Page 265 - Avian Virology: Current Research and Future Trends

258 | Schat

did not reveal clear phylogenetic clusters. Moreover, from a negatively controlled by the interactions between COUP-TF1
practical point of view, as far as is known, all strains belong to and δEF1 with the DR and E box and positively by the produc-
the same serotype. The only report that a second serotype exists tion of oestrogen. The implications of this regulation will be
(Spackman et al., 2002a,b) has never been confirmed and has not discussed in the section on Transmission of CAV in SPF flocks.
been reported afterwards. It is possible that this putative second
serotype is not even related to CAV (Schat, 2009). Viral nucleic acids
The single-stranded, negative-sense DNA genome is the encap-
Organization of the promoter/enhancer region sidated DNA strand (Noteborn et al., 1991, 1992; Phenix et al.,
The organization of the promoter/enhancer region was first 1994) and forms a double-stranded circular genome during virus
described in detail by Noteborn et al. (1991, 1994b) and replication. A single unspliced, polycistronic mRNA of approxi-
Meehan et al. (1992). Using the sequence of CIA-1 with four mately 2 to 2.1 kb is transcribed from the positive strand and
direct repeats (GenBank accession number L14767), the tran- is encoding the 3 VP by using alternate start codons. Using the
scription start point (TSP) is placed at nt 1 (Fig. 9.7A and B). TSP as nt 1 (Fig. 9.7B) a polyadenylation site was located at nt
The TATA box starts at nt –31 and the start codon is located 1963, which is 25 nt (Noteborn et al., 1992) or 21 nt (Phenix et
at nt +27. Three SP1-binding sites are starting at nt –49, –147 al., 1994) downstream from the unique poly(A) addition signal
and –238 and one NFY box starts at nt –92. The four 21 nt DR (AAUAAA). Phenix et al. (1994) also detected a minor 4 kb
are separated between DR 2 and 3, or DR 3 and DR 4 if there transcript, which is most likely the result of a failure to terminate
is a fifth DR, by 12 nt. The second SP1-binding site is located transcription efficiently by a small proportion of the RNA poly-
within this 12 nt sequence. Noteborn et al. (1994b) noted that merase molecules. The 2.1 kb transcript can be detected between
unidentified nuclear factors of T-cells are bound to each of the 4 and 12 hours pi of MSB1 cells (Noteborn et al., 1992; Phenix et
DR and to this 12 bp insert. Purified human SP1 had a strong al., 1994; Kamada et al., 2006).
affinity to the 12 nt insert. The first two or three DR followed Kamada et al. (2006) noted that TTV has three spliced tran-
by the 12 nt insert are essential for virus replication. Mutated scripts (Kamahora et al., 2000) and based on similarities between
CAV containing only the first two or three DR did not produce CAV and TTV decided to search for spliced transcripts during the
viable virus particles. Changing the 12 nt insert reduced but did replication of CAV. Spliced transcripts of 1.6, 1.3, 1.2 and 0.8 kb
not prevent virus replication (Noteborn et al., 1998b). The DR were indeed detected in CAV-infected MSB1 cells appearing
regions contain the 5′-ACGTCA sequence that binds CREB around 48 to 72 hours pi. The spliced transcripts were cloned
and ATF transcription factors, but Noteborn et al. (1994b) and sequenced with the exception of the 1.6 kb transcript, which
found that the CAV DR does not bind these transcription fac- for unknown reasons could not be cloned. The 1.3 kb transcript
tors. Miller et al. (2005) noticed that this sequence resembles could code for a truncated VP1_1 of 253 AA lacking the central
the oestrogen response element (ERE) consensus half-sites (A) 197 AA. The 1.2 transcript could code for a truncated 249 AA
GGTCA. The ERE consists of a palindrome of the half-sites VP1_2 and 259 AA VP2_3. Finally, the 0.8 kb interrupted VP2_1
with a 3 bp space between the half-sites. Oestrogen receptors at 280 nt from the VP2 start codon and connected to the middle
(ER) bind as homodimers to the ERE, but binding does not of the VP1 ORF yielding a hypothetical protein of 247 aa. The
require a perfect sequence of the ERE. Sequence scanning same 0.8 kb transcript would produce a VP3_2 of 59 A. It has to
indicated that additional ERE-like half-sites are located down- be emphasized that, thus far, these proteins have not been dem-
stream from the TATA box (Fig. 9.7A and B). To determine if onstrated and the relevance of the transcripts for virus replication
the promoter/enhancer region of CAV can bind to ER, Miller has not been elucidated, nor have these spliced transcripts been
et al. (2005) used the oestrogen receptor-enhanced LMH/2A reported in infected chickens.
cell line, which overexpresses ER 150 times compared with the
parent LMH cell line (Sensel et al., 1994). Using the short Viral proteins
promoter sequence (Fig. 9.7A) to drive expression of enhanced Three viral proteins (VP1, VP2, and VP3) have been identified
green fluorescent protein (EFGP) it was shown that the addi- in MSB1 cells infected with CAV (reviewed by Schat, 2009).
tion of oestrogen increased the expression of EGFP significantly Using immunofluorescence VP3 was detected in a few cells as
in the LMH/2A cell line. When a long promoter sequence (Fig. early as 6 hours pi of MSB1 cells, while VP2 was present in very
9.7A) was used that included the potential ERE half-sites down- few cells at 12 hours pi. For both proteins maximum fluorescence
stream from the TATA box, there was no significant increase in was reached 30 hours pi. At that time, VP1 and viral capsids were
EGFP expression. In a subsequent study, Miller et al. (2008) also present in the infected cells (Todd et al., 1994; Douglas et al.,
identified two inhibitors of transcription. Co-transfection assays 1995). Recently, Trinh et al. (2015) showed the presence of VP1
using the short promoter sequence and the nuclear receptor in infected MSB1 cells as early as 12 hours pi. The reason(s) for
chicken ovalbumin upstream promoter transcription factor 1 the discrepancy is not clear.
(COUP-TF1), which can bind to ERE-like motifs, decreased
expression of EGFP significantly. The inhibition of transcription Viral protein 1
using the long promoter was caused by binding of the transcrip- VP1 is the only viral protein associated with virus particles (Todd
tion regulator delta-EF1 protein (δEF1) to the E box region et al., 1990, 1994). The N-terminal region of approximately 40 aa
at the TSP (Miller et al., 2008). Thus, transcription of CAV is has a significant degree of similarity to histone proteins (Claessens