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. 2001 Nov;75(22):10856-69.
doi: 10.1128/JVI.75.22.10856-10869.2001.

Insertions in the gG gene of pseudorabies virus reduce expression of the upstream Us3 protein and inhibit cell-to-cell spread of virus infection

G L Demmin  1 , A C Clase, J A Randall, L W Enquist, B W Banfield
Affiliations

Insertions in the gG gene of pseudorabies virus reduce expression of the upstream Us3 protein and inhibit cell-to-cell spread of virus infection

G L Demmin et al. J Virol. 2001 Nov.

Abstract

The alphaherpesvirus Us4 gene encodes glycoprotein G (gG), which is conserved in most viruses of the alphaherpesvirus subfamily. In the swine pathogen pseudorabies virus (PRV), mutant viruses with internal deletions and insertions in the gG gene have shown no discernible phenotypes. We report that insertions in the gG locus of the attenuated PRV strain Bartha show reduced virulence in vivo and are defective in their ability to spread from cell to cell in a cell-type-specific manner. Similar insertions in the gG locus of the wild-type PRV strain Becker had no effect on the ability of virus infection to spread between cells. Insertions in the gG locus of the virulent NIA-3 strain gave results similar to those found with the Bartha strain. To examine the role of gG in cell-to-cell spread, a nonsense mutation in the gG signal sequence was constructed and crossed into the Bartha strain. This mutant, PRV157, failed to express gG yet had cell-to-cell spread properties indistinguishable from those of the parental Bartha strain. These data indicated that, while insertions in the gG locus result in decreased cell-to-cell spread, the phenotype was not due to loss of gG expression as first predicted. Analysis of gene expression upstream and downstream of gG revealed that expression of the upstream Us3 protein is reduced by insertion of lacZ or egfp at the gG locus. By contrast, expression of the gene immediately downstream of gG, Us6, which encodes glycoprotein gD, was not affected by insertions in gG. These data indicate that DNA insertions in gG have polar effects and suggest that the serine/threonine kinase encoded by the Us3 gene, and not gG, functions in the spread of viral infection between cells.

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Figures

FIG. 1
FIG. 1
PRV infection of embryonic chicken retinal cells. (A) Infection of primary retinal cultures. Mixed retinal cultures 24 h after infection with PRV151 and PRV152. The EGFP signal is shown on the left, and the same cells stained with Hoechst 33258 are shown on the right. (B) Optical sections midway through PRV-infected chicken embryo retinal whole mounts 48 h after intravitreal infection with PRV151 (left) or PRV152 (right). The EGFP signal was visualized by confocal fluorescence microscopy. The arrowhead indicates fused cells.
FIG. 2
FIG. 2
Plaque morphologies of PRV strains with lacZ insertions in gG on GBK and PK15 cells. Low- and high-magnification images of infected GBK cells at 48 h after a low-multiplicity infection. Cells were fixed in 4% formaldehyde for 10 min, rinsed, and incubated in an X-Gal solution to identify infected cells. (A) GBK cells. (B) PK15 cells. BeBlu, Becker-Blu; BaBlu, Bartha-Blu. Results similar to those obtained on GBK cells were observed on monolayers of MDBK cells, another bovine kidney cell line (Fig. 8).
FIG. 3
FIG. 3
Growth of Bartha and Bartha-Blu on GBK cells. (A) Plaque morphologies of Bartha and Bartha-Blu on GBK cell monolayers. Forty-eight hours after infection, cells were fixed and stained with 0.5% methylene blue in 70% methanol. Single plaques are shown. (B) Single-step growth of Bartha and Bartha-Blu on monolayers of GBK cells. Kinetics of infectious virus production in cells is plotted in the top graph, and the rate of release of infectious virus into the medium is plotted on the bottom graph.
FIG. 4
FIG. 4
A Bartha derivative with a nonsense mutation in gG is not affected in cell-to-cell spread. (A) Autoradiogram of tissue culture supernatants from infected cells pulse-labeled with [35S]methionine for 30 min at 2 h postinfection and chased in the presence of nonlabeled methionine for 1 h. Samples were analyzed by SDS-PAGE on an 8% gel. Asterisk denotes internal loading control. gG is secreted from cells in three predominant forms of approximately 180, 75, and 50 kDa. (B) Plaque morphologies of Bartha, PRV156, PRV157, and PRV156R on GBK cell monolayers. Forty-eight hours after infection, cells were fixed and stained with 0.5% methylene blue in 70% methanol.
FIG. 5
FIG. 5
Expected transcripts from Us3, gG (Us4), and gD (Us6) in Becker or Bartha (A), viruses with lacZ insertions (B), and viruses with egfp insertions (C). Horizontal arrows denote the position of a promoter. The Us3/Us4 transcripts are a 3′nested family of three mRNAs. The Us3 gene encodes two transcripts, designated Us3a and Us3b, that both terminate at a polyadenylation site located downstream of the gG (Us4) open reading frame. The slightly larger (by ∼150 bases) Us3a transcript represents about 5% of the total Us3 transcription, and the smaller Us3b transcript represents about 95% (83). In PRV, these mRNAs encode two different proteins using the same reading frame. It follows that both proteins have identical carboxy termini. The larger Us3a transcript encodes a protein with an additional 54 N-terminal amino acids. Both of these proteins are expressed in PRV-infected cells, as shown by Western blot analysis (83). It is unclear if the two products of the Us3 gene have different functions. The mRNA that directs the translation of gG is initiated just upstream of the gG (Us4) open reading frame and terminates at a polyadenylation site located immediately downstream of the gG (Us4) open reading frame. Located immediately downstream of gG (Us4) is the essential gD (Us6) gene. gD (Us6) belongs to a family of transcripts different from that of Us3 and gG. SV40, simian virus 40; CMV IE, cytomegalovirus immediate-early promoter.
FIG. 6
FIG. 6
Analysis of Us3 transcription in PRV strains containing lacZ insertions in gG. (A) Cartoon illustrating the predicted size of Us3 transcripts from parental and lacZ insertion viruses and the location of the double-stranded DNA probe used to detect Us3 RNA by Northern blotting. Only the Us3 transcript is shown. (B) Northern blot of total RNA isolated from MDBK cells infected with Becker, Bartha, Becker-Blu (BeBlu), or Bartha-Blu (BaBlu) at 3 and 6 h postinfection (top panel). The blot was hybridized to a biotinylated double-stranded DNA probe corresponding to the Us3b open reading frame and hybridizing RNAs detected, as described in Materials and Methods. The positions of RNA size markers (in kilobases) are shown on the left of the gel. The bottom panel shows the ethidium bromide-stained gel prior to transfer of RNA to the blot and is meant to serve as a loading control. hpi, hours postinfection.
FIG. 7
FIG. 7
Reduced Us3 expression in cells infected with PRV strains that have insertions in gG. (A) Western blot of proteins extracted from MDBK cells (Mock) or MDBK cells infected with Bartha (Ba) or Bartha-Blu (BaBlu) probed with rabbit polyclonal antisera to PRV gD. (B) Western blot of proteins extracted from MDBK cells (Mock) or MDBK cells infected with Bartha (Ba) or Bartha-Blu (BaBlu) probed with goat polyclonal antisera to PRV gB. (C) Western blot of proteins extracted from MDBK cells (Mock) or MDBK cells infected with Bartha (Ba), Bartha-Blu (BaBlu), PRV152, Becker (Be), Becker-Blu (BeBlu), or PRV151, probed with an affinity-purified rabbit polyclonal antiserum raised against a synthetic peptide corresponding to the carboxy terminus of Us3. All infected cell extracts were prepared at 6 h postinfection. The positions of protein size markers (in kilodaltons) are shown to the left of the gels.
FIG. 8
FIG. 8
Plaque morphologies of PRV strains with insertions in gG in the presence or absence of gE and gI. Forty-eight hours after infection with the indicated virus strain, MDBK cells were either fixed and stained with 0.5% methylene blue in 70% methanol or examined by fluorescence microscopy for EGFP expression. (A) egfp insertions in gG (Us4) in the presence of gE and gI. (B) egfp insertions in gG (Us4) in the absence of gE and/or gI.
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