The UL7 gene of pseudorabies virus encodes a nonessential structural protein which is involved in virion formation and egress

Abstract

Homologues of the UL7 gene of herpes simplex virus type 1 are conserved in alpha-, beta-, and gammaherpesviruses. However, little is known about their functions. Using a monospecific rabbit antiserum raised against a bacterial fusion protein, we identified the UL7 gene product of the neurotropic alphaherpesvirus pseudorabies virus (PrV). In Western blot analyses of infected cells and purified PrV particles the serum specifically detected a 29-kDa protein, which matches the calculated mass of the 266-amino-acid translation product of PrV UL7. For functional analysis, UL7 was deleted by mutagenesis of an infectious full-length clone of the PrV genome in Escherichia coli. The obtained recombinant PrV-DeltaUL7F was replication competent in rabbit kidney cells, but maximum virus titers were decreased nearly 10-fold and plaque diameters were reduced by ca. 60% compared to wild-type PrV. Electron microscopy of infected cells revealed that in the absence of UL7, formation and nuclear egress of nucleocapsids were not affected, whereas secondary envelopment of cytoplasmic nucleocapsids appeared to be delayed and release of mature virions was less efficient. The observed replication defects were corrected by repair of the viral UL7 gene or by propagation of PrV-DeltaUL7F in UL7-expressing cells. PrV-DeltaUL7F was moderately attenuated in mice. Compared to wild-type virus, mean survival times were prolonged from 2 to 3 days after intranasal infection. However, neuroinvasion and transneuronal spread of PrV were not abolished in the absence of UL7. Thus, UL7 encodes a virion protein of PrV, which plays a role during virion maturation and egress both in vitro and in vivo.

FIG. 1.

Construction of plasmids and PrV recombinants. (A) The PrV genome consists of two unique regions (U_L and U_S) and of inverted repeat sequences (IR_S and TR_S) flanking the U_S region. The positions of BamHI restriction sites are indicated. An enlarged section shows the analyzed genome part which was cloned in pBl-B3N. Open reading frames (ORFs) are drawn as pointed rectangles. (B) In plasmid pBl-ΔUL7KF the UL7 ORF was replaced by a kanamycin resistance gene (KanR) flanked by Flp-recombinase target sites (FRT). The insert of pBl-ΔUL7KF was amplified by PCR and used for mutagenesis of an infectious full-length clone of the PrV-genome in E. coli. The resistance gene was removed by Flp-mediated recombination, and PrV-ΔUL7F was isolated after transfection of rabbit kidney cells. The UL7 rescuant PrV-UL7R was obtained after transfection of cells with genomic DNA of PrV-ΔUL7F and pBl-B3N. (C) Plasmid pcDNA-UL7 permits constitutive expression of UL7 in eucaryotic cells under control of the HCMV immediate-early promoter (P_HCMV-IE) and was used for generation of the trans-complementing cell line RK13-UL7. The bacterial UL7 fusion protein with glutathione S-transferase (GST) expressed from pGEX-UL7 was used for rabbit immunization followed by antiserum preparation. Codon ranges of expressed or retained UL7 gene fragments as well as relevant restriction sites are indicated (artificial sites in parentheses). aa, amino acids.

FIG. 2.

Expression kinetics and virion incorporation of the PrV UL7 protein. Rabbit kidney cells were infected with PrV-Ka at a multiplicity of infection of 5 and incubated at 37°C for 0 to 18 h. Lysates of infected and noninfected cells (N) and purified virions (V) were separated by SDS-PAGE. Western blots were incubated with monospecific antisera against the UL7 gene product (A), the nonstructural UL34 protein (B), or envelope glycoprotein gH (C). Molecular masses of marker proteins are indicated.

FIG. 3.

Protein expression of UL7 mutants. Western blots of rabbit kidney cells harvested 24 h after infection (multiplicity of infection, 5) with PrV-Ka, PrV-ΔUL7F, or PrV-UL7R of noninfected cells (RK13) and of RK13-UL7 cells were incubated with monospecific antisera against the UL7 protein (A), the UL34 protein (B), or gH (C). Molecular masses of marker proteins are indicated.

FIG. 4.

Plaque sizes of UL7 mutants. RK13 (A) or RK13-UL7 (B) cells were infected with PrV-Ka, PrV-ΔUL7F, or PrV-UL7R and incubated for 48 h under semisolid medium. For each virus the average diameters of 30 plaques were determined and calculated in percentages compared to the plaques induced by PrV-Ka. Standard deviations are also shown.

FIG. 5.

One-step growth kinetics of UL7 mutants. RK13 (A and C) or RK13-UL7 (B) cells were infected with PrV-Ka, PrV-ΔUL7F, or PrV-UL7R at a multiplicity of infection of 5 and incubated at 37°C for 1, 4, 8, 12, 24, 36, and 48 h. Cells and medium were then harvested either together (A and B) or separately (C). After freeze-thawing, progeny virus titers (PFU/ml) in whole-cell lysates or cell pellets (P) and supernatants (S) were determined by plaque assays in RK13-UL7 cells. Crossover points of intra- and extracellular virus titers are indicated by arrowheads. The mean results of at least three independent experiments are shown.

FIG.6.

Egress of PrV-ΔUL7F. RK13 cells were fixed 14 h after infection with PrV-ΔUL7F (A, B, and C) or PrV-UL7R (D and E) at a multiplicity of infection of 1 and stained with uranyl acetate. Ultrathin sections were analyzed by electron microscopy. In the absence of UL7, nucleocapsids were formed and released from the host cell nucleus (A). However, secondary envelopment of cytoplasmic nucleocapsids appeared to be delayed (B and C), and only a few mature virus particles were detectable in the extracellular space (A and C). In contrast, virions of PrV-UL7R were efficiently released from cells (D and E). Bars represent 2.0 mm (A and D) or 1.0 mm (B, C, and E).