Identification of an in Vitro Interaction between an Insect Immune Suppressor Protein (CrV2) and Gα Proteins

Abstract

The protein CrV2 is encoded by a polydnavirus integrated into the genome of the endoparasitoid Cotesia rubecula (Hymenoptera:Braconidae:Microgastrinae) and is expressed in host larvae with other gene products of the polydnavirus to allow successful development of the parasitoid. CrV2 expression has previously been associated with immune suppression, although the molecular basis for this was not known. Here, we have used time-resolved Förster resonance energy transfer (TR-FRET) to demonstrate high affinity binding of CrV2 to G␣ subunits (but not the G␤␥ dimer) of heterotrimeric G-proteins. Signals up to 5-fold above background were generated, and an apparent dissociation constant of 6.2 nM was calculated. Protease treatment abolished the TR-FRET signal, and the presence of unlabeled CrV2 or G␣ proteins also reduced the TR-FRET signal. The activation state of the G␣ subunit was altered with aluminum fluoride, and this decreased the affinity of the interaction with CrV2. It was also demonstrated that CrV2 preferentially bound to Drosophila G␣ o compared with rat G␣ i1 . In addition, three CrV2 homologs were detected in sequences derived from polydnaviruses from Cotesia plutellae and Cotesia congregata (including the immune-related early expressed transcript, EP2). These data suggest a potential mode-of-action of immune suppressors not previously reported, which in addition to furthering our understanding of insect immunity may have practical benefits such as facilitating development of novel controls for pest insect species.

Polydnaviruses: From discovery to current insights

“…Several of these genes were also related to factors in eukaryotic signaling pathways, which suggested they were virulence factors that caused some of the physiological alterations that occur in parasitized hosts. Subsequent studies supported this by demonstrating experimentally how certain BV and IV gene products interact with host immune molecules or cells (Beck and Strand, 2005; Thoetkiattikul et al, 2005; Beck and Strand, 2007; Ibrahim and Kim, 2008; Labropoulou et al, 2008; Kwon and Kim, 2008; Cooper et al, 2011; Magkrioti et al, 2011; Bitra et al, 2012; Gueguen et al, 2013), while implicating other genes in altering host growth, metabolism or endocrine physiology (Provost et al, 2004; Falabella et al, 2006; Kim et al, 2013; Presad et al, 2013). …”

Fluorescence/Bioluminescence Resonance Energy Transfer Techniques to Study G-Protein-Coupled Receptor Activation and Signaling

“…The studies of species more suitable for microscopy will certainly facilitate such measurements. It is noteworthy that FRET imaging has been achieved in zebrafish (Fan et al, 2007;Tao et al, 2011) as well as Drosophila melanogaster (Lissandron et al, 2007;Shafer et al, 2008;Gervasi et al, 2010;Cooper et al, 2011), and the latter studies have helped to elucidate the role of adenylyl cyclases and cAMP in learning processes in the D. melanogaster brain.…”

Polydnaviruses: Evolution and Function

“…Further support for the latter came from studies showing that most genes on DNAs in virions are transcribed in hosts but are not transcribed in wasps (Rasoolizadeh et al, 2009;Bitra et al, 2011;Provost et al, 2011;Burke and Strand, 2012b;Chevignon et al, 2014;Dorémus et al, 2014;Bitra et al, 2015). Experimental studies also showed that genes/ microRNAs in BV or IV virions disrupt specific host immune defences against wasp offspring (Beck and Strand, 2005;Thoetkiattikul et al,, 2005;Beck and Strand, 2007;Pruijssers and Strand, 2007;Ibrahim and Kim, 2008;Labropoulou et al,, 2008;Kwon and Kim, 2008;Cooper et al, 2011;Magkrioti et al, 2011;Bitra et al, 2012;Gueguen et al, 2013), alter host growth (Provost et al, 2004;Falabella et al, 2006;Pruijssers et al, 2009;Kim et al, 2013;Prasad et al, 2014;Ignesti et al, 2018;Wang et al, 2018), and potentially affect host interactions with food plants and other organisms (Zhu et al, 2018;Tan et al, 2018).…”