Characterization of pseudorabies virus (PrV) cleavage-encapsidation proteins and functional complementation of PrV pUL32 by the homologous protein of herpes simplex virus type 1

Abstract

Cleavage and encapsidation of newly replicated herpes simplex virus type 1 (HSV-1) DNA requires several essential viral gene products that are conserved in sequence within the Herpesviridae. However, conservation of function has not been analyzed in greater detail. For functional characterization of the UL6, UL15, UL28, UL32, and UL33 gene products of pseudorabies virus (PrV), the respective deletion mutants were generated by mutagenesis of the virus genome cloned as a bacterial artificial chromosome (BAC) in Escherichia coli and propagated in transgenic rabbit kidney cells lines expressing the deleted genes. Neither of the PrV mutants was able to produce plaques or infectious progeny in noncomplementing cells. DNA analyses revealed that the viral genomes were replicated but not cleaved into monomers. By electron microscopy, only scaffold-containing immature but not DNA-containing mature capsids were detected in the nuclei of noncomplementing cells infected with either of the mutants. Remarkably, primary envelopment of empty capsids at the nuclear membrane occasionally occurred, and enveloped tegument-containing light particles were formed in the cytoplasm and released into the extracellular space. Immunofluorescence analyses with monospecific antisera of cells transfected with the respective expression plasmids indicated that pUL6, pUL15, and pUL32 were able to enter the nucleus. In contrast, pUL28 and pUL33 were predominantly found in the cytoplasm. Only pUL6 could be unequivocally identified and localized in PrV-infected cells and in purified virions, whereas the low abundance or immunogenicity of the other proteins hampered similar studies. Yeast two-hybrid analyses revealed physical interactions between the PrV pUL15, pUL28, and pUL33 proteins, indicating that, as in HSV-1, a tripartite protein complex might catalyze cleavage and encapsidation of viral DNA. Whereas the pUL6 protein is supposed to form the portal for DNA entry into the capsid, the precise role of the UL32 gene product during this process remains to be elucidated. Interestingly, the defect of UL32-negative PrV could be completely corrected in trans by the homologous protein of HSV-1, demonstrating similar functions. However, trans-complementation of UL32-negative HSV-1 by the PrV protein was not observed.

FIG. 1.

Plasmids and virus mutants. (A) Maps of the PrV and HSV-1 genomes containing long (U_L) and short (U_S) unique regions, which are partly flanked by inverted-repeat sequences (IR_L, TR_L, IR_S, and TR_S). The investigated virus genes and BamHI fragments of PrV DNA are indicated. Shown below are the plasmids used for characterization of UL6 (B), UL15 (C), UL28 (D), UL32 (E), and UL33 (F) of PrV or of UL32 of HSV-1 (G). ORFs are shown as pointed rectangles, and relevant restriction sites, as well as expressed amino acids (aa) or retained codon ranges, are indicated. For constitutive expression in mammalian cells, the ORFs were cloned between the HCMV immediate-early promoter (P-HCMV) and a polyadenylation signal (pA). In pcDNA-PUL15C, a genomic BmgBI/BamHI fragment was replaced by a cDNA fragment (hatched). Plasmids permitting the expression of LexA fusion proteins in yeast were used for two-hybrid studies, and rabbits were immunized with bacterial GST fusion proteins to obtain monospecific antisera. For mutagenesis of the cloned virus genomes in E. coli, parts of the investigated genes were replaced by a kanamycin resistance (KanR) gene flanked by FRT sites.

FIG. 2.

Indirect immunofluorescence reactions of antisera. RK13 cells transfected with pcDNA3 expression plasmids for the indicated PrV proteins were fixed with methanol and acetone (1:1) after 24 h. Binding of the corresponding monospecific antisera was detected by Alexa Fluor 488-conjugated secondary antibodies (488 nm; green), and nuclear chromatin was stained with propidium iodide (543 nm; red). Fluorescence was analyzed by confocal laser scanning microscopy. Bars, 10 μm.

FIG. 3.

One-step growth kinetics of PrV mutants. RK13 cells (continuous lines) or trans-complementing cells (dotted lines) were infected with PrV-Ka (circles) and the deletion mutant (triangles) PrV-ΔUL6F (A), PrV-ΔUL15F (B), PrV-ΔUL28F (C), PrV-ΔUL32F (D), or PrV-ΔUL33F (E) at an MOI of 5, harvested together with the supernatant at the indicated times, and lysed by freeze-thawing. Progeny virus titers were determined by plaque assays on RK13-PUL6 (A), RK13-PUL15 (B), RK13-PUL28 (C), RK13-PUL32 (D), or RK13-PUL33 (E) cells. The mean progeny virus titers (log PFU/ml) of four experiments are shown.

FIG. 4.

DNA replication of PrV mutants. RK13 cells were infected with PrV-ΔUL6F, PrV-ΔUL15F, PrV-ΔUL28F, PrV-ΔUL32F, PrV-ΔUL33F, or wild-type PrV-Ka at an MOI of 5, and total DNA was prepared 24 h p.i. After BamHI cleavage Southern blots were hybridized with the labeled terminal BamHI fragment 13 of the PrV genome, which permits differentiation of packaged unit size DNA (B 13) and the concatemeric primary replication products (B 13+14′). A fragment (B 8′) from the internal inverted-repeat sequences was also detected by the probe (Fig. 1A). The sizes of marker DNAs are indicated.

FIG. 5.

Electron microscopy of cells infected with PrV-ΔUL15F. RK13 (A) or RK13-PUL15 (B) cells were fixed 14 h p.i. at an MOI of 1, and uranyl-acetate-stained ultrathin sections were analyzed. The inset in panel A shows numerous scaffold-containing B capsids in the nuclei of infected cells. Bars, 3 μm or 250 nm (inset in panel A).

FIG. 6.

Electron microscopy of cells infected with PrV-ΔUL32F. RK13 (A and B) or RK13-PUL32 (C) cells were fixed 14 h p.i. at an MOI of 1, and uranyl-acetate-stained ultrathin sections were analyzed. Bars, 3 μm (A and C) or 250 nm (B).

FIG. 7.

Physical interactions of DNA cleavage-encapsidation proteins of PrV. (A) DNA-binding fusion proteins were expressed from pLex-PUL6, pLex-PUL15, pLex-PUL28, pLex-PUL32, and pLex-PUL33 in transformed yeast cells and used as baits for two-hybrid screening of an expression library of PrV gene products cloned in pB42AD, which provides a trans-activation domain. The minimum amino acid ranges of reproducibly detected viral prey proteins and the numbers of positive clones are indicated. (B) Induction of β-galactosidase reporter gene expression by the fusion protein complexes expressed by pLex-UL15 and pB42-PUL28 or pLex-PUL28 and pB42-PUL33 was quantified and compared to positive (pLexA pos) and negative (empty expression vectors pLexA and pB42AD) controls. The mean results of three experiments and standard deviations are shown.

FIG. 8.

trans-Complementation of UL32-negative PrV by the homologous HSV-1 protein. One-step growth kinetics were determined after infection of RK13 (A) or RK13-HUL32 (B) cells with PrV-Ka, PrV-ΔUL32F, pHSV-1ΔgJ, or pHSV-1ΔUL32KF at an MOI of 5. At the indicated times, progeny virus titers were determined by plaque assays on RK13-HUL32 cells. Shown are the mean results of three experiments. (C) Cell-to-cell spread was investigated 48 h after infection of RK13 or RK13-HUL32 cells at low MOI. The average diameters of 30 plaques each were calculated, and the plaque sizes of PrV-ΔUL32F and pHSV-1ΔUL32 were displayed as percentages of those of PrV-Ka and pHSV-1ΔgJ on the same cell line, which were set at 100%.