Antivirals reduce the formation of key Alzheimer's disease molecules in cell cultures acutely infected with herpes simplex virus type 1

Abstract

Alzheimer's disease (AD) afflicts around 20 million people worldwide and so there is an urgent need for effective treatment. Our research showing that herpes simplex virus type 1 (HSV1) is a risk factor for AD for the brains of people who possess a specific genetic factor and that the virus causes accumulation of key AD proteins (β-amyloid (Aβ) and abnormally phosphorylated tau (P-tau)), suggests that anti-HSV1 antiviral agents might slow AD progression. However, currently available antiviral agents target HSV1 DNA replication and so might be successful in AD only if Aβ and P-tau accumulation depend on viral DNA replication. Therefore, we investigated firstly the stage(s) of the virus replication cycle required for Aβ and P-tau accumulation, and secondly whether antiviral agents prevent these changes using recombinant strains of HSV1 that progress only partly through the replication cycle and antiviral agents that inhibit HSV1 DNA replication. By quantitative immunocytochemistry we demonstrated that entry, fusion and uncoating of HSV1, are insufficient to induce Aβ and P-tau production. We showed also that none of the "immediate early" viral proteins is directly responsible, and that Aβ and P-tau are produced at a subsequent stage of the HSV1 replication cycle. Importantly, the anti-HSV1 antiviral agents acyclovir, penciclovir and foscarnet reduced Aβ and P-tau accumulation, as well as HSV1, with foscarnet being less effective in each case. P-tau accumulation was found to depend on HSV1 DNA replication, whereas Aβ accumulation was not. The antiviral-induced decrease in Aβ is attributable to the reduced number of new viruses, and hence the reduction in viral spread. Since antiviral agents reduce greatly Aβ and P-tau accumulation in HSV1-infected cells, they would be suitable for treating AD with great advantage unlike current AD therapies, only the virus, not the host cell, would be targeted.

Figure 1. Initiation of Aβ accumulation requires the initiation of early and/or late protein synthesis.

Vero cells were infected for 16 hours with HSV1 strain 17 or with HSV1 recombinants, each at an MOI of 5, and tested for Aβ by immunocytochemistry. Infection with in1374 did not lead to Aβ accumulation (A) whereas infection with wild type HSV-1 (in1863) did show Aβ accumulation (B). Infection with recombinant tsK/lacZ showed no Aβ accumulation at the restrictive temperature (C). Infection with recombinant in1404 did show Aβ staining (D). Mock-infected cells at 37°C (E) or at 38.5°C (F) showed no staining. Scale bar: 50 µm. To confirm infection of cells, monolayers were stained for the expression of β-gal. Infection with in1863 (G) or tsK/lacZ (H) resulted in β-gal expression in most cells. In cultures infected with in1374 only a small proportion of cells expressed β-gal (I), whereas many more were positive after co-infection with in1374 and WT HSV1 (MOI 5 for each virus), demonstrating that in1374 had entered a high proportion of cells (J). Cells show typical HSV1-cytopathic effects, i.e., they become rounded and contracted.

Figure 2. Initiation of abnormal tau phosphorylation requires the initiation of early and/or late protein synthesis.

Vero cells were infected for 16 hours with HSV1 strain 17 or with HSV1 recombinants, each at an MOI of 5, and tested for abnormal tau phosphorylation (pS214) by immunocytochemistry. Infection with in1374 did not lead to abnormal tau phosphorylation (A) whereas infection with in1863 did show phosphorylation (B). Infection with recombinant tsK/lacZ showed no abnormal tau phosphorylation at the restrictive temperature (C). Infection with recombinant in1404 did show staining (D). Mock-infected cells at 37°C (E) or at 38.5°C (F) showed no staining. Scale bar: 50 µm.

Figure 3. Acyclovir inhibits HSV1 replication.

Vero cells were infected with HSV1 SC16 at an MOI of 1 for 16 hours. Cells were treated with 0 µM, 50 µM, 100 µM or 200 µM acyclovir (ACV), which was present throughout infection. After fixation the slides were tested for HSV1 proteins using immunocytochemistry. Reactivity with HSV1 proteins was significantly reduced in the presence of ACV. Scale bar: 50 µm.

Figure 4. Acyclovir inhibits HSV1-induced β-amyloid accumulation.

Vero cells were infected with HSV1 SC16 at an MOI of 1 for 16 hours. Cells were treated with 0 µM, 50 µM, 100 µM or 200 µM acyclovir (ACV), which was present throughout infection. After fixation the slides were tested for β-amyloid (Aβ) accumulation using immunocytochemistry. These results clearly show that ACV reduces Aβ significantly. Scale bar: 50 µm.

Figure 5. Acyclovir inhibits HSV1-induced abnormal tau phosphorylation.

Vero cells were infected with HSV1 SC16 at an MOI of 1 for 16 hours. Cells were treated with 0 µM, 50 µM, 100 µM or 200 µM acyclovir (ACV), which was present throughout infection. After fixation the slides were tested for abnormal tau phosphorylation (AT100) using immunocytochemistry. ACV significantly reduces AT100 staining. Scale bar: 50 µm.

Figure 6. Quantification of HSV1 proteins, β-amyloid and abnormal tau phosphorylation in HSV1-infected cells after acyclovir treatment.

Vero cells were infected with HSV1 SC16 at an MOI of 1 for 16 hours. Cells were treated with 0 µM, 50 µM, 100 µM or 200 µM acyclovir (ACV), which was present throughout infection. After fixation the slides were tested for HSV1 proteins, β-amyloid (Aβ) accumulation and abnormal tau phosphorylation (AT100) using immunocytochemistry and the amount of staining was quantified using Image J. Values are expressed as a percentage of staining produced when no antiviral is used. For HSV1 proteins (A) and abnormal tau phosphorylation (C), there is a statistically significant decrease in staining for all concentrations of ACV tested compared to the staining in cells infected but not treated with ACV (p<0.0001 in both cases). For Aβ (B), there is a statistically significant decrease in staining with 100 µM ACV and 200 µM only (p<0.0001). ANOVA was used to test for significance. The results are a combination of two independent experiments.

Figure 7. Comparison of different antiviral agents on proteins accumulating during HSV1 infection.

Vero cells were infected with HSV1 SC16 at an MOI of 1 for 16 hours. Cells were treated with 0 µM, 50 µM, 100 µM or 200 µM penciclovir (PCV) or foscarnet (FOS) which was present throughout infection. After fixation the slides were tested for (A) β-amyloid (Aβ) accumulation, (B) abnormal tau phosphorylation (AT100), and (C) HSV1 proteins using immunocytochemistry and the amount of staining was quantified using Image J. Values are expressed as a percentage of staining produced when no antiviral is used. These values were used to determine the antiviral concentration required to reduce staining to 50%. The results are a combination of two independent experiments. Values for mock-infected cells were between 0 and 8% (data not shown).

Figure 8. Antiviral agents reduce the HSV1-induced increases in β-amyloid-related enzymes.

Vero cells were infected with HSV1 SC16 at an MOI of 1 for 16 hours. Cells were treated with 0 µM, 50 µM, 100 µM or 200 µM acyclovir (ACV), penciclovir (PCV) or foscarnet (FOS) which was present throughout infection. After fixation the slides were tested for (A) β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and (B) nicastrin. Images for no antiviral, 50 µM ACV, 50 µM PCV and 200 µM FOS are shown. Scale bar: 50 µm.

Figure 9. Quantification of the effect of antiviral agents on the HSV1-induced increases in amyloid- and tau-related enzymes.

The immunocytochemistry results in Figures 8 and 9 were quantified using Image J. (A) β-site amyloid precursor protein cleaving enzyme 1, (B) nicastrin, (C) protein kinase A and (D) glycogen synthase kinase 3β.

Figure 10. Antiviral agents reduce the HSV1-induced increases in tau-related enzymes.

Vero cells were infected with HSV1 SC16 at an MOI of 1 for 16 hours. Cells were treated with 0 µM, 50 µM, 100 µM or 200 µM acyclovir (ACV), penciclovir (PCV) or foscarnet (FOS) which was present throughout infection. After fixation the slides were tested for (A) protein kinase A (PKA) and (B) glycogen synthase kinase 3β (GSK3β). Images for no antiviral, 50 µM ACV, 50 µM PCV and 100 µM FOS are shown. Scale bar: 50 µm.