PTPs

Gene families encoding PTPs are widespread in PDVs, having been identified in two bracovirus subfamilies (Microgastrinae and Cardiochilinae) and recently in a PDV associated with Banchinae wasps (Provost et al, 2004; Webb et al, 2006; Lapointe et al., 2007). Furthermore, these genes have been subject to intensive expansion, leading to the largest gene families of PDV genomes (27 PTP genes in CcBV, 13 PTP genes in MdBV and TnBV). PTPs, together with protein tyrosine kinases, are known to play key roles in the control of signal transduc-tion by controlling the levels of cellular protein phosphorylation (Andersen et al., 2001). Each PTP dephosphorylates phosphotyrosine residues on a specific substrate. Bracovirus PTPs show considerable diversity in their amino acid sequences suggesting that each PTP has the potential to interact with a different specific substrate (Provost et al., 2004). PDV PTPs are therefore likely to target signal transduction involved in multiple processes such as host immunity and development.

Mammalian immune-cell actin dynamics depends on the phosphorylation of proteins localized in focal adhesions. Certain mammalian bacterial pathogens have been shown to inhibit phagocytosis by injecting PTPs, which disrupt these actin rearrangements (DeVinney et al., 2000). PDV PTPs were therefore also proposed to be able to disrupt signalling pathways controlling haemo-cyte cytoskeleton dynamics, thereby inhibiting encapsulation. In accordance with this prediction, certain bracovirus PTPs were found to be expressed in haemocytes and in certain cases bracovirus-infected host haemocytes showed more PTP activity than mock-infected controls (Provost et al., 2004; Gundersen-Rindal and Pedroni, 2006; Ibrahim et al., 2007; Pruijssers and Strand, 2007). Furthermore, transient expression of biochemically active MdBV PTP-H2 or PTP-H3 in Drosophila S2 cells led to a reduction of phagocytosis of Escherichia coli by these cells. This reduction was even more drastic if cells were co-transfected with PTP contructs and Glc1.8 (see below). Both PTP-H2 and PTP-H3 were shown by immunofluorescence to localize to focal adhesions in Drosophila S2 cells (Pruijssers and Strand, 2007). Taken together these results indicate that PTP-H2 and -H3 have antiphagocytic activity. Bracoviruses have been shown by in vitro biochemical assays to encode both catalytically active and inactive PTPs (Provost et al., 2004; Pruijssers and Strand, 2007; Ibrahim and Kim, 2008). The inactive forms may have a different biochemical activity such as trapping phosphorylated tyrosine proteins. Transient expression in host (Spodoptera exigua) haemocytes of active CvBV PTP1 and inactive PTP5 resulted in reduced cell spreading and encapsulation of beads, suggesting that both PTPs are

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Figure 9.4 Gene content of the double-stranded DNA circles included in bracovirus particles: C. congregata bracovirus (CcBV) contains nine gene families, four of which encode proteins with well-known conserved domains (IKB-like, cysteine-motif (CRP), protein tyrosine phosphatase (PTP), cystatin), another four of which encode proteins of unknown function conserved in bracoviruses associated with wasps of the Cotesia genus (EP1 -like, f1, f2, f3), and one of which is not present in the available sequences of other bracoviruses (hp2). Approximately 40% of the genes encode proteins showing no similarity to proteins in the databases (hypothetical) or having similarities with predicted proteins of different vertebrate or invertebrate species (putative). Some encoded proteins have similarities with products of mobile elements (retro-like), different viruses (viral protein), and unique proteins conserved in bracoviruses associated with wasps of the Cotesia genus (braco-like). Several lines of evidence indicate that at least some iKB-like and PTP gene products contribute to impairing host immunity.

involved in altering haemocyte behaviour, either by direct dephosphorylation of tyrosine residues or through competition with host PTPs (Ibrahim and Kim, 2008).

In certain host-parasitoid interactions haemo-cyte cell death and even apoptosis have been described (Schmidt et al, 2001; Lavine and Strand, 2002). For instance, MdBV infection of P. includens and Spodoptera frugiperda leads to a large proportion of granulocytes (but not plasmatocytes) dying by apoptosis (Strand and Pech, 1995a; Suderman et al, 2008). It has been recently shown that transient expression of MdBV PTP-H2 in the Sf21 cell line induces caspase-dependent apoptosis of these cells, in contrast to seven other MdBV genes and a PTP-H2 phosphatase-inactive mutant, all of which lacked apoptosis-inducing activity (Suderman et al, 2008). If Sf21 cells were cultured under conditions in which apopotosis was inhibited, PTP-H2 was found both to inhibit the ability of the cells to engulf bacteria, and also to reduce proliferation. PTP-H2 has therefore been suggested to induce apoptosis by directly or indirectly perturbing the cell cycle. The MdBV PTP-H2 protein therefore appears to have different effects depending on target cells.

9.4.2 Mucin-like glycoprotein, Glc1.8

PTPs are not the only PDV proteins targeting haemocyte function. MdBV Glc1.8, a PDV gene that encodes a cell-surface mucin-like glycoprotein, has

Table 9.1 PDV genes for which the functional evidence of involvement in host immune disruption is the most advanced.

Protein PDV and host

Predicted function

Observed physiological disruption

Functional data

Methodology

References

PTP CcBV, Manduca

Disruption of signalling

PTPA is a functional tyrosine phosphatase.

Activity of Sf21 cell lysates

Provost et al.

sexta

pathways involved in

PTPM is non-functional.

infected with recombinant

(2004)

hormone biosynthesis or

baculovirus

haemocyte cytoskeleton

dynamics

CvBV, Plutella

As above

Loss of haemocyte

PTP 1 expression in Spodoptera exigua haemocytes leads

In vivo transient expression in S.

Ibrahim and Kim

xylostella

adhesion

to increased PTP activity, reduction of cell spreading, and

exigua haemocytes

(2008)

Loss of

reduction of encapsulation.

phagocytosis

MdBV, Noctuid

As above

As above

MdBV-infected haemocytes have higher PTP activity.

Parasitism of Pseudoplusia

Pruijssers and

moths

MdBV infection

PTP-H2, -H3 are functional tyrosine phosphatases.

includens

Strand (2007)

of S. frugiperda

PTP-H2 in combination with Glc1.8 reduces S2 cell

Bioassays in Drosophila S2 cell

Suderman et al.

induces apoptosis

phagocytosis.

lines expressing PTP

(2008)

of granulocytes

PTP-H2 expression in Sf21 cells induces apoptosis by caspase

Protein expression in Sf21 cell

activation.

lines

Glc1.8 MdBV

Disruption of capsule-

Loss of haemocyte

Loss of adhesion of Hi5 cell lines infected with MdBV

RNAi in MdBV-infected cell

Beck and Strand

Noctuid moths

forming haemocytes

adhesion

RNAi using Glc1.8 restores adhesion.

cultures

(2003)

Loss of

Expression of Glc1.8 causes loss of adhesion and reduced

Recombinant expression in cell

Beck and Strand

phagocytosis

phagocytosis in Hi5 and S2 cell lines.

cultures

(2005)

CrV1 CrBV, Pieris rapae

Inactivation of

Injection of recombinant CrV1 in P. rapae modifies

Recombinant protein injection in

Asgari et al. (1997)

haemocytes

haemocyte spreading and disrupts actin filaments

natural host

Actin-filament

disorganization

CcV1 CcBV, M. sexta

Disruption of haemocyte

CcV1 interacts with hemolin.

Yeast two-hybrid and

Labropoulou et al.

cytoskeleton

CcBV1 inhibits hemolin binding to lipopolysaccharide.

co-immunoprecipitation

(2008)

Hemolin-induced bacterial agglutination is abolished in the

In vitro assays using purified

presence of CcBVI.

recombinant protein

B. mori haemocytes, S2 cells, and Sl2b cells show reduced

Immunofluorescence

phagocytosis ability in the presence of rec CcBVI.

co-localization experiments

CcV1 and hemolin interact at the cell surface.

Protein PDV and host Predicted function Observed physiological disruption

VHv1.1 Cys-motif

CslV, Heliothis virescens

Disruption of immunity/ development

Translation inhibition during parasitism

Vinnexins

Lectin 15b

CslV, H. virescens

CvBV, P. xylostella CvBV, P. xylostella

Disruption of cellular immunity by altering gap junctions

Interruption of haemocyte recognition

Disruption of cellular immunity

Disruption of cellular immunity

H4 histone CvBV, P. xylostella

TnBV1 EP1 -like Egf

Smapin

TnBV, H. virescens

CvBV, P. xylostella Immunosuppressant

MdBV, Noctuid moths

Inhibition of melanization

Cell death

Suppression of melanization during parasitism PO cascade reduces MdBV and wasp survival.

Functional data

Methodology

References

H. virescens infection with VHv1.1-expressing baculovirus reduces encapsulation response to washed wasp eggs.

Injection of recombinant VHv1.1 increases susceptibility to baculovirus infection.

Recombinant VHv1.1 and VHv1.4 inhibit translation of host RNA.

cs-Vnxd and cs-Vnxg expression in Xenopus laevis oocytes form functional gap junctions.

In vivo expression in the natural host using baculovirus Recombinant protein injection in natural host In vitro translation assays

Protein expression in X. laevis

Li and Webb (1994) Fath-Goodin et al. (2006) Kim (2005)

Turnbull et al. (2005)

Recombinant CvBV-lectin reduces bacterial attachment to haemocytes.

Recombinant CvBV15b: induces impaired spreading of P. xylostella and S. exigua haemocytes and markedly reduces protein release from haemocytes.

Transfection of P. xylostella larvae with CvBVH4 recombinant expression vector induces loss of host haemocyte spreading ability.

Infection of Sf21 and Hi5 cell lines with recombinant TnBV1 baculovirus induces apoptosis-like programmed cell death

Transfection of P. xylostella larvae with ELP1 induces reduction in haemocyte numbers.

Recombinant Egfl.O reduces M. sexta PO activity.

Conditioned medium from Hi5 cells treated with double-stranded RNAi Egf1.0/1.5 lost antimelanization capacity.

Egfl.O inhibits M. sexta PAP-3 and PAP-1 activity.

Recombinant Egfl.O blocks processing of pro-PAP1, pro-PAP3, proPO, and serine proteinase homologues 1 and 2.

In vitro effect of recombinant protein on host haemocytes In vitro effect of recombinant protein on host haemocytes

In vivo transfection in natural host using eukaryote expression vector Recombinant baculovirus expression in insect cell lines In vivo transfection of natural host

In vitro assays using recombinant protein and M. sexta plasma

Nalini and Kim

Gad and Kim

Lapointe etal. (2005) Kwon and Kim (2008) Beck and Strand (2007)

licB-like CslV, H. virescens Irreversible inhibitors of nuclear factor kB (NF-kB) transcription factors.

Disruption of signalling pathways involving NF-kB transcription factors MdBV, Noctuid As above moths P. includens and Trichoplusia ni

Increased susceptibility in PDV-infected hosts

TnBV, H. virescens As above

Cystatin

CcBV, M. sexta

Immune disruption and/or developmental arrest

In H. virescens parasitism affects NF-kB nuclear localization.

IkB nuclear localization post parasitism

Immunofluorescence assays on haemocytes and fat body

Kroemer and Webb (2005)

IkB reduces expression of antimicrobial protein (AMP)

reporter constructs. IkB binds to Drosophila Rel proteins. IkB inhibits Rel binding to kB sites in AMP promoters.

IkB reduces expression of tumour necrosis factor a (TNFa) reporter-gene constructs.

Parasitism affects NF-KB-like protein nuclear localization.

Recombinant cystatin 1 is a functional C1A cysteine protease inhibitor in vitro.

Cotesia spp. cystatins are subject to strong diversifying selection.

In vitro recombinant protein expression and reporter gene assays in Drosophila S2 cells Co-immunoprecipitation Electrophoresis mobility shift assays

In vitro recombinant protein expression and reporter gene assays in Hela cells Immunofluorescence In vitro enzymic assays Molecular evolution models

Thoetkiattikul et al. (2005)

Falabella et al. (2007)

Espagne et al. (2005) Serbielle et al. (2008)

been shown using RNAi to be involved in inhibition of cell adhesion. Infection of High Five (Trichoplusia ni) cells with MdBV resulted in loss of cell adhesion to culture plates but adhesion was restored when PDV-infected culture cells were treated with double-stranded RNA specifically targeted against Glc1.8 (Beck and Strand, 2003). Furthermore, transient expression of Glc1.8 in High Five cells reduced their ability to adhere to foreign surfaces and to phagocytose E. coli in a similar manner to that after MdBV infection, showing that Glc1.8 is an important viral factor involved in disruption of adhesion and phagocytosis in these cell types (Beck and Strand, 2005). Glc1.8 is composed of an extracellular domain with amino acid repeats arranged in tandem, and a C-terminal transmembrane domain. Transient expression of Glc1.8 mutants lacking the membrane anchor had no effect on cell adhesion or phagocytosis. Sequential deletion of the Glc1.8 repeats led to progressive reduction in adhesion blocking activity. Collectively the data indicate that membrane localization is essential for Glc1.8 function, and that PDV mucins form structures which may physically block adhesion by hindering lig-and-receptor interactions (Beck and Strand, 2005).

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