Role of phosphatidylinositol-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) signalling pathways in porcine reproductive and respiratory syndrome virus (PRRSV) replication
Introduction
Porcine reproductive and respiratory syndrome virus (PRRSV), the etiological agent for porcine reproductive and respiratory syndrome (PRRS), is an emerging viral infection in pigs that exerts severe economic losses to the pork industry worldwide. Recent economic estimates of annual losses to the US swine industry due to PRRSV infection amount to more than $660 million (Neumann et al., 2005). The virus causes late-term reproductive failure in pregnant sows and respiratory distress among young piglets. PRRSV emerged in the late 1980s with serious outbreaks of reproductive failure, pneumonia, and reduced growth performances among pigs in the US and Europe (Snijder et al., 2013). Within a couple of years it had spread worldwide. The causative agent was isolated and identified to be a positive sense RNA virus for the first time in The Netherlands and later named porcine reproductive and respiratory syndrome virus, reflecting the main clinical manifestations of infection (Hanada et al., 2005, Wensvoort et al., 1991).
PRRSV along with lactate dehydrogenase-elevating virus (LDV) of mice, equine arteritis virus (EAV), and simian hemorrhagic fever virus (SHFV) are placed in the family Arteriviridae. Irrespective of the diversity in the genome size, similarities in genome organization and transcriptional strategies, Coronaviridae and Roniviridae families along with Arteriviridae form the order Nidovirales (Cavanagh, 1997). PRRSV is an enveloped virus with a +ssRNA genome of approximately 15 kb in length. The European, or type 1 virus, and the North American, or type 2 virus, are the two designated genotypes for PRRSV with worldwide distribution (Snijder et al., 2013). The sequence identity is restricted to 50–60% between these two subtypes (Nelsen et al., 1999, Nelson et al., 1993).
The RNA genome is polyadenylated at the 3′ end and possesses a 5′ cap. It encodes for 10 open reading frames (ORF). Two thirds of the genome in the 5′ end encode the non-structural genes ORF1a and ORF1b, while the rest of the genome in the 3′ end codes for the structural genes ORF2a, ORF2b ORF3, ORF4, ORF5a, ORF5b, ORF6 and ORF7 (Snijder et al., 2013). Translation of ORF1a yields the polyprotein pp1a. ORF1b is expressed through a −1 ribosomal frame shift, leading to the formation of a large polyprotein, pp1ab. In addition, PRRSV uses a −2 ribosomal frame shift to translate a short ORF (TF) located within ORF1a (Fang et al., 2012). The polyproteins pp1a and pp1ab are processed by viral proteases to release 14 non-structural proteins, which include four proteases (NSP1α, NSP1β, NSP2 and NSP4), the RNA-dependent RNA polymerase (NSP9), a helicase (NSP10) and an endonuclease (NSP11) while the structural proteins include GP2a, E, GP3, GP4, GP5, 5a, M and N (Dokland, 2010). Interestingly, NSP2 has also been reported as a structural PRRSV protein (Kappes et al., 2013).
Like other arteriviruses, PRRSV also has a particular tropism, and its replication is largely restricted to macrophages. Porcine alveolar macrophage (PAM) and MARC-145 (MA-104 clone, African green monkey kidney cell line) both support PRRSV replication and are used as models to understand virus infection and the replicative cycle (Kim et al., 1993). Virus entry starts with the initial contact of heparin sulphates on the macrophage cell membrane with the virus (Fig. 1). Subsequently, the virus binds to sialoadhesin and is internalized via the process of clathrin-mediated endocytosis. Upon internalization, the viral genome is released from the early endosome into the cytoplasm, and scavenger receptor CD163 is essential for this process (Van Breedam et al., 2010).
With limited resources of their own, viruses exploit host machinery for their propagation and maintenance. Upon review, it becomes evident that viruses manipulate different host signalling pathways in their favour. The process of signal transduction via protein phosphorylation events has been very successfully and precisely hijacked by many viruses including PRRSV, in order to promote their own replication, while inhibiting anti-viral responses (Rothenburg et al., 2009).
It is interesting to note the commonality of many of the host pathways being targeted by many viruses, which allows them to propagate in the host and evade the host immune response. Control of kinase activity is one of the best examples of this, as it regulates cellular protein translation, metabolic activity, cell division and susceptibility to programmed cell death. Phosphatidylinositol-3-kinase (PI3K) functions both as a protein kinase and a lipid kinase, thus regulating the majority of the cellular activities mentioned above, in association with protein kinase B, also known as Akt (Cantrell, 2001, Toker, 2000). This is one of the best characterized pathways, which a number of viruses have been shown to usurp in order to enhance their own replication (Diehl and Schaal, 2013).
The conserved serine/threonine kinase mTOR (mammalian target of rapamycin), a downstream effector of the PI3K/Akt pathway, forms two distinct multiprotein complexes: mTORC1 and mTORC2 (Loewith et al., 2002). mTORC1 is sensitive to a macrocyclic antibiotic rapamycin, activates S6 kinase (S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1), which are involved in mRNA translation. It is activated by several stimuli, such as growth factors, nutrients, energy and stress signals and by some signalling pathways, such as PI3K, MAPK (mitogen-activated protein kinase) and AMPK (5′-adenosine monophosphate-activated protein kinase), to control cell growth, proliferation, survival and autophagy. mTORC2 is considered resistant to rapamycin, activates PKC-α (protein kinase C) and Akt and regulates the actin cytoskeleton (Oh and Jacinto, 2011). Viruses appear to regulate the mTOR signalling pathway for their own benefit (Peng et al., 2010).
Activated mTORC1 down-regulates autophagy. Autophagy can be considered the recycling unit of the cell, as it ensures the degradation of unwanted materials including defective organelles and proteins (Parzych and Klionsky, 2014). Autophagosomes (double-membrane vesicles containing their cytosolic cargo) and their fusion with lysosomes, forming autolysosomes, are the hallmark structures of autophagy. Importantly, the autophagy machinery has been shown to play a critical role in the degradation of pathogens of bacterial, viral and parasitic origins (Mizushima et al., 2008). A number of viruses have evolved strategies to exploit the autophagy pathway for their replication, while others have developed strategies to evade it (Heaton and Randall, 2010, Suhy et al., 2000, Taylor and Kirkegaard, 2008, Wong et al., 2008, Zhou et al., 2009).
In this review, we summarize the results of recent research on the role of PI3K/Akt and autophagy pathways in PRRSV infection and present our recent results on regulation of mTOR in PRRSV infected cells.
Section snippets
Modulation of PI3K/Akt signalling by PRRSV
PI3K is a heterodimer of catalytic (p110) and regulatory (p85) subunits (Cantrell, 2001). It is activated upon binding of either autophosphorylated receptor tyrosine kinase or non-receptor tyrosine kinase to the Src homology (SH2) domain of the p85 regulatory subunit (Vogt et al., 2010). The activated form of PI3K converts phosphophatidylinositol 4, 5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3). PIP3 acts as a second messenger (Fig. 1), which activates proteins that
PRRSV infection triggers mTOR signalling
Mammalian target of rapamycin (mTOR) is linked to PI3K/Akt signalling and integrates both intracellular and extracellular signals. It is an essential regulator of cell metabolism, growth, proliferation and survival (Ma and Blenis, 2009). The mTOR exerts its kinase activity within the cytoplasmic compartment as two different complexes (mTORC1 and mTORC2) with discrete functions (Fig. 1). mTORC1 positively regulates protein synthesis, phosphorylating eukaryotic initiation factor 4E (eIF4E)
PRRSV and autophagy
It has been shown that PRRSV induces autophagy to promote virus replication (Chen et al., 2012, Liu et al., 2012, Sun et al., 2012). Microtubule associated protein 1 light chain 1 (LC3) and lysosome associated membrane protein (LAMP1) are amongst the markers for autophagosomes and autolysosomes, respectively (Kabeya et al., 2000, Tabata et al., 2013). The conversion of LC3-I to its lipidated form LC3-II as this protein is incorporated into autophagosomes, is a well-established indicator for the
Conclusions
PI3K/Akt/mTOR is a critical signalling pathway for both the host and pathogen (Bordon, 2013, Laplante and Sabatini, 2009). Besides recognising the availability of glucose, amino acids and growth factors for maintaining the anabolic activities of the cell, it alerts, and acts to eliminate the threat from invading microorganisms including viruses. Like other enveloped, positive sense viruses and other nidoviruses, PRRSV enters the host cell through receptor-mediated endocytosis. It proceeds to
Acknowledgements
We are thankful to the Saskatchewan Ministry of Agriculture (Agriculture Development Fund) for support of the PRRSV research in our laboratories. This paper was published with the permission of the Director of VIDO-InterVac, Journal series no. 715.
References (52)
- et al.
Role of translocation in the activation and function of protein kinase B
J. Biol. Chem.
(1997) - et al.
Induction of autophagy enhances porcine reproductive and respiratory syndrome virus replication
Virus Res.
(2012) The structural biology of PRRSV
Virus Res.
(2010)- et al.
Dengue virus-induced autophagy regulates lipid metabolism
Cell Host Microbe
(2010) - et al.
Autophagy sustains the replication of porcine reproductive and respiratory virus in host cells
Virology
(2012) - et al.
Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control
Mol. Cell
(2002) - et al.
Human cytomegalovirus protein UL38 inhibits host cell stress responses by antagonizing the tuberous sclerosis protein complex
Cell Host Microbe
(2008) - et al.
Hepatitis C virus NS5A activates the mammalian target of rapamycin (mTOR) pathway, contributing to cell survival by disrupting the interaction between FK506-binding protein 38 (FKBP38) and mTOR
J. Biol. Chem.
(2010) - et al.
The mRNA 5′ cap-binding protein eIF4E and control of cell growth
Curr. Opin. Cell Biol.
(1998) Infectious disease: hushing mTOR boosts immunity to pathogens
Nat. Rev. Immunol.
(2013)
Intramolecular and intermolecular interactions of protein kinase B define its activation in vivo
PLoS Biol.
Phosphoinositide 3-kinase signalling pathways
J. Cell Sci.
Role of AKT kinase in measles virus replication
J. Virol.
Nidovirales: a new order comprising Coronaviridae and Arteriviridae
Arch. Virol.
Vesicular stomatitis virus infection alters the eIF4F translation initiation complex and causes dephosphorylation of the eIF4E binding protein 4E-BP1
J. Virol.
Structural basis for competitive inhibition of eIF4G-Mnk1 interaction by the adenovirus 100-kilodalton protein
J. Virol.
Make yourself at home: viral hijacking of the PI3K/Akt signaling pathway
Viruses
The mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C
Eur. Mol. Biol. Org. J.
Efficient-2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein
Proc. Natl. Acad. Sci. USA
Activation of the translational suppressor 4E-BP1 following infection with encephalomyocarditis virus and poliovirus
Proc. Natl. Acad. Sci. USA
The origin and evolution of porcine reproductive and respiratory syndrome viruses
Mol. Biol. Evol.
Hepatitis C virus inhibits AKT-tuberous sclerosis complex (TSC), the mechanistic target of rapamycin (MTOR) pathway, through endoplasmic reticulum stress to induce autophagy
Autophagy
Coordinate regulation of ribosome biogenesis and function by the ribosomal protein S6 kinase, a key mediator of mTOR function
Growth Factors
LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing
Eur. Mol. Biol. Org. J.
Highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of nonstructural protein 2 into virions
J. Virol.
Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line
Arch. Virol.
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