Cellular unfolded protein response against viruses used in gene therapy

Abstract

Viruses are excellent vehicles for gene therapy due to their natural ability to infect and deliver the cargo to specific tissues with high efficiency. Although such vectors are usually "gutted" and are replication defective, they are subjected to clearance by the host cells by immune recognition and destruction. Unfolded protein response (UPR) is a naturally evolved cyto-protective signaling pathway which is triggered due to endoplasmic reticulum (ER) stress caused by accumulation of unfolded/misfolded proteins in its lumen. The UPR signaling consists of three signaling pathways, namely PKR-like ER kinase, activating transcription factor 6, and inositol-requiring protein-1. Once activated, UPR triggers the production of ER molecular chaperones and stress response proteins to help reduce the protein load within the ER. This occurs by degradation of the misfolded proteins and ensues in the arrest of protein translation machinery. If the burden of protein load in ER is beyond its processing capacity, UPR can activate pro-apoptotic pathways or autophagy leading to cell death. Viruses are naturally evolved in hijacking the host cellular translation machinery to generate a large amount of proteins. This phenomenon disrupts ER homeostasis and leads to ER stress. Alternatively, in the case of gutted vectors used in gene therapy, the excess load of recombinant vectors administered and encountered by the cell can trigger UPR. Thus, in the context of gene therapy, UPR becomes a major roadblock that can potentially trigger inflammatory responses against the vectors and reduce the efficiency of gene transfer.

Keywords: ER-homeostasis; ER-stress; UPR; chaperones; gene therapy; viral vectors.

Figure 1

Unfolded protein response signaling. The signaling is initiated by the activation of the proximal sensors of the unfolded protein response (UPR) namely, (1) protein kinase R (PKR)-like ER kinase (PERK), (2) activating transcription factor (ATF) 6 and (3) inositol-requiring enzyme 1 (IRE1). A protein called immunoglobulin heavy chain binding protein (BiP) functions as the master regulator. BiP under normal conditions remains attached to all the three sensors in the luminal domain of the endoplasmic reticulum (ER). Upon encountering any stress like accumulation of misfolded/unfolded proteins or a massive inflow of any exogenous proteins into the ER, the stress sensors, PERK, IRE1, and ATF6, are activated by the release of BiP from the sensors leading to any of the three distinct pathways. (1) When PERK is activated, it dimerises and autophosphorylates leading to phosphorylation of the eukaryotic translation initiation factor (elF) 2α. Activated elF2α represses global protein translation of the cell. However the downstream protein called ATF 4 can escape translational repression since it has upstream open reading frames leading to its activation. The activated ATF4 translocates into the nucleus activating a set of target genes to restore cellular homeostasis (adaptive response). However in situations when the cellular homeostasis cannot be restored, C/EBP homologous protein (CHOP) is activated leading to apoptosis. (2) When IRE1 is activated, it dimerizes and autophosphorylates leading to the activation of its endoribonuclease activity. This leads to an unusual splicing of XBP1 (X-box binding protein 1) cleaving 26 nucleotide intron within. The Spliced XBP1 (sXBP1) protein translocates to nucleus transcribing chaperones and unfolded protein response elements (UPREs) to restore cellular homeostasis. In some cases, the IRE1 activates the cellular JNK through phosphorylation. This activated JNK either leads to apoptosis by activaton of caspase 19 or leads to autophagy. Alternatively, IRE1 activates IKK by interacting with tumor necrosis factor receptor-associated factor 2 (TRAF2) which phosphorylates Iκ B. This releases nuclear factor (NF)-κ B. The activated NF-κ B translocates into the nucleus and transcribes inflammatory genes. (3) Activation of the third sensor of UPR, ATF6 leads to its translocation into the Golgi complex. In the golgi complex, ATF6 will be cleaved by proteases such as site-1 protease (S1P) and S2P. This cleaved ATF6 fragment further transcribes chaperones and UPRE to cope with the cellular stress and restore homeostasis (Yoshida et al., ; Lee et al., ; Harding et al., ; Novoa et al., ; Wu et al., ; Yamamoto et al., ; Raven et al., 2008).

Figure 2

(A) Herpes simplex virus (HSV-1) and UPR. HSV-1 produces proteins such as glycoprotein B (gB) and US11 that have been shown to evade the host UPR mechanism (Mulvey et al., 2003, 2007). In particular, the protein gB specifically binds to the PERK proteins preventing their phosphorylation. This leads to PERK inactivation and hence the downstream effector protein elF2α could not get activated leading to ATF4 repression. Alternatively another viral protein called US11 represses the elF2α phosphorylation by directly binding to it. The late HSV viral protein γ₁34.5 also induces dephosphorylation of eIF2α with the help of the cellular phosphatase PP1α (He et al., 1996). This leads to early repression of ATF4 and CHOP genes downstream. Thus the host UPR response is attenuated and leads to successful viral transduction. (B) Adenovirus (Ad) and UPR. Adenoviruses during their late phase of their infection, try to overcome the cellular stress response by preventing the shutdown of protein translation through PKR mediated inhibition of elF2α phosphorylation via viral associated RNA molecule I (VAI RNA) as well as double-stranded RNA-activated inhibitor (DAI) (Huang and Schneider, ; Mathews and Shenk, ; McKenna et al., 2006). Other Ad proteins such as E1B and E4 has also been found to directly bind to the elF2α, thus preventing its phosphorylation and activation of downstream UPR related genes like ATF4 and CHOP (Spurgeon and Ornelles, 2009). (C) Adeno associated virus (AAV) and UPR. When the cellular ER encounters AAV particles, specific stress sensors, PERK and IRE1 gets activated (Balakrishnan et al., 2013). PERK phosphorylation leads to the activation of the elF2α through phosphorylation. The phosphorylated elF2α further activates the activating transcription factor 4 (ATF4) the protein of which translocates into the nucleus transcribing UPR responsive genes necessary to cope up with the cellular stress. The phosphorylated elF2α also arrests the translation of cellular proteins to maintain homeostasis. It has been noted that the AAV particles also activates IRE1 which induces the unusual splicing of X-box binding protein 1 (XBP1) mRNA downstream. The XBP1 protein translocates into the nucleus activating a set of UPR responsive elements. The IRE1 also activates the IKK leading to NF-κ B upregulation. The activated NF-κ B further activates the inflammatory genes thus inducing an inflammatory response (Jayandharan et al., ; Balakrishnan et al., 2013). (D) Murine leukemia virus (MLV) and UPR. MLV based γ-retroviral vectors, which are the most common used in gene therapy, has been showed to induce neuropathogenecity in astrocytes (Dimcheff et al., 2003). Later in NIH3T3 cells it was shown that the murine retroviruses induce the ER stress related genes such as CHOP/GADD153 which leads to apoptosis (Dimcheff et al., 2003). On the other hand, the lentiviral proteins such as Tat and Nef have been shown to activate unfolded protein response elements (UPRE) by increasing ROS (Tiede et al., ; Abbas et al., 2012).