Conceived and designed the experiments: IJM TM. Performed the experiments: IJM. Analyzed the data: IJM. Contributed reagents/materials/analysis tools: IJM. Wrote the paper: IJM TM.
Current address: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America
The authors have declared that no competing interests exist.
The envelope of HSV-1 contains a number of glycoproteins, four of which are essential for virus entry. Virus particles lacking gB, gD, gH or gL are entry-defective, although these viruses retain the ability to bind to the plasma membrane via the remaining glycoproteins. Soluble forms of gD have been shown to trigger the nuclear translocation of the NF-κB transcriptional complex in addition to stimulating the production of Type I interferon. By taking advantage of the entry-defective phenotype of glycoprotein-deficient HSV-1 virus particles, the results presented here show that binding of virions to cellular receptors on the plasma membrane is sufficient to stimulate a change in cellular gene expression. Preliminary microarray studies, validated by quantitative real-time PCR, identified the differential expression of cellular genes associated with the NF-κB, PI3K/Akt, Jak/Stat and related Jak/Src pathways by virions lacking gB or gH but not gD. Gene induction occurred at a few particles per cell, corresponding to physiological conditions during primary infection. Reporter assay studies determined that NF-κB transcriptional activity is stimulated within an hour of HSV-1 binding, peaks between two and three hours post-binding and declines to background levels by five hours after induction. The immediate, transient nature of these signalling events suggests that HSV-1 glycoproteins, particularly gD, may alter the cellular environment pre-entry so as to condition the cell for viral replication.
Subjugation of the intracellular environment by viruses is essential to ensure the effective expression and replication of the viral genome to allow production of progeny virions. One such viral strategy involves hijacking signalling pathways that ultimately control host gene transcription. Interactions between intracellular viral proteins and cellular kinases responsible for signal transduction are a means by which to achieve this. However, there is growing evidence to support the premise that glycoproteins on the surface of virus particles may trigger intracellular signalling pathways by interacting with their cognate receptors on the host cell membrane
Binding of HSV-1 to permissive cells occurs through viral glycoproteins on the viral envelope interacting with specific receptors on the cell surface, triggering fusion of the plasma membrane with the outer envelope. Five of these glycoproteins are known to be involved in virion binding to the cell surface: gB, gC, gD and the heterodimer gH-L. Of the five, only gC is dispensable for allowing productive infection as deletion of gB, gD or gH-L results in an entry-defective phenotype
One of the first studies to examine whether HSV-1 glycoproteins play a role in the induction of signalling found that gD was able to block Fas-mediated apoptosis
There are reports showing that HSV-1 triggered the translocation of NF-κB by six hours post-infection
In comparison to wild-type gD, a mutated form of gD that was unable to bind to HVEM did not stimulate NF-KB activity in co-cultured monocytoid cells
Soluble glycoproteins from beta- and gammaherpesviruses can also stimulate intracellular signalling pathways; gB of CMV has been shown to stimulate the differential expression of cellular transcription factors and interferon-stimulated genes (ISGs), and soluble gp350 of EBV can trigger NF-κB activation
The treatment of cells with soluble glycoproteins or UV-inactivated virus has provided strong evidence that HSV-1 binding to the plasma membrane triggers signal transduction in the host cell as a prelude to infection, but interpretation of these experiments is not straightforward. The use of soluble glycoproteins does not mimic the binding of individual virus particles and the physiological significance of the results is difficult to assess. The use of UV-inactivated particles in signalling studies simulates infection but these particles are able to enter cells and deliver virion proteins to the cytoplasm. Experiments with UV-inactivated particles do not therefore distinguish between events arising from virus binding from those resulting from virus entry.
The main focus in these studies was to examine signalling events using virus particles that are capable of binding to the cell surface but are incapable of entry due to the absence of one of the three essential glycoproteins, gB, gD or gH
All cells other than Human Foreskin Fibroblasts (HFF) (ATCC cell line CRL-2522) were grown in Glasgow Modified Eagles Medium supplemented with 10% foetal bovine serum, 4 mM glutamine, 100 units/ml penicillin and 100 ug/ml streptomycin. HFF cells were grown in Dulbecco modified Eagles medium supplemented as described above but with the addition of 1x MEM non-essential amino acids.
Working stocks of HSV-1 SC16 were grown on HaCaT cells and assayed on Vero cells
Purified preparations of WT virus, or of virions lacking individual glycoproteins, were produced as previously described
Human foreskin fibroblast cells (HFF) were seeded at 1.5×106 in a 1752 cm flask and grown to 90% confluence then growth-arrested in medium supplemented with only 100 U/ml Penicillin-G and 100 µg/ml Streptomycin for five days. Real-time PCR experiments were conducted with growth-arrested HFFs that were inoculated in triplicate with 1000 particles/cell of entry-defective HSV-1 in warm, serum-free medium, with parallel mock-infected cells that received an equivalent volume of PBS in warm serum-free medium. Inoculations took place at 37°C. At the indicated times after the addition of virus, the medium was removed, the cells were lysed with TRI Reagent (Sigma) and total RNA was immediately purified. For each entry-defective HSV-1 mutant, three independent biological replicates were carried out. Microarray experiments were conducted under the same conditions using wild-type HSV-1 (SC16) at an MOI of 20.
Total RNA was isolated from TRI reagent lysates as per the manufacturer's instructions. Contaminating genomic DNA was digested with 2 U DNase I (Invitrogen) and RNA was re-purified with TRI reagent. cDNA was derived from 30 µg total RNA using anchored oligo (dT)20 primers (Invitrogen) as described in the manufacturer's instructions. For each entry-defective HSV-1 mutant, the cDNA from three independent infections were used in technical triplicates for real-time PCR reactions.
Microarray analysis was carried out using the Signal Transduction PathwayFinder cDNA array as per the manufacturer's instructions (Superarray). Nylon cDNA microarrays membranes were pre-hybridised with supplied sheared salmon sperm DNA at 60°C for one to two hours. The pre-hybridisation solution was replaced with the biotin-dUTP labelled cDNA probe, which was diluted in sheared salmon sperm DNA. The solution was left to hybridise at 60°C overnight. The following morning the cDNA probe was discarded and the membrane was washed twice then blocked in Solution Q, as provided by the manufacturer. After 40 minutes, the blocking solution was discarded and the membrane was incubated with a binding buffer, Solution F. After washing, the chemiluminescent substrate was incubated with the membrane for two to five minutes at room temperature. The microarray membrane was then exposed to X-ray film. Analysis was performed using GEArray Expression Analysis Suite as provided by the manufacturer (Superarray). All the data sets used in the microarray analysis are available in
cDNA derived from cells that were mock-infected or inoculated with 1000 particles/cell of entry-defective ΔgB, ΔgD and ΔgH virions was used for real-time PCR analysis. Primers were designed, using the Primer3 software
A relative change in expression of two-fold was set as a threshold for determining whether differential expression of a gene had occurred. The
An NF-κB reporter assay system was designed using a pGL4-NFκB construct (Promega). Quadruplicate repeats of the NF-κB binding consensus sequence
gD and the major tegument protein, VP16, were detected by immunoblotting using the monoclonal antibodies LP2 and LP1, respectively
Signalling-specific microarrays were chosen as an initial route to identifying signalling pathways that may be stimulated early in HSV-1 infection, and more precisely, by virion binding. The Signal Transduction PathwayFinder cDNA array from Superarray incorporates 96 targets covering 19 associated signalling pathways, in addition to two negative controls corresponding to pUC18 plasmid DNA and a ‘blank’ spot, as well as four positive controls representing the housekeeping genes for GAPDH, beta actin, cyclophilin A and ribosomal protein L13a.
Serum-starved HFFs were inoculated with 1000 particles/cell of ΔgB, ΔgD or ΔgH entry-defective HSV-1 virions. Total RNA was isolated immediately after inoculation, corresponding to 0 hpi, and 6 hpi. Changes in gene expression associated with the activation of signalling pathways were identified by comparison to the transcript abundance at 0 hpi. The results from duplicate experiments indicated that downstream gene targets of a number of signalling pathways are differentially expressed above the two-fold threshold after virion binding (
Gene | Description | ΔgB | ΔgD | ΔgH |
BCL2A1 | hematopoietic BCL2-related protein A1 | −3.85 * | 5.56 | 1.42 |
BIRC1 | baculoviral IAP repeat-containing 1 | −2.33 | −1.12 | −3.13 ** |
BIRC2 | baculoviral IAP repeat-containing 2 | 3.85 | 3.98 | 1.72 |
BIRC3 | baculoviral IAP repeat-containing 3 | 2.88 | 1.00 | 7.71 *** |
CCL2 | macrophage chemoattractant protein-1 | 2.32 * | 1.34 | 2.13 * |
LTA | lymphotoxin alpha (TNF superfamily, member 1) | −1.22 | 2.31 | −1.20 |
NFKB1 | nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105) | 2.98 * | −1.92 | 3.09 ** |
NFKBIA | nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha | −1.19 | 1.23 | 2.54 ** |
PECAM1 | platelet/endothelial cell adhesion molecule (CD31 antigen) | 4.60 * | 1.00 | 3.09 * |
A2M | alpha-2-macroglobulin | 5.07 * | 1.79 | 2.23 |
CSN2 | casein beta | 5.23 ** | −1.54 | 1.48 |
CXCL9 | chemokine (C-X-C motif) ligand 9 | 2.22 | −1.59 | 1.06 |
IRF1 | interferon regulatory factor-1 | 1.90 | −2.94 | 1.00 |
MMP10 | matrix metallopeptidase 10 | 1.09 | −1.12 | 2.50 |
NOS2A | nitric oxide synthase 2A | 1.26 | 1.21 | 8.01 ** |
BCL2 | B-cell CLL/lymphoma 2 | 3.54 | −1.54 | 1.86 ** |
CCND1 | cyclin D1 | 1.54 | −9.09 *** | 3.22 * |
FN1 | fibronectin 1 | 1.50 | 1.03 | 6.28 ** |
MMP7 | matrix metallopeptidase 7 | 2.05 | 1.91 | 3.05 ** |
MYC | v-MYC myelocytomatosis viral oncogene homolog | 2.83 | 1.09 | 4.55 |
WISP2 | WNT1 inducible signaling pathway protein 2 | −7.69 ** | 1.50 | −4.55 *** |
BCL2 | B-cell CLL/lymphoma 2 | 3.54 | −1.54 | 1.86 ** |
BCL2L1 | BCL2-like 1; Bcl-X | −4.92 | −5.00 ** | −3.33 |
GADD45A | growth arrest and DNA-damage-inducible, alpha | 5.22 * | −1.15 | 1.96 |
IGFBP3 | insulin-like growth factor binding protein 3 | −6.67 * | −1.04 | 1.18 * |
MDM2 | Mdm2 p53 binding protein homolog (mouse) | 6.83 * | −1.82 | 2.89 |
EGR1 | early growth response 1 | −2.70 | −2.50 * | 1.09 |
FAS | fas (TNF receptor superfamily, member 6) | −2.38 | 1.15 | −2.63 * |
FOS | FBJ murine osteosarcoma viral oncogene homolog | −2.78 * | 1.56 | −1.59 |
JUNB | jun B proto-oncogene | −1.01 | −2.56 | −1.33 |
PTGS2 | rostaglandin-endoperoxide synthase 2 | 1.26 | 1.21 | 8.01 |
CDKN1A | cyclin-dependent kinase inhibitor 1A | 1.12 * | 4.01 * | 1.10 |
CDKN2A | cyclin-dependent kinase inhibitor 2A | 1.89 | 1.35 | −4.17 ** |
CDKN2B | cyclin-dependent kinase inhibitor 2B | −1.19 | 2.72 | −2.27 |
CDKN2D | cyclin-dependent kinase inhibitor 2D | 1.31 | −4.55 ** | 1.05 |
CDX1 | caudal type homeobox 1 | 1.31 | 2.05 | 1.09 |
CTSD | cathepsin D | −1.05 | −3.70 | −1.12 |
EN1 | engrailed homeobox 1 | −1.10 | −2.22 | 1.00 |
RBP1 | retinol binding protein 1, cellular | 1.68 | −3.03 * | 1.00 |
CDK2 | cyclin-dependent kinase 2 | 3.86 | −1.45 | 3.44 ** |
KLK2 | kallikrein-related peptidase 2 | 6.83 *** | −1.82 | 2.89 |
TMEPA1 | prostate transmembrane protein, androgen induced 1 | 3.69 | 1.41 | −3.13 *** |
BMP4 | bone morphogenetic protein 4 | −2.22 * | −2.38 | −1.67 |
HFFs were stimulated with 1000 particles/cell of ΔgB, ΔgD or ΔgH HSV-1 for six hours and a cDNA microarray corresponding to targets of 19 signalling pathways was used to detect changes in cellular gene expression when compared to mock-infected. Infections and mock infections were carried out in duplicate. A number of signalling pathways were shown to be stimulated by one or more of the entry-defective mutants. These data were used to independently verify changes in expression using real-time PCR.
Using the same methodology, serum-starved HFF cells were exposed to 1000 particles per cell of virions lacking either gD, gB or gH and RNA was extracted for cDNA synthesis immediately (0 h) or after 6 h.
Gene | Description | ΔgB | ΔgD | ΔgH |
BIRC2 | baculoviral IAP repeat-containing 2 | 3.66 |
1.20 | 2.92 |
BIRC3 | baculoviral IAP repeat-containing 3 | 4.45 |
1.27 | 3.71 |
CCL2 | macrophage chemoattractant protein-1 | 2.09 | −1.51 | 3.16 |
NFKB1 | nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105) | 3.54 |
1.13 | 2.61 |
NFKBIA | nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha | 2.35 |
1.07 | 1.48 |
NFKBIB | nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, beta | 2.72 |
1.16 | 1.40 |
PECAM1 | platelet/endothelial cell adhesion molecule (CD31 antigen) | 3.49 |
1.22 | 4.44 |
REL | v-REL reticuloendotheliosis viral oncogene homolog (C-REL) | 2.05 | −1.25 | 2.63 |
A2M | alpha-2-macroglobulin | 3.54 |
1.77 | 2.55 |
MMP10 | matrix metallopeptidase 10 | 2.87 |
1.52 | 2.62 |
NOS2A | nitric oxide synthase 2A | 5.00 |
−1.09 | 4.41 |
BCL2 | B-cell CLL/lymphoma 2 | 2.86 |
−2.33 |
2.13 |
MMP7 | matrix metallopeptidase 7 | 3.67 |
−1.03 | 2.04 |
MYC | v-MYC myelocytomatosis viral oncogene homolog | 4.53 |
−1.04 | 2.67 |
BCL2 | B-cell CLL/lymphoma 2 | 2.86 |
−2.33 |
2.13 |
BCL2L1 | BCL2-like 1; Bcl-X | 2.56 |
−1.92 |
2.09 |
CDK2 | 3.01 |
1.18 | 3.08 |
Note.
*p<0.05.
**p≤0.01.
***p≤0.001.
HFFs were stimulated with 1000 particles/cell of entry-defective HSV-1 and changes in cellular gene expression were detected by real-time PCR at 6 hours post-inoculation. NF-κB responsive genes appear to be up-regulated by binding of either ΔgB or ΔgH virions to the cell. The increase in NF-κB-associated transcripts appears specific as not all targets were differentially regulated. A similar pattern of gene expression is seen for Jak/Stat pathway targets in that ΔgD virions appear to be unable to stimulate signalling whereas ΔgB and ΔgH binding can. MMP7 is only associated with the PI3 Kinase/Akt pathway and coupled with the up-regulation of BCL2, CCND1 and MYC, it would appear that binding may be sufficient to also stimulate this pathway, although not by virions lacking in gD. The Jak/Src pathway only contains two targets on the signalling specific microarray, one of which, BCL2, was associated with other pathways.
Gene | ΔgB | ΔgD | ΔgH | |||
Microarray | Real-Time | Microarray | Real-Time | Microarray | Real-Time | |
BIRC2 | 3.66 | 3.85 | 1.20 | 3.98 | 2.92 | −1.01 |
BIRC3 | 4.45 | 2.88 | 1.27 | 1.00 | 3.71 | 7.71 |
CCL2 | 2.09 | 2.32 | −1.51 | 1.34 | 3.16 | 2.13 |
NFKB1 | 3.54 | 2.98 | 1.13 | −1.92 | 2.61 | 3.09 |
NFKBIA | 2.35 | −1.19 | 1.07 | 1.23 | 1.48 | 2.54 |
NFKBIB | 2.72 | - | 1.16 | - | 1.40 | - |
PECAM1 | 3.49 | 4.60 | 1.22 | 1.00 | 4.44 | 3.09 |
REL | 2.05 | - | −1.25 | - | 2.63 | - |
A2M | 3.54 | 5.07 | 1.77 | 1.79 | 2.55 | 2.23 |
MMP10 | 2.87 | 1.09 | 1.52 | −1.12 | 2.62 | 2.50 |
NOS2A | 5.00 | 1.26 | −1.09 | 1.21 | 4.41 | 8.01 |
BCL2 | 2.86 | 1.86 | −2.33 | −1.54 | 2.13 | 1.86 |
MMP7 | 3.67 | 2.05 | −1.03 | 1.91 | 2.04 | 3.05 |
MYC | 4.53 | 2.83 | −1.04 | 2.85 | 2.67 | 2.48 |
BCL2 | 2.86 | 1.86 | −2.33 | −1.54 | 2.13 | 1.86 |
BCL2L1 | 2.56 | 1.42 | −1.92 | −5.00 | 2.09 | −1.18 |
CDK2 | 3.01 | 3.86 | 1.18 | −1.45 | 3.08 | 3.44 |
There was a degree of correlation between the change in expression determined by microarray studies and those confirmed by real-time PCR, particularly for genes under the control of NF-κB. Note:
The JAK/STAT and related JAK/Src pathway show a similar response to that seen for NF-κB. Of the JAK/STAT-responsive genes, only NOS2A is regulated by more than one pathway, with ΔgB and ΔgH virions inducing their transcription and ΔgD particles having no significant effect (
Induction of this pathway may be responsible for the upregulation of CCND1, MYC, BCL2 and MMP7 after inoculation with ΔgB and ΔgH but not ΔgD HSV-1 (
Both ΔgB and ΔgH virions are capable of stimulating an NF-kB response (
The downstream signalling appears specific as not all NF-κB targets were up-regulated. The NF-κB repressors, NFKBIA and NFKBIB, were only up-regulated by ΔgB virion binding (
To confirm that virion binding induces NF-κB and to examine the kinetics, an NF-κB promoter-driven luciferase construct was transfected into HFF cells and cells were treated with ΔgH virions (1000 particles per cell) or were mock-treated. Cells were harvested every hour until 9 hr post treatment, lysed and assayed for luciferase activity in triplicate.
(A) An NF-κB luciferase construct was transfected into HFF, which were left to serum-starve for five days. Cells were then mock-infected or inoculated with 1000 particles/cell of entry-defective ΔgH HSV-1 then lysed at the indicated times and assayed for luciferase activity. Error bars are the +/− standard error across triplicate luciferase assays for each biological duplicate. (B) NF-κB, JAK/STAT, JAK/Src and PI3K/Akt-responsive genes that were previously shown to be differentially expressed at 6 hours post-inoculation with ΔgB and ΔgH virions were examined for changes in expression at two-and-a-half hours after inoculation with 1000 particles/cell of ΔgH virions. The induction of JAK/STAT and PI3K/Akt targets is not significantly up-regulated above the two-fold threshold by two-and-a-half hours post-inoculation. A number of NF-κB-associated transcripts are significantly up-regulated, with
As induction of the NF-κB reporter construct occurred within one hour of inoculation with ΔgH virions and peaked at around two-and-a-half hours post-inoculation, then the transcripts previously shown in
The initial microarray screen was uninformative with respect to interferon signalling, but this was of interest because soluble gD has been reported to induce IFN
Name | Description | ΔgB | ΔgD | ΔgH | MOI 0.01 |
IFNA1 | interferon, alpha 1 | 2.63 |
−1.11 | 2.25 |
−1.23 |
IFNB1 | interferon, beta 1, fibroblast | 3.01 |
2.15 |
−1.07 | −1.18 |
IL29 | interleukin 29 (interferon, lambda 1) | 1.51 | −1.33 | −1.69 | 1.11 |
ISG54 | IFN-induced protein 54 | 1.20 | 1.57 | −1.54 | −1.45 |
IRF1 | interferon regulatory factor 1 | −1.21 | 1.07 | 1.36 | 1.00 |
IRF3 | interferon regulatory factor 3 | 1.14 | −1.10 | 1.05 | 1.08 |
IRF7 | interferon regulatory factor 7 | 1.19 | 1.03 | −1.05 | −1.23 |
IRF9 | interferon regulatory factor 9 | −1.23 | 1.03 | 1.44 | −1.33 |
Note.
*p<0.05.
Binding by ΔgB and ΔgH virions up-regulated the expression of IFN-α whereas ΔgB and ΔgD virions were able to stimulate an increase in IFN-β. Infection with wild-type HSV-1 at an MOI of 0.01 was insufficient to cause similar increases in interferon expression. Interferon-stimulated genes were not up-regulated above the two-fold threshold by binding of any of the entry-defective HSV-1 mutants. Wild-type infection at a low MOI was also insufficient to cause in increase in ISG transcripts when compared to mock-infected cells.
IFN-® transcript levels were up-regulated 2.22 fold by ΔgB virions and 2.06 fold (p = 0.01) by ΔgD virions, again with a high degree of statistical significance. Inoculation with virus particles lacking gH led to a −1.07 fold change (p = 0.44) in IFN-® expression when compared to mock-infected cultures. All three entry-defective mutants were unable to modulate the expression of IFN-λ above the arbitrary two-fold threshold.
The glycosylation status of IFN-inducing viral glycoproteins is a potential determinant of induction efficacy
HFF were inoculated as above with ΔgH virions that had been incubated for 18 hrs in the presence or absence of PNGase. Glycosylated and deglycosylated ΔgH virions stimulated a 2.23 and 2.28 fold increase in IFN-α transcription, respectively. These results suggest that glycosylation has no impact on the interferogenic properties of ΔgH virions, unlike the glycoproteins of MHV and TGEV, which appear to stimulate Type I IFN through a lectin-like action, HSV-1 gD may trigger IFN up-regulation through a different mechanism, possibly analogous to that used to stimulate other signalling pathways.
Five interferon-stimulated genes were chosen to examine whether they were differentially expressed by virion binding. IFN-induced protein 54 (ISG54) transcripts are routinely used as a marker of ISG induction. Binding by ΔgB, ΔgD or ΔgH virions was insufficient to cause changes in the transcript abundance of interferon regulatory factor-1, -3, -7, -9 or ISG54 (
To ensure that these observations could not result from a background of infection by competent virus, parallel cultures were infected with wild type virus at an MOI of 0.01, a level of infection some tenfold higher than would result from the ‘worst case’ scenario using preparations of entry defective virions (see
All the experimental work described above was performed using treatments with 1000 virus particles per cell, a condition that would be unlikely to pertain in natural primary infections. To establish whether much lower virion numbers would be effective, HFFs were serum-starved for five days then mock-inoculated or inoculated with 1, 10, 100 or 1000 particles per cell of ΔgH HSV-1 for six hours. Infections were carried out in duplicate with mock- inoculated controls that received an equivalent volume of PBS. Total RNA was purified and reverse transcribed, with the cDNA from each duplicate inoculation used in triplicate for real-time PCR.
The NF-κB responsive genes NFKB1, REL and CCL2, in addition to the JAK/STAT target NOS2A, were used as they had previously been shown to be differentially expressed after inoculation with 1000 particles/cell of ΔgH HSV-1. Recalling that the more abundant a transcript the fewer number of cycles it takes to cross a threshold set in the exponential phase of PCR product accumulation (the CT value),
Low multiplicities of infection, which may represent physiological conditions, were sufficient to trigger an intracellular signalling response. (A) As the number of inoculated ΔgH particles increases, the number of cycles taken to reach the cycle threshold decreases for the NF-KB-responsive genes
Arrows in
Evidence from previous studies using soluble HSV-1 glycoprotein and UV-inactivated virus suggested that binding of HSV-1 virions to the cell surface might be sufficient to stimulate intracellular signalling pathways. We undertook preliminary microarray studies with entry-defective HSV-1 virions and identified a number of pathways that were stimulated at early time points during infection.
Microarray experiments are inevitably vulnerable to false positives and negatives due to non-specific binding of labelled cDNA probes, necessitating a more robust follow-up with highly sensitive methods. Due to such confounders, these data were used exclusively as a guide to select, for real-time PCR, the transcriptional targets of intracellular signalling pathways stimulated after inoculation with entry-defective HSV-1.
Gene targets associated with the NF-κB, JAK/STAT/Src, and PI3K/Akt pathways were shown to be differentially expressed after inoculation with glycoprotein-deficient virions, with the majority of signalling events being associated with the presence of gD on the envelope. Nineteen other signalling pathways present on the preliminary microarray experiments, and confirmed by real-time PCR, were not stimulated (data not shown). These results are compatible with our model, shown in
Glycoprotein D acts as the main signalling molecule on the surface of the HSV-1 envelope. gH interacts with αvβ3 integrins to potentially trigger the production of IFN-β, which is known to involve IRF-3 and 7
Real-time PCR confirmed that changes in transcription associated with the NF-κB, JAK/STAT, JAK/Src and PI3K pathways were modulated as a result of virion binding, all of which required gD on the envelope surface To demonstrate that signalling occurred at physiologically relevant multiplicities of infection, HFFs were inoculated with either 1000, 100, 10 or 1 particles per cell of entry-defective HSV-1. Changes in gene transcription occurred in a dose-dependent manner and were detectable at 10 virus particles per cell. Given the particle∶infectivity ratios usually quoted for HSV1, we argue that this corresponds to physiological conditions (i.e. less than one infectious unit per cell), but it is impossible to know the circumstances that pertain
HSV-1, as well as beta- and gammaherpesviruses, are capable of stimulating the NF-κB pathway in a bi-phasic manner, with our data supporting that an early, transient induction is reliant on virions expressing gD
Intracellular signalling induced by soluble gD can protect against Fas-mediated apoptosis with inhibition of NF-κB signalling leading to a loss of this protection
Aspects of the HSV-1 life cycle, such as stimulating the progression of the cell cycle in the absence of serum, may be sufficient to induce a stress response and trigger apoptosis. Both
It is less apparent as to the biological relevance of an innate immune response stimulated through HSV-1 binding. It may be that there is a “cost” associated with altering the intracellular environment, which leads to the differential expression of cytokines, such as
Signalling by secreted Type I IFNs occurs through the Jak/Stat pathway results in the expression of various ISGs; a response that is also triggered by virus entry
The methodology used here required the serum-starvation of primary human fibroblasts for five days. In the absence of serum, primary fibroblasts rapidly enter a quiescent state. As a DNA virus that requires host nuclear factors to replicate its genome, it is therefore not surprising that HSV-1 would stimulate cells from a G0 state into one that would favour DNA replication and possibly promote the transcription of viral genes.
Quiescent cells
Cyclin-dependent kinase 2 (CDK2) is one such downstream target of c-Myc activity, as well as the Androgen pathway, highlighting the signalling cross-talk that may occur
Epithelial cells at the initial site of HSV-1 infection
Normalised datasets for genes differentially expressed on a signalling-specific microarray after infection with entry-defective HSV-1.
(0.14 MB XLS)
Primers used in real-time PCR to determine the relative abundance of corresponding mRNA transcripts.
(0.28 MB DOC)
Characteristics of virions treated with PNGase.
(0.76 MB PDF)
We thank Susanne Bell for invaluable technical assistance and Helena Browne for helpful discussions and critical reading of the manuscript.