We carried out initial experiments on SHSY5Y cells to determine the level of HSV1 needed to ensure that on inoculation most cells were infected. We then examined levels of APP in lysates prepared from the cells 6 h after infection with this dose of HSV1. Western blots stained with anti C-terminal APP antibody (Fig. 1A) revealed, as expected, various full-length APP bands in uninfected cells corresponding to the three main isoforms in multiple glycosylation states. Deglycosylation experiments have shown that the lower single band (100 kDa) is immature APP695, that the doublet above this (approx molecular weight 115 kDa) is mature APP695 and immature APP751/APP770 and that the highest molecular weight group (approx. 125 to 135 kDa) comprises a mixture of mature APP751 and mature APP770 (Parkin et al., unpublished observations).
The intensity of these bands decreased appreciably in cells harvested only 3 h after inoculation with HSV1, as did those from cells treated with the protein synthesis inhibitor cycloheximide. This decline after infection may reflect loss of ability of the cells to generate new APP due simply to virus-induced shut-down of protein synthesis [18].
Of particular interest was the relative intensity of a 55 kDa band, again apparent after C-terminal APP antibody staining. This band was similarly intense in control-treated and cycloheximide-treated cells, but had far greater intensity after HSV1 infection. This increase in HSV1-infected cells alone was highly reproducible, occurring in many experiments, each carried out independently, from the infection to the blotting stage. Band intensity for cells treated for 6 hours was quantified using Syngene Genetools software and averaged over five such experiments is shown in Figure 1B; value for HSV1-infected cells was significantly different from control, being elevated by 124% (95%CI 101 – 148%; p < 0.002), whereas that for cycloheximide-treated cells was similar to the control value.
To exclude the possibility that the band reflected synthesis of a viral protein which cross-reacts non-specifically with the antibody, we immunostained blots produced from gels of viral proteins from our HSV1 preparations. No evidence for any cross-reaction of the Sigma C-terminal APP antibody with these proteins could be found, suggesting that the strengthened band was cell-derived (results not shown). This result does not preclude any cross-reactivity with an ICP, as the latter would not be present within virions, but the presence of the 55 kDa protein in uninfected cells, and also, at a higher level, in APP-transfected cells (see below), strongly supports a cellular, non-viral origin of the protein. Interestingly, APP has been detected in HSV1 virions by Western blotting [17], but this probably reflects the authors' usage of a much more concentrated and purified virus preparation than that of the inoculate we use for infecting cells.
We next examined the influence of duration of infection on these viral-induced changes (Fig. 1C). As anticipated, with increasing duration before harvesting, the depletion of APP became more pronounced (this was found also with cycloheximide treatment); in contrast, the intensity of the C-terminal 55 kDa fragment seen in the infected cells was not higher than in uninfected cells at 3 hr, but increased at 6 hr and 9 hr. However there was minimal further change as incubation increased to 24 hr (not shown). This result also was found to be reproducible on repeating the experiment several times from infection to blotting. It suggests that infection prevents synthesis of APP, leading to its gradual depletion, but causes also the abnormal accumulation of the 55 kDa fragment due, presumably, to a decrease in degradation of the latter. However, for longer incubation times, we cannot exclude the possibility of sequestration of some APP within newly synthesised viral particles, as reported elsewhere [17], which might reduce the amount of APP in the virus-free cell lysates used for the preparation of cell proteins.
We repeated these experiments with herpes simplex virus type 2 (HSV2), to see whether these effects were specific to HSV1. Fig. 2 shows that very similar findings were obtained, with 55 kDa band intensity for HSV2-infected cells 9 hr after infection being 101% greater than that for control cells (95%CI 94 – 107%; p < 0.005) suggesting that this phenomenon is not uniquely associated with HSV1 infection.
On the assumption that a corresponding N-terminal fragment of approximately similar size to that of the 55 kDa C-terminal fragment would be produced as a result of this abnormal processing (the size of APP being 110 kDa to 135 kDa), we probed our blots with the Sigma N-terminal antibody (Fig 3A). However we found no clear evidence for an increased amount of an N-terminal fragment of around this size after infection. Possibly, such a fragment would be either degraded or released into the cell culture medium, thus precluding its detection here.
To confirm further that the C-terminal fragment is derived from APP (and not for example from an APLP, i.e. of a type of protein related to APP, though with a less clear involvement in AD) we obtained human SYSY5Y cells that had been transfected with a human APP gene (APP695), and which over-express APP. We stained blots of proteins from non-transfected, APP transfected, and mock-transfected (i.e. with vector alone) cells with the C-terminal antibody, and found the 55 kDa band in all three cases, though its intensity was greater in the APP transfected cells alone (see Fig. 3B). This indicates that the 55 kDa fragment is indeed an APP product.