PKR

PKR[1] is an intracellular innate immune factor found in many higher animals, including birds and mammals. Because chickens possess this factor, our experiments employed human PKR in human cultured cells (HEK293) and chicken PKR in chicken cultured cells (DF-1), respectively.

PKR consists of two functional domains: the double-stranded RNA Binding Motif (dsRBM) at the N-terminus and the kinase domain at the C-terminus, which becomes catalytically active upon polymerization[2].

During viral infection, PKR recognizes long double-stranded RNA (greater than 30 base pairs) through its dsRBM and undergoes self-oligomerization[3]. Because most endogenous dsRNAs present in the cytoplasm are typically shorter than 23 base pairs, this mechanism allows PKR to specifically recognize viral dsRNA.

The subsequent polymerization of the kinase domain leads to the phosphorylation and inhibition of eIF2α, a key factor required for mRNA translation[3]. This process halts viral protein synthesis and thereby suppresses viral replication within the host cell.

In our system, COCCO, as well as in the preceding study DRACO, we aim to achieve a more definitive inhibition of viral replication by bypassing the translational arrest step and directly inducing apoptosis following PKR-mediated dsRNA detection.

Fig 2-A. PKR Domain

The PKR protein contains two dsRBMs at its N-terminus and a kinase domain at its C-terminus. Both the DRACO and COCCO systems employ the N-terminal region, specifically aa 1–181, as the dsRNA-sensing module[11].

Fig 2-B. PKR Structure

DsRBM1 and dsRBM2 together form the complete dsRBD(green), which is followed by the enzymatic kinase domain(yellow).

RIG-I

RIG-I is an intracellular innate immune factor. Mammals and mallard ducks possess this factor; however, interestingly, no gene corresponding to this factor has been identified in chickens. Recently, a study reported the creation of transgenic chickens by cloning and introducing duck RIG-I into the chicken genome [4]. According to this report, chickens transplanted with RIG-I exhibited an altered immune response to infection by the H7N1 type of avian influenza. Although this "change" did not necessarily result in the acquisition of influenza resistance in chickens, it provided important evidence that RIG-I is capable of detecting the dsRNA of influenza viruses within chicken cells.

Encouraged by this report, we decided to employ RIG-I, in addition to PKR, as a dsRNA sensor. RIG-I preferentially recognizes and oligomerizes on dsRNA shorter than 1000 base pairs that possesses a triphosphate group at the 5′ end. After binding to dsRNA, RIG-I is predicted to slide along the dsRNA while oligomerizing and to dissociate after forming a tetramer [5]. Following dissociation, it connects to the downstream MAVS protein and induces the production of interferon [6].

RIG-I is composed of three domains [6]. The N-terminus contains two CARD (caspase activation and recruitment domain) motifs that activate the downstream protein MAVS. The C-terminal side contains the CTD (C-terminal Domain), which recognizes the triphosphate group of dsRNA. Between these domains lies the Helicase Domain, which recognizes and binds to dsRNA. In the absence of dsRNA, the CARD domains of RIG-I are bound to the Helicase Domain and remain unexposed. This interaction prevents CARD from oligomerizing and activating downstream interferon production in the absence of dsRNA [7]. When dsRNA is present, it binds to the Helicase Domain, causing the CARD domains to dissociate and be released into the solution. The exposed CARD domains then interact with the downstream factor MAVS, thereby promoting interferon expression.

In the COCCO system, the region of RIG-I excluding the CARD domains (the CTD and Helicase Domain) is used as the dsRNA sensor. In experiments using human cultured cells (HEK293), human RIG-I was employed, whereas in experiments using chicken cultured cells (DF-1), mallard duck RIG-I was used [4].

Fig 3. RIG-I Domain Information

At the N-terminus, RIG-I contains two CARD motifs that connect to MAVS, followed by the Helicase Domain and the CTD. In the COCCO system, a CARD-deleted form of RIG-I (aa: 187–925) is utilized as the dsRNA sensor. An Apoptosis Inducer is fused to the region corresponding to the original CARD domains of RIG-I [8].

APAF1

APAF1 undergoes cytochrome c-dependent self-polymerization when mitochondria-mediated intrinsic apoptosis is induced. This polymerization forms an apoptosome, which subsequently induces apoptosis. During polymerization, the apoptosome promotes the oligomerization of Procaspase-9, thereby converting it into the active form, Caspase-9[9]. Activated Caspase-9 then initiates the apoptotic cascade.

APAF1 comprises two domains: a cytochrome c receptor domain and a CARD, which recruits and polymerizes Procaspase-9. The previously mentioned RIG-I also contains two CARD motifs. While these CARDs share sequence similarity, it is critical to note that they interact with different binding partners. In contrast to the RIG-I CARD, which recruits MAVS, the APAF1 CARD recruits Procaspase-9.

In both DRACO and COCCO, only the CARD domain of APAF1 is isolated and cloned for use as the Apoptosis Inducer[10].

Fig 4. APAF1 Structure

The N-terminus contains the Cytochrome c receptor (blue), and the C-terminus contains the CARD (yellow).

ΔCaspase9

ΔCaspase9 is Caspase9 with the self-oligomerization domain removed. It is composed solely of the enzymatically active portion responsible for inducing downstream apoptosis. This molecule was selected for a strategy that involves directly oligomerizing Caspase itself, which differs from the approach of promoting Caspase oligomerization by oligomerizing the CARD domain of APAF1.

ΔCaspase9 is an apoptosis-inducing protein also utilized in iCaspase9 (Inducible Caspase9). Due to its reduced capacity for self-oligomerization, it can be used with a favorable safety profile [11].

Fig 5. iCaspase9 Structure

The Drug-binding Domain is located at the N-terminus. When this domain binds to a specific drug, it undergoes oligomerization, causing ΔCaspase9 to oligomerize as well, which ultimately induces apoptosis.

Fig 6. Overview diagram summarizing the intracellular function of APAF1 and Caspase9

APAF1 forms the apoptosome in a cytochrome C-dependent manner, which then promotes the oligomerization of Procaspase9.