Persistence and expression of the herpes simplex virus genome in the absence of immediate-early proteins

Abstract

The immediate-early (IE) proteins of herpes simplex virus (HSV) function on input genomes and affect many aspects of host cell metabolism to ensure the efficient expression and regulation of the remainder of the genome and, subsequently, the production of progeny virions. Due to the many and varied effects of IE proteins on host cell metabolism, their expression is not conducive to normal cell function and viability. This presents a major impediment to the use of HSV as a vector system. In this study, we describe a series of ICP4 mutants that are defective in different subsets of the remaining IE genes. One mutant, d109, does not express any of the IE proteins and carries a green fluorescent protein (GFP) transgene under the control of the human cytomegalovirus IE promoter (HCMVIEp). d109 was nontoxic to Vero and human embryonic lung (HEL) cells at all multiplicities of infection tested and was capable of establishing persistent infections in both of these cell types. Paradoxically, the genetic manipulations that were required to eliminate toxicity and allow the genome to persist in cells for long periods of time also dramatically lowered the level of transgene expression. Efficient expression of the HCMVIEp-GFP transgene in the absence of ICP4 was dependent on the ICP0 protein. In d109-infected cells, the level of transgene expression was very low in most cells but abundant in a small subpopulation of cells. However, expression of the transgene could be induced in cells containing quiescent d109 genomes weeks after the initial infection, demonstrating the functionality of the persisting genomes.

FIG. 1

Structures of isogenic mutants defective in combinations of IE genes. (A) The viral genome is represented with the unique long (U_L) and short (U_S) regions bounded by terminal and internal repeats (white boxes). The genomic locations and directions of transcription of the IE genes are indicated (arrowheads). (B) The TAATGARAT deletion, TGTΔ (bracketed), introduced into the promoter regions of ICP22/47. The positions of the TAATGARAT elements (open arrows), binding sites for the transcription factor SP1 (arrowheads), TATA boxes (open squares), and oriS (open circle) are shown relative to the transcription start sites of ICP4 and ICP22/47. (C) Expanded map of the right end of the d109 genome, showing the coding sequences of the IE genes (solid arrows) and the deletion mutations (white bars) in ICP4 (d120), ICP0 (0Δ), and the ICP22/47 promoter (TGTΔ). The transgene cassette containing the GFP reporter gene under the control of the HCMV IE promoter substituted into the deletion in ICP27 is represented by a shaded bar, with the arrow inside indicating the direction of transcription. The relevant restriction sites are indicated and are a bbreviated as follows: H, HpaI; P, PstI; S, SacI; B, BamHI; and N, NcoI. The line of asterisks represent the nick-translated DNA fragments used as probes for Southern blot analysis. (D) Structures of the isogenic mutants. The deletion mutations in ICP4, ICP0, and the ICP22/47 promoter present in the different virus mutant strains are indicated by the shaded bars inside the white boxes representing the repeat sequences. The GFP substitution in ICP27 is also shown (inverted triangles). The mutant designations are indicated on the right along with a list of the IE proteins not synthesized by the viruses in the absence of complementation. Viral DNA isolated from cells infected with the indicated viruses were digested with HpaI (E), PstI-SacI (F), BamHI-SacI (G), or NcoI (H) and analyzed by Southern hybridization. The probes used are shown in panel C and described in Materials and Methods.

FIG. 2

Effect of the TGTΔ mutation on expression of ICP22. Total cell RNA, isolated at 6 h p.i. from Vero or E5 cells (ICP4⁺) infected (MOI = 10) with the indicated viruses, was processed for Northern blot analysis. For infections of Vero cells done in the presence of CHX, the medium was supplemented with the inhibitor 1 h prior to and during infection. The blots were hybridized to probes specific for ICP22 and ICP27 mRNAs as indicated (22 and 27, respectively). KOS is the wt virus control, and d96 (ICP4⁻ ICP22⁻) (117) carries the d120Δ deletion in ICP4 and the n199 nonsense mutation in ICP22 (93).

FIG. 3

Synthesis of viral proteins in cells infected with d109. Vero cells were infected (MOI = 20) with the indicated viruses in the presence or absence of CHX, pulsed with [³⁵S]methionine at 6 to 9 h p.i., and processed for SDS-PAGE. The CHX-treated cultures were incubated in the presence of actinomycin D during labeling. The samples were run on 9% (A) and 18% (B) SDS-polyacrylamide gels (the latter to better visualize the smaller viral proteins). The positions of the IE proteins and GFP, as well as some early and late proteins, are indicated. (C) The same samples were also transferred to a membrane for Western blot analysis with an α-GFP monoclonal antibody. Mock, uninfected cells.

FIG. 4

Accumulation of GFP mRNA and protein. (A) Total cell RNA, isolated at 6 and 24 h p.i. from Vero cells infected (MOI = 10) with the indicated viruses, was processed for Northern blot analysis with a GFP-specific probe. (B) Proteins extracted at 24 h p.i. from infected cells (MOI = 10) were separated on an SDS–18% polyacrylamide gel and transferred to a membrane for Western blot analysis with an α-GFP monoclonal antibody. Mock, uninfected cells.

FIG. 5

Survival of cells infected with IE mutants. Vero cell monolayers were infected with the IE mutant viruses at the indicated MOI. The monolayers were harvested at 6 h p.i. and plated for determination of CFU. Colonies were counted 10 days after cell plating. Points plotted represent the surviving fractions of the infected cells relative to uninfected cells.

FIG. 6

Nuclear distribution of PML in cells infected with IE mutants. Human fetal lung cells infected (MOI = 10) with the indicated viruses were processed at 6 and 24 h p.i. for immunofluorescence with an α-PML antibody. Shown are stained infected nuclei (magnification, ×100).

FIG. 7

Persistence and induction of d109. Mock-infected (Mock) and d109-infected (MOI = 20) Vero cells were maintained for a period of 28 days in 2% serum at 34°C to limit cell division. At different time points (in days) after infection (indicated on the left), the monolayers were photographed under phase-contrast and fluorescence microscopes. A separate set of d109-infected cultures was superinfected with d95 (MOI = 20) 1 day prior to the time points indicated on the left.

FIG. 8

Persistence of d109 genomes in infected cells. A separate set of d109-infected monolayers was maintained in parallel to those described in the legend to Fig. 7. At the same time points indicated, total infected cell DNA was isolated. Equivalent aliquots of the DNA samples, along with standards, were used in PCRs to amplify sequences from the gC gene (top). The amounts of total infected cell DNA (in micrograms) isolated at different time points (in days) after infection with d109 are plotted along with the number of viral genomes derived from quantitation of the gC PCR products.

FIG. 9

Absence of ICP0 and ICP4 in GFP-expressing d109-infected cells. Vero cells were infected with d109 (MOI = 10) and, at 24 h p.i., processed for immunofluorescence with α-ICP0 and α-ICP4 monoclonal antibodies. Also included were d106- and d99-infected cells as positive controls for ICP0 and ICP4 staining, respectively. The secondary antibody used was conjugated to rhodamine. Photomicrographs in each column represent the same field viewed by phase-contrast microscopy (top) or fluorescence microscopy with the appropriate filter blocks for observing green (middle) or red (bottom) fluorescence.

FIG. 10

Efficiency of plating of capsid gene mutants on d109-infected cells. Vero cell monolayers were infected with d109 (MOI = 10), and at 24 h p.i. the infected monolayers were used to determine the titers of virus mutants with lesions in capsid genes. The structure of the d109 genome is shown, along with the genomic locations of the capsid genes mutated in the viruses whose titers were measured. Virus stock titers of the mutants, determined with the appropriate complementing cell lines, were provided by S. Person. See the legend to Fig. 1 for definitions of symbols and abbreviations.