average values and standard deviations from three independent experiments are shown.

FIG. 5.

Ultrastructural analysis of PrV-ΔUL25. RK13 cells were infected at an MOI of 1 with transcomplemented PrV-ΔUL25 and analyzed 14 h after infection. (A) Overview of an infected cell; (B) higher-magnification view of intranuclear capsids. Scale bars: A, 2 μm; B, 500 nm.

FIG. 6.

Ultrastructural analysis of HSV1-ΔUL25. RK13 cells were infected at an MOI of 1 with transcomplemented HSV1-ΔUL25 and analyzed 20 h after infection. (A) Overview of an infected cell; (B) higher-magnification view of intranuclear capsids; (C) vesicles within the perinuclear cleft. Scale bars: A, 1.5 μm; B, 500 nm; C, 500 nm.

FIG. 7.

Ultrastructural analysis of PrV-ΔUL25 deleted on HSV-1 pUL25-expressing cells. RK13-UL25(HSV-1) cells were infected at an MOI of 1 with transcomplemented PrV-ΔUL25 and analyzed 14 h after infection. (A) Overview of an infected cell; (B) primary enveloped virion; (C) secondary envelopment in the cytosol; (D) extracellular L-particles and an enveloped virion. Scale bars: A, 3 μm; B, 300 nm; C, 1 μm; D, 500 nm. Virions in panels C and D are marked by asterisks.