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Host-cell Interaction Partners for IBV Accessory Proteins

1.1.1      Betacoronaviruses

Betacoronaviruses are an important genus of coronaviruses regarding human health and include human coronavirus HKU1 (HCoV-HKU1), severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) along with the mouse hepatitis coronavirus (MHV) (Table 1) (To, Hung et al. 2013). These viruses can encode numerous accessory proteins with a variety of functions. SARS-CoV encodes eight known accessory proteins, 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9 (Narayanan, Huang et al. 2008), while MERS-CoV expresses four accessory proteins, 3, 4a, 4b, and 5, encoded between gene S and E. SARS-CoV accessory proteins have been studied extensively and have been implicated in a wide variety of functions. SARS-CoV 3a is a minor structural protein and has been detected in VLPs by electron microscopy (Ito, Mossel et al. 2005). Deletion of ORF3a showed a log drop in viral titres compared to wild-type suggesting although 3a is non-essential for replication, it may be beneficial (Yount, Roberts et al. 2005). SARS-CoV 3b is also expressed from mRNA3 and localises to both the nucleus and mitochondria where it inhibits MAVS signalling and IRF3 activation, respectively (Spiegel, Pichlmair et al. 2005, Freundt, Yu et al. 2009). Protein 3b has an apoptotic phenotype when expressed alone and further modulates the immune response by binding to the transcription factor, Runt Related Transcription Factor 1b (RUNX1b) (Khan, Fielding et al. 2006, Kopecky-Bromberg, Martinez-Sobrido et al. 2007). SARS-CoV protein 6 is not required for replication but does play a role in virulence (Zhao, Falcon et al. 2009). GFP-tagged protein 6 has been shown to localise to the ER and Golgi apparatus (Geng, Liu et al. 2005, Pewe, Zhou et al. 2005). Overexpression of protein 6 inhibits interferon-beta (IFNβ) expression and prevents signal transducer and activator of transcription 1 (STAT1) nuclear translocation in IFN treated cells by sequestering the STAT1 nuclear import factor karyopherin alpha 1 (KPNA1) (Zhao, Falcon et al. 2009). Deletion of protein 7a has no effect on SARS-CoV replication in both in vitro and in vivo (Pekosz, Schaecher et al. 2006). Overexpression of protein 7a has been shown to induce cell-cycle arrest and induce caspase-dependent apoptosis as well as inhibit cellular translation (Tan, Fielding et al. 2004, Kopecky-Bromberg, Martinez-Sobrido et al. 2006, Tan, Tan et al. 2007). Protein 7b is also dispensable for viral replication in vitro (Yount, Roberts et al. 2005, Schaecher, Touchette et al. 2007). Conversely, recombinant virus lacking ORF7b resulted in higher titres in vivo suggesting this protein may play a role in attenuation (Pfefferle, Krahling et al. 2009). MERS-CoV encodes four accessory proteins between gene S and E, proteins 3, 4a, 4b and 5. Protein 3 and 5 localise to the ER-GIC while protein 4a and 4b have a diffuse cytoplasmic distribution as well as a nuclear localisation. Protein 4a, 4b, and 5 are all IFN antagonists. Protein 4a binds to dsRNA to prevent detection by MDA-5 and RIG-I (Niemeyer, Zillinger et al. 2013). In addition, protein 4a has been shown to inhibit the stress granule pathway, increasing cellular translation (Rabouw, Langereis et al. 2016). MERS-CoV also contains an ORF known as 8b in the N gene that has not yet been characterised (van Boheemen, de Graaf et al. 2012, Raj, Osterhaus et al. 2014).

1.1.2      Gammacoronaviruses

IBV is the prototype coronavirus within this genus, with TCoV considered a close relative (Guy 2000). IBV is known to express four accessory proteins during infection, 3a, 3b, 5a and 5b (Casais, Davies et al. 2005, Hodgson, Britton et al. 2006). TCoV also expresses these four accessory proteins which all have a high sequence identity with IBV (Britton P 2007).

1.1.3      Deltacoronaviruses

Deltacoronaviruses is the most recently described genus within the coronavirinae subfamily and as such little is known about this genus of coronaviruses, including the function of the accessory proteins. These viruses mainly infect wild birds and pigs (Woo, Lau et al. 2012). The prototype virus, porcine deltacoronavirus (PDCoV), is predicted to express four accessory proteins, ORF6 located between gene M and N, and ORF7, encoding 7a, 7b, and 7c, located downstream of N (Woo, Lau et al. 2012, Fang, Fang et al. 2016). Currently, only accessory proteins 6 and 7a have been shown to be translated during infection. Production of sgRNA7a is regulated by a non-canonical TRS (Fang, Fang et al. 2017).

Table 3 The role of coronavirinae accessory proteins

Coronavirus Name Role
FIPV 3a, b, c Restricts cell tropism to the intestine (Adam Balint 2014)
7a, 7b IFN antagonist (dependent on ORF3) (Annelike Dedeurwaerder 2014)
TGEV 3a Limited role in virulence and cell tropism (Kim, Hayes et al. 2000)
3b Limited role in virulence and cell tropism (Galan, Sola et al. 2009)
7 Deletion decreases cell death and cytopathic effect. 

Interacts with phosphatase protein 1c (Ortego, Sola et al. 2003, Cruz, Sola et al. 2011)

HCoV 229E 4a Viroporin (Zhang, Wang et al. 2014)
PRCoV 3a, 3b Unknown
MERS-CoV 3 Unknown
4a IFN antagonist(Siu, Yeung et al. 2014) 

Stress granule inhibitor (Rabouw, Langereis et al. 2016)

4b IFN antagonist (Yang, Ye et al. 2015)
5 IFN antagonist (Yang, Zhang et al. 2013)
8b Unknown
MHV HE Sialate-O-acetylesterase (Siddell, Wege et al. 1983) 

Non-essential structural protein (Lissenberg, Vrolijk et al. 2005)

2a Cleaves 2′-5′-oligoadenylate synthase (OAS) (Zhao, Jha et al. 2012)
4a Role in pathogenicity  (de Haan, Masters et al. 2002) 

IFN antagonist

Stress granule inhibitor (Raaben, Groot Koerkamp et al. 2007)

5a IFN antagonist (Koetzner, Kuo et al. 2010)
SARS-CoV 3a Induces cellular membrane rearrangement {Ito, 2005 #412} 

Activates the PERK pathway [37]

3b IFN antagonist by inhibiting IRF3 activity 

Interacts with activator protein 1 (AP-1)

Interacts with runt-related transcription factor 1b (RUX1b) (McBride and Fielding 2012)

6 IFN antagonist 

Interacts with N-Myc and karyopherin α2

Essential for replication and present in virions

Induces cellular DNA synthesis(McBride and Fielding 2012)

7a Induces apoptosis in a caspase-3 and p38 MAPK dependent manner  Interacts with virions (McBride and Fielding 2012) (Tan, Tan et al. 2007)
7b Non-essential for replication 

Role in apoptosis (Schaecher, Touchette et al. 2007)

8a Forms an ion-channel in lipid bilayers (Chen, Kruger et al. 2011)
8b Inhibits expression of the E viral protein (McBride and Fielding 2012)
9b Apoptosis inducer 

Interacts with chromosomal maintenance (CRM1)

Associated with virions (McBride and Fielding 2012)

IBV 3a,3b No role in replication in vitro (Kint, Fernandez-Gutierrez et al. 2015) 

Regulates expression of IFNβ(Casais, Davies et al. 2005, Hodgson, Britton et al. 2006)

5a No role in replication in vitro (Casais, Davies et al. 2005, Hodgson, Britton et al. 2006)
5b Host-cell translational shut-off (Kint, Langereis et al. 2016)
PDCoV 6 Detected during infection (Fang, Fang et al. 2016)
ORF7 Potentially encodes three accessory proteins, only 7a has been detected (Woo, Lau et al. 2012, Fang, Fang et al. 2017)

Alphacoronavirinae (red), Betacoronavirinae (blue), Gammacoronavirinae (green), Deltacoronavirinae (yellow), FIPV (feline infectious peritonitis virus), FCoV (feline coronavirus), TGEV (transmissible gastroenteritis coronaviruses), MERS-CoV (Middle East respiratory syndrome coronavirus), MHV (mouse hepatitis virus), SARS-CoV (serve acute respiratory syndrome coronavirus), IBV (infectious bronchitis virus), PRCoV (porcine respiratory coronavirus), PDCoV (Porcine deltacoronavirus)

1.2      IBV Accessory Proteins

The 3′-end of the coronavirus genome encodes for accessory proteins as well as the structural proteins. IBV expresses four known accessory proteins during infection, 3a, 3b, 5a, and 5b. These accessory proteins are dispensable for replication in vitro (Britton, Casais et al. 2006). Along with these four proteins, IBV has also been shown to produce an additional transcript referred to as 4b, although translation of this transcript has not yet been demonstrated (Bentley, Keep et al. 2013). Coronavirus accessory proteins are non-essential for in vitro replication but are commonly highly conserved and often play a role in regulating IFN expression, cellular translation and apoptosis (Table 3) (Liu, Fung et al. 2014). While coronavirus accessory proteins may share similar functions, they have limited sequence homology to one another.

1.2.1      Gene 3 accessory proteins

Messenger RNA 3 is polycistronic and encodes three proteins, accessory proteins 3a, 3b and structural protein E. IBV 3a and 3b have been detected during IBV infection in chicken kidney (CK) cells utilising antibodies raised against the predicted peptide sequence (Liu, Cavanagh et al. 1991). Recombinant IBVs that do not express 3a and 3b due to a scrambled start codon, grow to similar titres in vitroin ovo, and in ex vivo organ culture, suggesting these proteins are not required for in vitro replication(Hodgson, Britton et al. 2006).

IBV 3a is a 6 kDa protein and the first protein expressed from mRNA 3. IBV 3a is highly conserved among the many different IBV strains, with an 81 – 86.2% similarity in polypeptide sequences (Jia and Naqi 1997). The relatively high degree of sequence preservation suggests an important role for this protein. IBV 3a contains a signal peptide which directs 3a to the ER membrane, but due to the small size of 3a, the signal peptide sequence is not effectively recognised by signal recognition particles (Pendleton and Machamer 2005). This explains the dual localisation pattern seen for 3a during infection and transfection in Vero cells, which is either membrane-bound at the smooth ER or diffuse in the cytoplasm (Pendleton and Machamer 2005).  This theory is further supported by the fact that extension of 3a with a GFP-tag resulted in more membrane-bound 3a, compared to 3a expression alone. During infection in Vero cells, membrane-bound 3a has been shown to span the membrane once, with the C-terminus exposed to the cytoplasm and the N-terminus to the lumen of the smooth ER (Pendleton and Machamer 2005). IBV 3a has been shown to closely localise with MxA, which is a small GTPase that plays a role in vesicle formation and has anti-viral activity (Pendleton and Machamer 2005). Protein 3a has also been shown to inhibit IFN mRNA transcription at 24 h.p.i, while also inducing IFN protein expression at 36 h.p.i (Kint, Fernandez-Gutierrez et al. 2015). The mode of action of IBV 3a on IFN expression is unknown.

IBV 3b is a 7.4 kDa protein and the second protein translated from sgRNA 3 via leaky ribosomal scanning (Liu and Inglis 1992). IBV 3b predominantly localises to the nucleus in mammalian cells while in avian cells appears predominantly in the cytoplasm (Pendleton and Machamer 2006). Furthermore, IBV 3b turnover is proteasome-dependent in mammalian cells and proteasome-independent in avian cells. The differences seen in avian and mammalian cells highlights the importance of using an appropriate cell line for experiments. In addition, the half-life of 3b is short, making it very difficult to detect the protein during both infection and transient expression (Pendleton and Machamer 2006). A truncated form of 3b has been detected in a strain of Beau-CK serially passaged in Vero cells. This truncation was an advantageous mutation that conferred higher growth kinetics in vitro and higher virulence in ovo (Shen, Wen et al. 2003). Interestingly, this truncation changed the localisation of 3b from the nucleus to a diffuse cytoplasmic pattern in Vero cells, suggesting the C-terminal proportion is responsible for nuclear localisation in mammalian cells (Pendleton and Machamer 2006). There is little understanding of the function and mechanism of this small protein, although previous work has shown that 3b plays a role in conferring IBV resistance to IFN expression (Kint, Fernandez-Gutierrez et al. 2015).

1.2.2      Transcript 4b

Positioned between the M gene and gene 5 is the intergenic region (IR), which contains a putative ORF known as 4b. Due to the lack of an upstream TRS, this ORF was previously thought to be a pseudogene (Stern and Kennedy 1980). However, Bentley et al (2013)showed by northern blot that a sgRNA is expressed from the intergenic region during Beau-R infection, referred to as sgRNA 4b (Bentley, Keep et al. 2013). The presence of sgRNA 4b was also identified in RNA extracted from M41 infected bird trachea. While other proteins have an upstream canonical TRS (CUUAACAA), gene 4b is regulated by a non-canonical TRS (CAA). This non-canonical TRS results in transcription of mRNA 4b at a lower level than expected for its genome location. Furthermore, the related coronavirus TCoV also expresses this previously unidentified 4b transcript. The 4b transcript is not required for in vitro replication, suggesting if a protein is translated, it is most likely an accessory protein (Bentley, Keep et al. 2013). Replacement of ORF4b with GFP did result in GFP expression, suggesting that the transcript can act as a mRNA. The gene is also present in the Beau-R strain of IBV, although the potential protein is truncated to 5 kDa due to a stop codon present in the middle of the ORF (Bentley, Keep et al. 2013). Whether this transcript is translated during IBV infection is not known, as is the function of this putative protein.

1.2.3      Gene 5 accessory proteins

Messenger RNA 5 is dicistronic and encodes two accessory proteins, 5a and 5b, 8 kDa and 9 kDa respectively (Liu and Inglis 1992). These proteins have been detected during IBV infection in CK cells using antibodies raised against the predicted amino acid sequences (Liu and Inglis 1992). Recombinant IBV lacking 5a or 5b grew to similar titres to wild-type in vitroex vivo, and in ovo, suggesting they are not essential for replication (Casais, Davies et al. 2005). Protein 5a displays a diffuse pattern throughout the whole cell while 5b displays a more perinuclear granular pattern (Davies 2009). Kint et al (2014) showed that protein 5b is involved in host translational shut-off. Beau-R inhibits cellular translation while recombinant IBV lacking 5b expression does not. Translation of IBV proteins is not affected, suggesting 5b may target host-cell translation specifically. This reduction in host-cell translation can explain, in part, why there are high levels of IFN-β mRNA but low levels of IFN-β protein during IBV infection (Kint, Langereis et al. 2016).

1.3      Coronavirus–Host Interaction

1.3.1      Interferon response to viral infection

The IFN signalling cascade is a cellular response to infection and is one of the first lines of defence against invading pathogens. The innate immune response detects ‘non-self’ signals known as pathogen-associated molecular patterns (PAMPs). These PAMPs include dsRNA, 5′-C-phosphate-G-3′ (CpG), lipopolysaccharides and dsDNA, among others, and are recognised by specific pathogen recognition receptors (PRRs) (Thompson, Kaminski et al. 2011). Cellular detection of PAMPs activates the IFN signalling cascade to induce the expression of IFNs, cytokines, and chemokines (Wu and Chen 2014). IFN induces the expression of IFN stimulated genes (ISGs), which induces an anti-viral state in the cell and neighbouring cells to prevent further viral replication (Stark and Darnell 2012).  PRRs responsible for the detection of viral PAMPs such as dsRNA include RIG-I-like receptors (RLRs), and TOLL-like receptors (TLRs). So far 10 TLRs and 2 RLRs have been identified in domestic fowl compared to 11 and 3 in humans, respectively (Kannaki, Reddy et al. 2010). While chickens express the membrane-bound PPRs, TLR3 and TLR7, used to detect dsRNA and ssRNA, respectively, chickens do not express the cytosolic dsRNA sensing PPR, RIG-I (Zou, Chang et al. 2009). However, the other dsRNA sensing RLRs in humans, MDA-5 and Laboratory of Genetics and Physiology 2 (LGP2) are expressed (Barber, Aldridge et al. 2013). MDA-5 detects and binds to dsRNA, and subsequently interacts with and activates the mitochondrial anti-viral signalling protein (MAVS). MAVS is a membrane-bound protein that localises to the mitochondria, mitochondrial-associated membrane structures (MAMS) and peroxisomes (Seth, Sun et al. 2005). MDA-5 activation of MAVS causes aggregation of MAVS, which then recruits downstream signalling proteins including TANK-binding kinase 1 (TBK1), NF-kappa-B essential modulator (NEMO) and TNF receptor-associated factor 3 (TRAF3) (Kawai, Takahashi et al. 2005). Phosphorylation of TBK1 leads to the phosphorylation of interferon regulatory factors 3 (IRF3) and 7 (IRF7). Phosphorylated IRF3/7 dimerises and translocates to the nucleus inducing the transcription of IFNβ mRNA (Figure 1.4). Chickens express an IRF that closely resembles IRF7 but appear to have selectively lost IRF3 (Grant, Vasa et al. 1995, Cormican, Lloyd et al. 2009). After translation, IFNβ is secreted from the cell and binds to interferon receptors (IFNAR) on the same cell and neighbouring cells, activating the JAK-STAT pathway (Stark and Darnell 2012). This pathway leads to the dimerization of STAT3 and STAT7 which mediates expression of ISGs. ISGs include a wide variety of proteins involved in creating an anti-viral state within the cell leading to viral suppression along with immunomodulation. Overexpression of IFN can induce a hyper-immune state which can be deleterious to the body. For this reason, the IFN signalling cascade is highly regulated after activation by controlling levels of IFN signalling proteins including MAVS and RIG-I (Lin, Yang et al. 2006, Castanier, Zemirli et al. 2012, Fuchs 2012, Hu and Sun 2016). MAVS, as the gateway protein of the IFN signalling cascade, is targeted for degradation in a negative-feedback loop by E3-ligases Tripartite motif-containing protein 25 (TRIM25), glycoprotein 78 (gp78), and ring finger protein 5 (RNF5) to prevent further downstream stimulation. This helps to turn off the IFN signalling cascade after infection (Zhong, Zhang et al. 2010, Castanier, Zemirli et al. 2012, Jacobs and Coyne 2013).

1.3.2      Viruses and the interferon response

The innate immune response represents a significant barrier to viral replication. The selective pressure of the IFN response has created an evolutionary arms race, with viruses that inhibit this pathway having an advantage. Many viruses express proteins that antagonise IFN expression, with the aim to prevent the cell inducing an anti-viral state. These proteins can target the signalling cascade at any point in the pathway, from the detection of PAMPs to the translocation of IRFs into the nucleus, to the expression of IFN. A classic example of this is the multi-functional non-structural 1 (NS1) protein of Influenza A virus (IAV), which shields dsRNA from RLR detection, preventing MAVS activation and thus IFN expression (Hatada and Fukuda 1992). Accessory proteins, nsps and structural proteins in coronaviruses have been shown to inhibit IFN expression. SARS-CoV N protein is a potent IFN antagonist and inhibits IFN expression by interfering with TRIM25-mediated RIG-I ubiquitination (Hu, Li et al. 2017). MERS-CoV proteins M and 4b inhibit IFN expression by inhibiting TBK1 phosphorylation and MDA-5 dsRNA sensing, respectively (Niemeyer, Zillinger et al. 2013, Lui, Wong et al. 2016). Nsp3 from SARS-CoV and CoV-NL63 have also been shown to antagonise IFN expression independent of protease activity (Clementz, Chen et al. 2010, Sun, Xing et al. 2012). Alpha- and Betacoronaviruses nsp1 have been shown to induce host translation shut-off, limiting translation of IFN proteins (Kamitani, Narayanan et al. 2006) (Tohya, Narayanan et al. 2009) (Huang, Lokugamage et al. 2011) (Wang, Shi et al. 2010). IBV lacks nsp1, but most likely expresses multiple proteins that target the IFN signalling cascade. The IFN response to IBV infection has been investigated extensively by Kint et al (2015). MDA-5 is an essential PPR for the recognition of dsRNA and is required for the initiation of the IFN signalling cascade during IBV infection (Kint, Fernandez-Gutierrez et al. 2015). IFNβ expression is the main IFN subtype produced during IBV infection, with interferon-α (IFNα) undetectable (Kint 2015). IBV can inhibit IFNβ mRNA expression during infection but only up to 24 h.p.i. IBV accessory proteins 3a and 3b have both been shown to inhibit IFN expression by an uncharacterised mechanism. Recombinant IBV lacking 3a expression has also been further shown to induce IFN expression 36 h.p.i, suggesting 3a also induced IFN expression. The mechanism of 3a and 3b action on IFN expression is not understood. Interestingly, IBV infected cells once stimulated with poly(I:C) show increased IFN expression compared to either poly(I:C) transfection or IBV infection alone, suggesting IBV agonises IFN expression only after stimulation with poly(I:C) (Kint, Fernandez-Gutierrez et al. 2015).

1.3.3      Stress Granule pathway

The stress granule (SG) pathway is a cellular response to external and internal stimuli that induce cellular stress. The SG pathway is induced to preserve resources and energy until a return to cellular homeostasis is achieved (Buchan and Parker 2009). A range of stimuli can activate the stress granule pathway including ER stress, through the protein kinase R (PKR)-like ER kinase (PERK) pathway, nutrient starvation through General control non-derepressible 2 (GNC2) pathway, and heme deficiency and oxidative stress through the Heme-regulated eIF2α kinase (HRI) pathway (Beckham and Parker 2008, Lian and Gallouzi 2009, Narayanaswamy, Levy et al. 2009, Moutaoufik, El Fatimy et al. 2014). The SG pathway can also be activated during infection to inhibit viral protein translation. During infection, viral dsRNA produced during replication can be detected by protein kinase R (PKR) (Figure 1.5A), which in turn phosphorylates the serine residue in eukaryotic inhibition factor 2 (eIF2) at position 51 (Figure 1.5B) (Nanduri, Rahman et al. 2000, Dauber and Wolff 2009). Compared to the unphosphorylated form, the phosphorylated form of eIF2 has a higher affinity for the eukaryotic initiation factor 2B (eIF2B). This higher affinity binding to eIF2B prevents eIF2 from exchanging GDP for GTP, which is essential for recruitment of the initiator methionine transfer RNA (tRNA) (Figure 1.5C). Protein phosphatase 1 (PP1) is the primary regulatory protein of eIF2α-induced translational arrest and can directly bind to and dephosphorylate eIF2α to return the protein to its active state (Figure 1.5G). Stalled initiation complexes aggregate forming large granular structures within the cytoplasm referred to as SGs (Kedersha, Gupta et al. 1999). SGs contain stalled mRNAs, translation initiation factors and small ribosomal subunits along with SG regulatory proteins, such as T cell intracellular antigen-1 (TIA-1) and TIA-1 related protein (TIAR) (Figure 1.5D). SG regulatory proteins play an integral role in SG formation and the shuttling of mRNA in and out of SGs (Kedersha, Cho et al. 2000). The Ras-GTPase activating protein-binding protein-1 (G3BP1) is essential for SG formation and is activated during cellular stress (Tourriere, Chebli et al. 2003). SGs are highly dynamic structures that can respond quickly to changes in the cellular environment, and the precise composition of SGs varies depending on the stress stimuli (Kedersha, Stoecklin et al. 2005). Viruses lack their own translational system and are thus dependent on the host-cell machinery for production of viral proteins (Beckham and Parker 2008). Stress-induced translational shut-off by the host, in theory, prevents translation of both cellular and viral proteins, preventing further viral replication. If the infection is not resolved mRNA within SGs can be shuttled to processing-bodies (p-bodies) where they are targeted for degradation (Figure 1.5E) (Balagopal and Parker 2009).  If the infection is cleared, SGs can disassemble and translation can be reinitiated (Figure 1.5F) (Mollet, Cougot et al. 2008).

1.3.4      Viruses and the stress granule pathway

Host triggered translational-shutdown, and SGs are a significant barrier to viral infection, preventing synthesis of viral proteins required for replication and assembly. For this reason, several viruses have been identified that express proteins that modulate SG formation and eIF2 phosphorylation. Sendai virus inhibits SG formation by interacting with TIA-1/TIAR through an AU-rich domain in viral RNA transcripts (Iseni, Garcin et al. 2002). While poliovirus (PV) 3c protease cleaves G3BP1 disrupting SG formation (White, Cardenas et al. 2007). Semliki Forest virus (SFV) and Mammalian orthoreovirus (MRV) inhibit SG formation in a time-dependent manner, with SGs only detectable early on in infection (Qin, Hastings et al. 2009, Qin, Carroll et al. 2011). Other viruses have been shown to target PKR and eIF2α. Indeed, the African swine fever virus (ASFV) DP71L protein can bind to and recruit PP1c to eIF2α to induce dephosphorylation, while IAV NS1 can bind to dsRNA preventing detection by PKR (Bergmann, Garcia-Sastre et al. 2000, Zhang, Moon et al. 2010). Alternatively, viruses can also induce SGs to inhibit cellular protein expression and to facilitate viral replication. Respiratory syncytial virus (RSV) replication benefits from the presence of SGs, while Newcastle disease virus (NDV) induces SGs to reduce global host-translation while maintaining viral protein translation by an as yet unknown mechanism (Sun, Dong et al. 2017).

Within the coronavirinae subfamily MERS-CoV, TGEV and MHV have been investigated for their ability to manipulate the SG pathway. MERS-CoV accessory protein 4a inhibits dsRNA-mediated PKR-dependent SG formation by binding to dsRNA directly. Conversely, MHV has been shown to induce eIF2α phosphorylation and SG formation (Raaben, Groot Koerkamp et al. 2007, Rabouw, Langereis et al. 2016). SGs also appear during TGEV infection with the occurrence of SGs linked to decreased viral replication (Sola, Galan et al. 2011). IBV accessory protein 5b has been shown to inhibit host-cell translation. Furthermore, previous work has shown that in a proportion of Beau-R infected cells SGs are assembled, although independent of 5b (Kint 2015). Simultaneously, Beau-R infected cells could strongly prevent sodium arsenite-induced SG formation, suggesting that while IBV infection can induce SGs, the virus also expresses an as yet unknown protein or proteins that can inhibit their formation too (Kint 2015). Collectively, this highlights the dynamic and responsive nature of mRNA movement between active ribosomes, SGs and P-bodies during IBV life-cycle (Decker and Parker 2012).

1.3.5      Apoptosis

Apoptosis or programmed cell death (PCD) is a cellular response to extreme stress, wherein cellular homeostasis is no longer viable, and cell death is preferable. During viral infection, the cell can induce apoptosis vto reduce viral progeny release, reducing disease outcome in the host. Apoptosis is regarded as the last resort for infected cells, although can be favourable to both prevent viral spread and to activate other parts the immune system (Campisi, Cummings et al. 2014). Apoptosis is a highly-regulated system that can be triggered by two main pathways; the extrinsic and intrinsic pathways, which are triggered by the activation of the death receptor or mitochondrial damage, respectively (Thorburn 2004). Each cascade leads to the cleavage of caspase-3, which results in DNA and protein degradation and irreversible apoptosis (Figure 1.8) (Cohen 1997). Although both pathways have a role in the immune response to infection, the intrinsic pathway is the primary cellular response to viral infection, for which mitochondria play an important role (Benedict, Norris et al. 2002). Mitochondria are the powerhouse of the cell and contain a matrix surrounded by an inner membrane (IM), and an intermembrane space surrounded by an outer membrane (OM). Under normal conditions, these membranes help to protect the cell from catabolic enzymes while also creating an electrical imbalance for ATP production (Bertram, Pedersen et al. 2006, Kroemer, Galluzzi et al. 2007). The integrity of the mitochondrial membranes is maintained by a balance of anti-apoptotic and pro-apoptotic regulatory factors, including members of the B-cell lymphoma 2 (Bcl-2) protein family. Viral infection can cause a shift in balance from anti- to pro-apoptotic factors through a range of cellular pathways, including Ca2+ release from ER stress and dsRNA detection, while production of reactive oxygen species (ROS) can directly affect mitochondrial membrane integrity (Figure 1.8A). ROS and pro-apoptotic factors lead to mitochondrial membrane permeabilisation (MMP) (Figure 1.8B), which causes the release of intermembrane proteins including cytochrome C. These factors are released through mitochondrial pores composed of Bcl-2-associated X (Bax) and Bcl-2 homologous antagonist killer (Bak) proteins, as well as mitochondrial channels such as the Voltage-dependent anion channel (VDAC) (Figure 1.8C). Once released into the cytoplasm, cytochrome C recruits the apoptosis protease-activating factor 1 (apaf-1) and pro-caspase 9 to form the apoptosome (Figure 1.8D). This multiprotein complex leads to stimulation of the irreversible apoptosis cascade resulting in activation of cysteine proteases such as caspase 3 and 7.  These proteases induce a range of cellular modifications, including membrane blebbing, DNA fragmentation, cellular shrinkage and finally cell death. Mitochondria can also release endonuclease G (EndoG) and apoptosis-inducing factor (AIF) to induce caspase-independent apoptosis (Li, Luo et al. 2001, Cande, Vahsen et al. 2004).

1.3.6      Viruses and Apoptosis

Viruses can express both anti-apoptotic proteins and/or pro-apoptotic proteins. Anti-apoptotic proteins increase the length of time that the cell is viable for virus replication while pro-apoptotic proteins can aid viral release and pathogenicity (Barber 2001, McLean, Ruck et al. 2008). Indeed, many oncogenic viruses encode anti-apoptotic proteins to establish persistent infections aiding disease progression and the formation of tumours (Fuentes-Gonzalez, Contreras-Paredes et al. 2013).  Epstein-Barr virus (EBV) expresses a viral Bcl-2 homologue, BHRF1, which localises to the mitochondrial membrane and stabilises membrane integrity by binding to and thus inhibiting pro-apoptotic factors (Kvansakul, Wei et al. 2010). While apoptosis is a cellular response to infection and stress, viruses do not necessarily solely inhibit this pathway. Influenza A virus (IAV) protein PB1-F2 and infectious bursal disease virus (IBDV) protein VP5 bind to the outer mitochondrial membrane-bound proteins VDAC1 and VDAC2 respectively, inducing MMP resulting in cytochrome C release and caspase-3 activation (Zamarin, Garcia-Sastre et al. 2005, Li, Wang et al. 2012). SARS-CoV accessory protein 7a induces caspase-dependent apoptosis by binding to the anti-apoptotic protein B-cell lymphoma extra-large (BcL-XL). However, the role of SARS-CoV protein 7a during infection is unknown as deletion of 7a does not affect replication or pathogenicity(Tan, Fielding et al. 2004)(Tan, Tan et al. 2007).No anti-apoptotic proteins have been identified for coronaviruses to date, although SARS-CoV has been shown to activate the anti-apoptotic AKT pathway (Mizutani, Fukushi et al. 2004). Caspase-dependent apoptosis has been observed during IBV infection in Vero cells. However, inhibition of apoptosis had no significant effect on IBV titres suggesting that apoptosis is not required for replication in vitro (Liu, Xu et al. 2001). Furthermore, IBV-induced apoptosis is in part inhibited and modulated by the pro-survival IRE1α-XBP1 ER stress pathway, suggesting IBV actively induces apoptosis during infection (Fung, Liao et al. 2014).


Figure 1.8 Schematic diagram of the intrinsic apoptosis pathway. (A) Internal stimuli including ER stress, viral infection, ER stress, DNA damage or pro-apoptotic proteins can increase levels of cytosolic Ca2+ and ROS levels or inactivate anti-apoptotic proteins. (B) ROS and Ca2+ can permeabilise the mitochondrial membrane, while pro-apoptotic proteins can form pores. (C) This permeabilisation releases apoptosis effector proteins including cytochrome C, AIF and EndoG. (D) Cytochrome C assembles with pro-caspase 9 and APAF-1 leading to the formation of the apoptosome. (E)  The apoptosome leads to activation of pro-caspase 3/7. (F) Caspase 3/7 along with caspase-independent effectors (AIF, EndoG) mediate a range of cellular changes including DNA fragmentation and membrane blebbing resulting in cellular apoptosis.

1.4      Aims

The aim of this project is to identify any host-cell interaction partners for IBV accessory proteins 3a and 3b that may allude to function and to determine the role of ORF4b. A series of objectives were established, to accomplish this aim.

Objective 1

The first objective is to utilise mass spectrometry to determine any cellular proteins that interact with IBV 3a and 3b that may allude to function.

Objective 2

The second objective is to characterise the role of 3a on the IFN response utilising IFN assays and mass spectrometry data.

Objective 3

To determine if ORF4b is translated into a protein during infection by optimising an antibody raised against the predicted peptide sequence or by using mass spectrometry.

Objective 4

To determine the role of ORF4b or the putative 4b protein using reverse genetics and/or mass spectrometry.

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