Page 267 - Avian Virology: Current Research and Future Trends

260 | Schat

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AA 97 is unusual for the signature motif. Mutation of C to serine resulting in a severe depletion of the thymus cortex at 13 days
abrogated the DSP activity (Peters et al., 2002), while mutation pi. In vitro infection of two cell lines (MSB1 and 1104-X5)
97
of C to serine resulted in a slight decrease of PTPAse activity also resulted in cell death by apoptosis. Apoptotic bodies were
but a drastic increase of S/T PPase activity (Peters et al., 2005). observed in thymocytes from infected chickens and in infected
Mutation of C and C resulted in the elimination of both cell lines. Subsequent studies using MAb specific for VP3 dem-
97
95
phosphatase activities. Virus with the C to S mutation at AA 95 onstrated that this protein was associated with the apoptotic
or 97 was replication competent but with a drastic reduction in bodies (Noteborn et al., 1994a). Transfection of MSB1 and
1.5
1.7
viral replication with titres between 10 to 10 TCID /0.1 ml LSCC-HD11, a myeloid cell line, with the expression vector
50
suggesting that the phosphatase activity is important for efficient pRSV-VP3 also resulted in the induction of apoptosis. Trunca-
virus replication. Viruses with AA mutations near or in the DSP tion of VP3 by deleting the 11 codons at the 3′ end of the ORF
catalytic motif (C87R, R101G, K102D and H103Y) and AA (VP3tr) resulted in a marked decrease in apoptosis. CEF were
affecting the region with a high degree of secondary structure resistant to the induction of apoptosis. Twenty-four to 48 hours
expected to affect the PTPAse activity (R129G, Q131P, R/K/ after infection or transfection VP3 was present in the nucleus as
K150/151/152G/A/A, D/E161/162G/G, L163P, D169G and finely distributed granules gradually increasing in size to form
E186G) were generated and examined in MSB1 cells (Peters aggregates associated with apoptotic bodies (Noteborn et al.,
et al., 2006). The mutation K102D yielded very low virus titres 1994a). Todd et al. (1994) demonstrated that, in addition to
comparable to the titres obtained with the mutations at AA 95 VP3, VP1 and VP2 are also present in the apoptotic bodies.
3
and 97. All other mutants yielded virus titres between 10 and VP3 is often referred to as Apoptin, which is derived from
10 TCID /0.1 ml compared with 10 5.5 TCID /0.1 ml for Apoptosis Induction (Pietersen and Noteborn, 2000). Apoptin
4
50
50
the wild-type CAU269/7 isolate. Differences in eclipse period, has been used at least since 1995 by the Noteborn research
latent period, cell-associated and extra cellular titres were noted group (Noteborn and Koch, 1995). In this chapter I will use
between the different mutant and the parent CAU269/7 viruses. both terms VP3 and Apoptin.
Two interesting differences are the absence of down regulation Noteborn et al. (1998c) quoted unpublished data that CAV
of MHC class I in CAV-infected MSB1 cells and the absence replication depends on the presence of intact VP3. Wei and
of VP3 in the nuclei of MSB1 cells infected with the mutant Schat (unpublished data, 1996) mutated the start codon of VP3
viruses. These observations suggest that VP2 may play a role in so that VP3 was no longer produced without changing the VP2
the transport of VP3 into the nucleus and is a mediator of MHC AA sequence and confirmed that the mutated virus was no
class I down-regulation. Using yolk-sac inoculation of 7-day-old longer able to replicate in vitro. In an elegant study, Prasetyo
embryos Peters et al. (2007) noticed that all mutants had reduced et al. (2009) also generated VP3 knock-out mutants [CAV/
pathogenicity at ED 21. In a follow-up study, Kaffashi et al. (2008) Ap(–)] by mutating the start codon of VP3. Transfection of
inoculated 1-day-old SPF chicks with different mutants. Chickens MSB1 cells with CAV/Ap(–) did not yield viral DNA or infec-
infected with mutant E186G had the highest VN antibody titres tious virus but virus-like particles were produced. The reverse
and the authors suggested that this mutant may be a vaccine can- mutant (CAV/ApRM), in which VP3 was restored, generated
didate. However, Schat (2009) raised some questions concerning the same titres as the wild-type virus. They also generated a
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the use of the mutants as vaccine strains. First of all, Scott et al. point mutation at AA 108 (T108I) generating CAV/ApT I
(2001) reported that E186G mutations had been found in the without changing VP2. This mutation reduced the production
high passage pathogenic and apathogenic clones 33 and 34. In of infectious virus significantly suggesting a major role for
addition, the stability of the mutants in cell culture or chickens is Threonine at AA 108 (see also Apoptin: use as an anti-cancer
not clear because a spontaneous mutant was isolated from MSB1 therapy). Co-transfection of MSB1 cells with CAV/Ap(–) with
cells infected with R129G (Kaffashi et al., 2008). a plasmid containing wild-type VP3 (pAp/WT) complemented
As mentioned above, VP2 may be important for the transfer DNA replication fully but did not yield infectious virus. Co-
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of VP3 into the nucleus of MSB1 cells or target cells in chickens transfection of CAV/ApT I with pAp/WT resulted in DNA
resulting in the induction of apoptosis. Noteborn and van der Eb replication and production of infectious virus without changing
(1998) and Noteborn (2004) suggested that VP2 may also cause the mutation.
apoptosis but did not publish the data. Because the VP3 ORF is Apoptin-like sequences have also been identified in other
located within the VP2 ORF a mutant VP2 construct is needed Anelloviridae. Miyata et al. (1999) reported that the sequence
which eliminates the expression of VP3. Kaffashi et al. (2015) of TTV TA278 (GenBank accession number AB017610) is
–
+
created a pCAT–VP2 VP3 construct which induced apoptosis similar to CAV in the arrangement of three partially overlap-
in transfected MSB1 cells. Interestingly a number of the cells ping ORFs. TTV VP3 showed apoptotic activity in three human
showed evidence of necrosis rather than apoptosis. The authors hepatocellular carcinoma cell lines and was named TTV-derived
suggested that VP2 can also have an anti-apoptotic activity per- apoptosis-inducing protein (TAIP) by Kooistra et al. (2004).
haps early during the infection cycle. Co-transfection of CAV/Ap(–) with TAIP complemented repli-
cation of CAV/Ap(–) DNA similar to the complementation with
Viral protein 3 pAp/WT (Prasetyo et al., 2009). VP3 of the first human gyro-
Jeurissen et al. (1992b) reported that in vivo infection with virus isolate (HGyV) (Sauvage et al., 2011) has also apoptotic
CAV caused apoptosis of thymocytes starting at 6 days pi properties in several human cancer cell lines comparable to CAV