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262 | Schat
Cytoplasm Nucleus

Importin β1 P
VP3
β1 NPC β1
GTP
NES1 NLS1 NES2 NLS2 Ran PML

Nuclear envelope Tumor P PML PML

cell

PML NB PML
CRM1
Normal
CRM1
NPC cell
GTP
GTP
Ran Ran
GDP RanGAP1
Ran

Figure 9.8 Schematic model for the differential regulation of VP3 nuclear targeting in tumour and normal cells. VP3 is transported into the
nucleus using the nuclear localization signals (NLS1 and NLS2) likely using the importin β1 protein. In the nucleus, VP3 is phosphorylated
Figure 8.
at T108, which in tumour cells inhibits export of Apoptin from the nucleus into the cytoplasm resulting in increased accumulation of
VP3. Phosphorylated VP3 binds primarily to the promyelocytic leukaemia (PLM) nuclear bodies. Unphosphorylated VP3 is recognized by
chromosome region maintenance 1 (CRM1) protein through the COOH-terminal nuclear export signal of VP3 resulting in export into the
cytoplasm in normal cells. Figure courtesy of David A. Jans. Adapted from Poon et al., Cancer Research 65, No 16, 2005, pp. 7059–7064,
with permission of the American Association for Cancer Research.

level of the cell line and the strain of virus (see Propagation, DNA synthesis and genome replication
cell culture). In an attempt to determine if specific phenotypes Small single-stranded DNA viruses with a circular genome lack
were susceptible or refractory to infection, Calnek et al. (2000) the ability to reproduce their own genome and need mitotically
examined 24 MD-derived CD3 T-cell lines representing six active cells to provide the enzymes involved in DNA replication.
+
different phenotypes. The results are summarized in Table 9.2. Although a rolling-circle replication (RCR) has been proposed for
Using an indirect immunofluorescence assay with MAb 51.3 the replication of CAV DNA, the actual process is incompletely
specific for VP3 (Chandratilleke et al., 1991), the number of understood. It differs in many aspects from the better understood
positive cells/50,000 ranged from 0 to 1664 at 3–4 days pi, RCR described for PCV and geminiviruses (reviewed by Todd
which increased to 4 to 47,000 at 5–7 days pi. At both time et al., 1991; Niagro et al., 1998; Faurez et al., 2009; Rizvi et al.,
+
–
points the CD4 CD8 lines had a higher percentage positive 2015). Briefly, PCV produces a double-stranded replicative form
–
–
–
+
cells than the CD4 CD8 and CD4 CD8 lines but the number (RF) by a still unknown mechanism. The RF produces the Rep
of cell lines was too small to draw major conclusions about proteins which bind to the stem loop structure and cleaves the
differences in susceptibility. Combined with the susceptibility stem loop to generate a free ‘OH extremity. Cellular DNA poly-
of selected ALV-transformed B cell lines, it is not yet possible merase initiates the production of the viral DNA from the free
to identify specific receptors for virus attachment. Two MSB1 ‘OH extremity. Once viral DNA replication is completed the Rep
–
+
(phenotype CD4 CD8 TCR2) sublines, used at different pas- complex closes the loop and new RF DNA or single-stranded
sage levels, were either negative at 5 days pi or had a low range genomic DNA is formed (Faurez et al., 2009).
of positive cells (8–52/50,000 cells). There is no information CAV does not code for clearly defined Rep proteins nor does
on the mechanisms of virus penetration and uncoating of the it have a clear stem loop to function as the origin of replication.
genome. It is my assumption that virus particles enter the To start the replication the circular negative strand DNA needs
nucleus where the genome is released from the virus particle, to be nicked and the complimentary strand needs to be gener-
but this will need to be demonstrated. ated to produce the RF. Todd et al. (1996) located a S1 nuclease