Herpes simplex virus 1 ICP22 but not US 1.5 is required for efficient acute replication in mice and VICE domain formation

Abstract

The herpes simplex virus 1 (HSV-1) immediate-early protein, infected cell protein 22 (ICP22), is required for efficient replication in restrictive cells, for virus-induced chaperone-enriched (VICE) domain formation, and for normal expression of a subset of viral late proteins. Additionally, ICP22 is important for optimal acute viral replication in vivo. Previous studies have shown that the US1 gene that encodes ICP22, produces an in-frame, N-terminally truncated form of ICP22, known as US1.5. To date, studies conducted to characterize the functions of ICP22 have not separated its functions from those of US1.5. To determine the individual roles of ICP22 and US1.5, we made viral mutants that express either ICP22 with an M90A mutation in the US1.5 initiation codon (M90A) or US1.5 with three stop codons introduced upstream of the US1.5 start codon (3×stop). Our studies showed that, in contrast to M90A, 3×stop was unable to replicate efficiently in the eyes and trigeminal ganglia of mice during acute infection, to efficiently establish a latent infection, or to induce VICE domain formation and was only mildly reduced in its replication in restrictive HEL-299 cells and murine embryonic fibroblasts (MEFs). Both mutants enhanced the expression of the late viral proteins virion host shutoff (vhs) and glycoprotein C (gC) and inhibited viral gene expression mediated by HSV-1 infected cell protein 0 (ICP0). When we tested our mutants' sensitivity to type I interferon (beta interferon [IFN-β]) in restrictive cells, we noticed that the plating of the ICP22 null (d22) and 3×stop mutants was reduced by the addition of IFN-β. Overall, our data suggest that US1.5 partially complements the functions of ICP22.

Fig 1

Construction of ICP22 and U_S1.5 mutant viruses. (A) Diagram of the HSV-1 genome showing the U_S1 gene, which encodes ICP22 and U_S1.5 protein. The open boxes denote the repeated sequences flanking the unique long (U_L) and unique short (U_S) segments. The ICP22 null mutant, d22, has the lacZ gene (which codes for β-galactosidase) inserted in place of the ICP22 ORF. M90A is mutated such that only ICP22, but not U_S1.5, is produced as the initiation codon of U_S1.5 is mutated to alanine. 3×stop produces only U_S1.5 and not ICP22 as this mutant has three stop codons inserted upstream of the initiation codon of U_S1.5. (B) The restriction enzyme digestion patterns with the fragment lengths (indicated in base pairs) on the left (B, BamHI; P, PciI; S, SacII) and Southern blots shown on the right. The M90A and 3×stop mutations were designed to eliminate a PciI site and add an SacII site, respectively. Lanes 1 and 2, KOS and d22, respectively, cut with BamHI; lanes 3 and 4, KOS and M90A, respectively, cut with PciI; lanes 5 and 6, KOS and 3×stop, respectively, cut with SacII. The star in the second blot denotes a 1.9-kb BamHI fragment of the opposite U_S/R_S junction (21). (C) ICP22 and U_S1.5 protein expression of KOS (lanes 1 and 5), d22 (lanes 2 and 6), M90A (lanes 3 and 7), and 3×stop (lanes 4 and 8) mutant viruses at 8 h postinfection in HEL-299 cells or Vero cells, as determined by Western blot analyses.

Fig 2

Acute replication of M90A and 3×stop mutants and MR viruses in vivo. (A and B) Acute ocular replication of M90A and 3×stop mutants and MR viruses in mice. CD-1 mice were infected with 2 × 10⁵ PFU per eye. At the indicated time points, the eyes of mice were swabbed, and viral titers were determined by standard plaque assays. (C to F) Acute TG replication of M90A and 3×stop mutants and MR viruses in mice. CD-1 mice were infected with 2 × 10⁵ PFU per eye. On day 3 (C and D) or day 5 (E and F) postinfection, mice were sacrificed, TG were removed and homogenized, and viral titers were determined by standard plaque assays. *, P < 0.05, compared to KOS (Student's t test). Error bars represent the standard errors of the means (n = 8 samples/virus/time point). In all cases, the dashed line is the limit of detection.

Fig 3

Gross pathological effects of M90A and 3×stop mutants and MR viruses. CD-1 mice were infected with 2 × 10⁵ PFU per eye. A representative picture from each group of infected mice at 8 days postinfection is shown.

Fig 4

Establishment of latency of M90A and 3×stop mutants and MR viruses. CD-1 mice were infected with 2 × 10⁵ PFU per eye. At 28 days postinfection, mice were sacrificed, TG were removed, and genomic DNA was isolated from samples. The amount of HSV-1 DNA present in each sample was quantified by real-time PCR (n = 10 to 12 TG per group). Results shown are the fold reductions compared to KOS levels. The fold reduction for d22 was comparable to that of the mock-infected TG (≥568-fold).

Fig 5

Growth of M90A and 3×stop mutant viruses on HEL-299 cells. HEL-299 cells were infected at an MOI of 0.1 with the indicated viruses. At 24 h postinfection, cells were harvested, and viral titers were determined by a standard plaque assay. *, P < 0.05 compared to KOS (Student's t test). Error bars represent the standard errors of the means.

Fig 6

Examination of late viral gene expression for M90A and 3×stop mutant viruses. HEL-299 cells were infected at an MOI of 2 with the indicated viruses. At 24 h postinfection, cells were harvested, and protein levels from cell extracts were determined by Western blot analysis.

Fig 7

ICP22 but not U_S1.5 induces VICE domain formation. (A) HEL-299 cells were mock infected or infected with the indicated viruses at an MOI of 0.1. At 24 h postinfection, cells were fixed and stained for ICP22 and Hsc70 and examined by fluorescence microscopy. (B) At least 100 cells that expressed ICP22 and/or US1.5 were examined for each virus, and the percentages of these cells that colocalized with Hsc70 are shown.

Fig 8

Inhibition of ICP0's transactivated gene expression by vectors that express either ICP22 or U_S1.5. Vero cells were transfected with an HSV-1 reporter plasmid (50 ng; pGL3-VP16) and a plasmid expressing wild-type ICP0 (pICP0), both ICP22 and U_S1.5 (pICP22), ICP22 alone (pM90A), or U_S1.5 alone (p3×stop) or a combination of pICP0 and pICP22, pM90A, or p3×stop for 48 h. Cell extracts were analyzed in luciferase assays to monitor ICP0's transactivating activity. *, P < 0.05 compared to pICP0 (Mann-Whitney U test). The error bars indicate the standard errors of the means.

Fig 9

Plaque reduction assays. HEL-299 cells or CD-1 MEFs cells were infected with serial dilutions of the indicated viruses in the absence or the presence of 1,000 U/ml IFN-β. At 3 days postinfection, cells were fixed and immunostained for plaque formation.

Fig 10

Growth of M90A and 3×stop with and without IFN-β. HEL-299 cells (A) or CD-1 MEFs (B) were infected at an MOI of 0.1 in the presence or in the absence of 1,000 U/ml IFN-β with the indicated viruses. At 24 h postinfection, cells were collected, and the viral titers were determined by a standard plaque assay. * P < 0.05, compared to KOS (Student's t test). Error bars represent the standard errors of the means.