Adeno-associated virus

AAV is a small human DNA virus with a single-stranded genome encoding two genes which give rise to four proteins with replication and regulatory roles (Rep78, ‑68, ‑52 and ‑40) and three capsid proteins. AAV is a defective virus in the sense that it generally needs co-infection with a helper virus to replicate efficiently. So far no disease has been associated with AAV. On the contrary, AAV was reported to have oncosuppressive activities and to play a protective role against the development of certain cancers. Infection by AAV was reported to be less frequent than average in women with cervical carcinoma. AAV also has an antiproliferative action on mammalian cells in culture. However the underlying mechanisms are poorly understood. We are therefore studying the effects of AAV and the Rep proteins on the cell division cycle. We discovered a novel and potentially important response of cells to the presence of AAV DNA, with its single-stranded stretch and hairpin-shaped termini (Fig. 1).

Figure 1: The terminal sequence of AAV DNA can be drawn as a stable base-paired hairpin.

Targeting genetic instability – AAV‑mediated killing of cells that lack p53

A major goal of molecular oncology is to identify means to kill cells lacking p53 function. Most current cancer therapy is based on damaging cellular DNA by irradiation or chemicals. There is evidence suggesting that, in the event of DNA damage, the p53 tumor suppressor protein is able to prevent cell death by sustaining an arrest of the cell cycle at the G2 phase. We found that AAV can selectively induce apoptosis in cells that lack active p53. Cells with intact p53 activity are not killed but undergo arrest in the G2 phase of the cell cycle. This arrest is characterized by an increase in p53 activity and p21 levels, but does not depend on the AAV-encoded proteins. Rather, the AAV DNA elicits a DNA damage response that leads to cell cycle arrest or, in the absence of active p53, to cell death.

In cells, the AAV DNA accumulates into nuclear foci with a damage signaling apparatus that includes the ATR kinase and other proteins characteristic of a cellular replication block response (Fig. 2). AAV therefore provides an opportunity to study the proteins and DNA structures mediating the cellular response to stalled replication forks, outside the context of chromatin.

AAV also inhibits tumor growth in mice. Our work therefore shows that viruses can be used to deliver DNA of unusual structure into cells to trigger a DNA damage response without damaging cellular DNA and to selectively eliminate those cells lacking p53 activity. Now we are studying exactly how the AAV DNA is recognized as damage, how the damage signal is transmitted, and the mechanisms that lead to cell death.

Figure 2: (A) UV-inactivated AAV2 DNA accumulates with damage-responsive proteins into intra-nuclear factories or “foci”, seen here by in situ hybridization as light blue dots, which signal cell cycle arrest. (B) Diagram of the proteins that are included in these foci. 

AAV Rep protein functions – how AAV Rep78 protein arrests cells completely in S phase

We are also investigating the inhibitory effect of the AAV Rep proteins on cell division, independently of AAV replication, using retroviral vectors to study the effects of the individual Rep proteins on cultured cells. AAV Rep78 protein inhibits cell cycle progression, and, in particular, Rep78 induces a complete arrest within S phase, a response rarely seen after cell DNA damage. We examined how Rep78 achieves such an efficient S phase block. Rep78 inhibits Cdc25A activity by a novel means in which binding between the two proteins stabilizes Cdc25A, thus increasing its abundance, while at the same time preventing access to its substrates cyclin-dependent kinase (Cdk) 2 and Cdk1. This effect alone does not induce a complete S phase block. In addition, Rep78, as well as Rep68, produces nicks in the cellular chromatin, inducing a DNA damage response mediated by ATM leading to G1 and G2 blocks. Mutational analysis shows that the zinc finger domain and nuclease activity of Rep78 are both required for the S phase block. The results suggest that a true S phase block cannot be achieved through a single pathway, and that AAV Rep78 protein arrests cells within S phase by interfering with two pathways that would normally lead to an S phase slow-down.

Another function of Rep78 is to promote the integration of AAV-based vector DNA into a specific site in human chromosome 19. We are therefore testing new ways to introduce Rep transiently into cells. (Fig. 3).

Figure 3: One consequence of AAV infection of tumor cells is centrosome overduplication.

Human papillomaviruses and cells containing episomal or integrated viral DNA

Most HPV infections do not lead to cancer. However, occasionally viral DNA integrates into the cell DNA, resulting in continued expression of viral oncogenes E6 and E7 and deletion of E2. This extends the life span of the infected cell and favors further genetic changes that lead to loss of differentiated characteristics and progression towards becoming a cancer cell. Our work is aimed at understanding and interfering with this process. Although integration and loss of E2 are considered important steps in tumorigenesis, their consequences are still unclear. A useful model system for these experiments is the W12 cell line. These cells are human keratinocytes that were derived from an early stage HPV-induced lesion. W12 cells in culture are feeder cell-dependent and carry several hundred copies of the HPV16 episome. They proliferate but maintain the potential to differentiate on appropriate signals, so they are considered to have properties of stem or transit amplifying cells of the epidermis. And the corresponding cells with the HPV DNA integrated are available for comparison.

AAV-host cell interaction and the DNA damage response

It now seems likely that, already in the initial stages of tumorigenesis, aberrant cell division cycles driven by activated oncogenes trigger a DNA damage response in cells. Loss of checkpoints and genetic instability could appear in these early, or precancerous, stages. The availability of AAV and W12 keratinocytes provides a valuable experimental tool to test the sensitivity of such precancerous cells to the virus. We are therefore looking at the ability of AAV to infect cell types that are particularly interesting from the point of view of its anti-tumor effects. And we are also interested in the role of the cellular DNA damage response on such a viral infection. 

Keywords

Tumor virus, tumor suppression, cell cycle