The US11 Gene of Herpes Simplex Virus 1 Promotes Neuroinvasion and Periocular Replication following Corneal Infection

Abstract

Herpes simplex virus 1 (HSV-1) cycles between phases of latency in sensory neurons and replication in mucosal sites. HSV-1 encodes two key proteins that antagonize the shutdown of host translation, US11 through preventing PKR activation and ICP34.5 through mediating dephosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). While profound attenuation of ICP34.5 deletion mutants has been repeatedly demonstrated, a role for US11 in HSV-1 pathogenesis remains unclear. We therefore generated an HSV-1 strain 17 US11-null virus and examined its properties in vitro and in vivo In U373 glioblastoma cells, US11 cooperated with ICP34.5 to prevent eIF2α phosphorylation late in infection. However, the effect was muted in human corneal epithelial cells (HCLEs), which did not accumulate phosphorylated eIF2α unless both US11 and ICP34.5 were absent. Low levels of phosphorylated eIF2α correlated with continued protein synthesis and with the ability of virus lacking US11 to overcome antiviral immunity in HCLE and U373 cells. Neurovirulence following intracerebral inoculation of mice was not affected by the deletion of US11. In contrast, the time to endpoint criteria following corneal infection was greater for the US11-null virus than for the wild-type virus. Replication in trigeminal ganglia and periocular tissue was promoted by US11, as was periocular disease. The establishment of latency and the frequency of virus reactivation from trigeminal ganglia were unaffected by US11 deletion, although emergence of the US11-null virus occurred with slowed kinetics. Considered together, the data indicate that US11 facilitates the countering of antiviral response of infected cells and promotes the efficient emergence of virus following reactivation.IMPORTANCE Alphaherpesviruses are ubiquitous DNA viruses and include the human pathogens herpes simplex virus 1 (HSV-1) and HSV-2 and are significant causes of ulcerative mucosal sores, infectious blindness, encephalitis, and devastating neonatal disease. Successful primary infection and persistent coexistence with host immune defenses are dependent on the ability of these viruses to counter the antiviral response. HSV-1 and HSV-2 and other primate viruses within the Simplexvirus genus encode US11, an immune antagonist that promotes virus production by preventing shutdown of protein translation. Here we investigated the impact of US11 deletion on HSV-1 growth in vitro and pathogenesis in vivo This work supports a role for US11 in pathogenesis and emergence from latency, elucidating immunomodulation by this medically important cohort of viruses.

Keywords: herpes simplex virus; innate immunity; pathogenesis.

FIG 1

Construction of strain 17 Us11 viruses. (A) Genomic map of Us11 and modification strategy. At top is a representation of the HSV-1 genome, indicating the long and short terminal repeats (TR_L and TR_S, respectively), long and short internal repeats (IR_L and IR_S, respectively), and unique long and short (U_L and U_S, respectively) regions. At the bottom, a magnified U_S-TR_S region shows the transcripts (bold black arrows), promoters (bent green arrows), and ORFs (hollow boxes) for the indicated U_S genes. An SphI restriction site at the beginning of the Us11 ORF served as the insertion site of a BglI linker, placing stop codons (in red) in all 3 reading frames. The introduced BglI site is underlined and the linker sequence is shown in uppercase letters. (B) Southern blot analysis of viral genomic DNA digested with SphI and hybridized to a radiolabeled probe derived from pSW1, containing the US11 coding sequence. The presence of the SphI site in strain 17 and 17Δ11R produced bands of 3,659 and 1,337 bp, while the 17Δ11 virus lacking the SphI site has a single 5,015-bp band. M, molecular mass markers.

FIG 2

Suppression of protein translation by Us11 is cell and virus strain dependent. (A) Immunoblot analyses of eIF2α phosphorylation following infection of HCLEs (left panels) or U373 cells (right panels) with the indicated viruses for 18 h. Detection antibodies are indicated between the upper panels. In the right panel, the red arrow denotes P-eIF2α and the asterisk marks a nonspecific protein. The infected/mock-infected ratio of phospho-eIF2α is shown below the blots for each lane. (B) Autoradiographs showing incorporation of [³⁵S]amino acids into total protein in HCLE and U373 cells during 1 h of pulse-labeling at 18 hpi with the indicated viruses. Molecular mass markers are at the far right. Red asterisks denote Us11. ³⁵S inc., quantitation of the level of ³⁵S incorporated into nascent proteins from the average of results from three replicates. This is shown as a ratio to infection with the wild-type parental virus. (C) Immunoblot analyses of ICP34.5 and US11 accumulation in U373 cells infected with either strain 17 or Patton HSV-1, as denoted above the panels. The time of infection (or mock infection) is shown above each lane. (D) The kinetics of gene expression of ICP34.5 and US11 from strain 17 or Patton were measured by densitometric analysis of a representative immunoblot (depicted in panel C). Data are displayed as the percentage of expression at each time point relative to that of maximal expression of that protein during the time course.

FIG 3

Us11 is not required for growth in vitro. HCLEs, either left untreated or pretreated with 30 IU/ml IFN-β (A), or U373s (B) were infected with the indicated viruses at MOI = 0.001. At 1 h postadsorption, viral inocula were replaced with media and wells (media and cells) harvested at the indicated time points and titers were determined on Vero cells. Data points represent means ± standard deviations (SD) of results from experiments performed three times. P = <0.01 to 0.0001 (two-way ANOVA) where shown. LOD, limit of detection.

FIG 4

Us11 contributes to virulence. (A) Wild-type 129SvEv mice were injected intracranially with 100 PFU of strain 17, strain 17Δ11, or strain 17Δ34.5 and euthanized when endpoint criteria were met. (B and C) Wild-type 129SvEv mice were subjected to corneal infection following scarification. Inoculation of either 2 × 10⁶ PFU/eye (B) or 10⁴ PFU/eye (C) of the indicated viruses was performed, and mice were euthanized when endpoint criteria were met. P values determined using the log rank (Mantel-Cox) test and numbers of animals per group are as indicated.

FIG 5

Viral replication in mouse tissues. Corneas of wild-type 129SvEv mice were scarified and infected with 2 × 10⁶ PFU/eye of strain 17, strain 17Δ11, or strain 17Δ11R. (A) At 24 and 48 hpi, eyes were swabbed and titers of virus in tear film were determined. The number of animals per group is indicated in the key. (B and C) Mice were sacrificed on days 3 and 5, and titers of virus were determined in trigeminal ganglia (B) and periocular tissue (C). Numbers within bars indicate the total number of animals in each group. Error bars show the standard deviations. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-way ANOVA and Bonferroni posttests).

FIG 6

Us11 promotes periocular pathogenesis. Corneas of wild-type 129SvEv and C57BL/6J mice were scarified and infected with 2 × 10⁴ PFU/eye (129SvEv) or 2 × 10⁶ PFU/eye (C57BL/6J) of strain 17, strain 17Δ11, or strain 17Δ11R. Mice were scored for disease daily from disease onset until resolution of periocular disease by an observer in a blind fashion. (A) Using the scoring metrics in the table (right), the mean numerical disease score on the day of maximal disease is shown for each virus/host strain combination (left). The number of animals per group is shown inside each bar. **, P < 0.01; ***, P < 0.001 (one-way ANOVA and Bonferroni posttests). (B and C) Data from the same experiment are shown as percentages of total days following infection in which the mean disease score equaled or exceeded 0.85 (129SvEv [SvEv] mice) (B) or 0.25 (C57BL/6J [Bl6] mice) (C).

FIG 7

Establishment of and reactivation from latency. Trigeminal ganglia were collected from wild-type 129SvEv and C57BL/6J mice 28 dpi with 2 × 10⁴ PFU/eye of strain 17, strain 17Δ11, or strain 17Δ11R. (A) DNA extracted from latently infected 129SvEv TGs was analyzed for viral thymidine kinase copy number by qRT-PCR. Data were normalized by comparison to data from a single-copy mouse adipsin gene. Data are expressed as numbers of genome copies per individual TG. (B) 129SvEv and C57BL/6J TG explants were plated with indicator Vero cells for 24 h during the interval 48 to 72 hpe. Wells were scored as CPE⁺ or CPE⁻ at 48, 72, and 96, and 120 h. Data shown represent levels of CPE⁺ wells as a percentage of total wells (shown in legend) within each infection cohort. *, P < 0.05 (two-way ANOVA and Bonferroni posttests). (C) At 72 hpe, TG from 129SvEv (SvEv) and C57BL/6J (Bl6) mice were homogenized and titers in the homogenate were determined on Vero cells. In panels A and C, bars indicate the mean titers, which were not significantly different (P > 0.05 [one-way ANOVA]).