Flaviviruses are positive sense single stranded RNA enveloped viruses which share aspects like size, symmetry, and are transmitted mostly by the bite of infected arthropod (mosquito or tick), hence classified as arboviruses. These viruses use the host cell’s RNA dependant RNA polymerase to replicate. Primarily, the virus will make a full length copy of the complementary, minus stranded genome which then serves as a template strand for further replication. The replication of flaviviruses occur in the intracellular compartments associated to the endoplasmic reticulum (ER) and Golgi complex, and results in the accumulation of viral RNAs and proteins, which are recognized by host cellular machinery to prevent the virus infection. In response to viral replication host cells activate multiple intracellular stress pathways such as unfolded protein responses (UPRs), ER stress and stress granules (SGs) assembly to limit the progression infection. In order to establish and progress the prolific infection, viruses have developed mechanisms to annihilate deleterious effects of these stresses. Emerging evidence suggests that flaviviruses manipulate ER stress pathways and stress granule assembly, and it has been reported that these viruses also modulate UPRs. Together these strategies employed by virus facilitates its replication or pathogenesis, thus prolongs the life cycle of flaviviruses. Here we review the current knowledge in this area and highlight the new strategies which Flaviviruses utilize to combat cellular mechanisms and escape host stress responses to ensure efficient replication and pathogenesis.
Keywords: Flaviviruses, UPR, stress granules, ER stress, replication
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The genus Flavivirus comprises of a number of medically important arthropod-borne viruses (arboviruses) like yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV), Usutu virus (USUV) or Zika virus (ZIKV), Louis encephalitis virus (SLEV), or West Nile virus (WNV), and tick-borne encephalitis virus (TBEV)1,2, affecting hundreds of millions of individuals annually3,4. These viruses belong to the family Flaviviridae and share features like size (45-65 nm), symmetry (enveloped, icosahedral nucleocapsid) and genome (positive sense single stranded RNA of about 10-11 Kilobases)5,6. Flavivirus pathogenesis ranges from mild illness such as fever, rash and joint pain, to more severe symptoms such as haemorrhagic fever and fatal encephalitis7,8. Despite the availability of vaccines against YFV, JEV and TBEV, diseases resulting from these viruses are still prevalent worldwide9. Protective vaccines or therapies are not yet available against the more pathogenic flaviviruses and prevention from insect bites remains the major defence against some of these viruses. It is believed that the knowledge about the host pathogen interactions is crucial for developing therapies and vaccines against flaviviruses8. Therefore, a better understanding of the flavivirus replication and its interaction with host cellular machinery would lead to development of effective antiviral intervention strategies and novel vaccine formulations.
Flaviviruses enter their host cells through a process of receptor-mediated endocytosis and subsequently its RNA genome also enters the host with the help of viral glycoprotein mediated membrane fusion10. The virus utilizes host cellular machinery to replicate itself and is translated as a single polyprotein11. The viral and host cellular proteases cleave the polyprotein into three structural proteins, capsid (C), precursor membrane (prM/M), envelope (E) and seven non-structural proteins (NSPs), NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5, which serve to coordinate the intracellular aspects of virus replication and assembly as well as modulation of host defence mechanisms12. Ordinarily, Flavivirus replication takes place at a membranous web, which is associated with the endoplasmic reticulum (ER)10.The NSPs are mainly involved in the formation of the membranous web, and an RNA-dependent RNA polymerase (named NS5) catalyses the replication via a negative sense RNA intermediate1314. The capsid proteins coat the genome copies and immature virions, containing surface E proteins, bud into the ER lumen and are transported via the trans-Golgi network (TGN). At the TGN, they undergo further glycan modifications and structural cleavage, forming mature infectious virions, which are transported out of the cell by exocytosis15.
Production of viral progeny interferes with different aspects of host cellular metabolism; therefore, viral infection may represent a stress condition to the host cell16. Distinct cell host processes are interrupted or co-opted during a viral infection, leading to the activation of cellular stress responses on many levels. The cells transiently inhibit protein synthesis and restrict the consumption of nutrients and energy to enhance restore homeostasis17. The virus triggered Stress responses include the mitochondria stress (oxidative stress),ER-associated stress, and cytoplasmic stress stress granules (SGs)10,18–20.
In response to different stresses, eukaryotic cells activate kinases HRI( Heme-regulated inhibitor ), GCN2(General control nonderepressible kinase 2, PKR(Protein kinase R) and PERK(PKR related ER kinase). All the kinases phosphorylate eIF2? (eukaryotic initiation factor 2 alpha) which stalls general translation to alleviate cellular injury or results in apoptosis Phosphorylation of eIF2? reduces global translation by impairing the formation of the complex ternary eIF2-GTP-tRNAMet, allowing cells to conserve resources and to initiate a reconfiguration of gene expression to manage stress conditions effectively21,22 . The arrest of Protein synthesis triggers the assembly of stress granules (SGs). SGs are large ribonucleoprotein (mRNP) aggregates formed by stalled translation preinitiation complexes 23. The major components of SGs are eukaryotic translation initiation factors (eIF4A, eIF2,eIF4E, eIF4G, ), the 40S ribosomal subunit ,untranslated mRNAs, and RNA-binding proteins such as , T-cell intracellular antigen 1 (TIA-1), TIA-1-related protein (TIAR), Ras GTPase activating protein-binding protein 1 (G3BP1) and the poly(A) binding protein (PABP)24. SG assembly forms a platform between virus and SGs that limits the cytosolic availability of components of the cellular translation machinery25 . In this sense, viruses have evolved several strategies to ensure their replication in host cells by avoiding or blocking SG assembly. 26.Nonetheless, cellular stress responses are essential in eliciting immune detection and in the cell’s ability to shut down viral gene expression in response to viral infection. This review presents an overview of the current knowledge of interactions between the viral and cellular stress responses and how flaviviruses modulate these stress responses to progress the infection.
ER Stress and UPR during flavivirus infections.
Endoplasmic reticulum (ER) is an organelle which performs multiple functions; it is involved in the synthesis, folding and processing of the transmembrane and secretory proteins. There should be a balance between the ER protein load and the folding capacity to process this load to ensure proper folding of proteins27. Therefore, the ER lumen maintains a unique environment to maintain a balance between the ER protein load and the capacity to handle this load. This ER homeostasis can be disturbed by physiological and pathological stresses such as high protein demand, environmental toxins, viral infections, inflammatory cytokines, and mutant protein expression. This results in the accumulation of misfolded and unfolded proteins in the ER lumen, which is termed as ER stress28. ER stress activates a complex signaling network which is called the Unfolded Protein response (UPR) to reduce ER stress and restore homeostasis. The UPR is designed to wipe out misfolded ER proteins either by arresting new protein synthesis or by amplifying processes of protein folding and degradation29. The enhanced expression chaperones, such as the glucose-regulated proteins GRP78 (also known as BiP) and GRP9430, and other ER-resident proteins are hallmarks of the UPR. On top of that, the UPR activates a cellular response, which results in translation initiation inhibition, thus preventing further accumulation of unfolded protein in the ER. UPR is initiated by three ER transmembrane proteins (ER stress sensors), viz Inositol Requiring1 (IRE1), Activating Transcription Factor 6 (ATF6) and PERK. These are also called branches of UPR signaling pathways 31(Figure Figure22). The UPR pathways are coordinated by a protein Binding immunoglobulin protein (BiP) to reinstate cell homeostasis. BiP is a heat shock protein that binds to correctly folded and misfolded proteins32. BiP in normal circumstances associates with the luminal domains of IRE1 , PERK, and IRE1 thus blocking the activation of UPR pathways. However Under stress conditions, like Flavivirus infection, BiP transiently associates with folding intermediates of viral glycoproteins releasing IRE1 , PERK, and IRE1 to activate UPR pathways33(Figure Figure22). The release of BiP from PERK or IRE1 allows the homodimerization of each protein through their luminal domain, which instigates their autophosphorylation, which leads to their activation. The activated PERK inhibits protein synthesis through phosphorylation of the eIF2-?. IRE1 leads to the transcription of a subset of genes encoding protein-degradation enzymes10. Concomitantly, the release of BiP from ATF6 advances the ATF6 translocation from ER to the Golgi apparatus, where it is cleaved and activated. Activated ATF6 promotes the transcription of genes encoding chaperones that help in refolding of misfolded proteins 34. The three branches of UPR mentioned above do not operate independently; an intricate signaling network is constituted by the tight temporal control and crosstalk among them. UPR pathway, leads to the production of proinflammatory cytokines, such as TNF-? and IL-6 by inducing NF-?B activation35 (Figure Figure22). Therefore, UPR pathways can promote inflammatory cytokine production serving as “danger” signal, accompanying cellular viral sensors in alarming a cell to invasion and boosting subsequent antiviral immune response36. However, viruses, on the other hand, have evolved mechanisms to counteract UPR pathways to progress their infection. This involves regulation of stress response proteins manipulation of UPR by several molecular chaperones and increases folding capacity that will be addressed below.
Several flaviviruses including JEV, WNV, HCV and DENV, activate the UPR pathway in a variety of mammalian cells 373810. The hallmarks observed UPR pathway activation by Flaviviruses infection was IRE1 mediated splicing of XBP-1, and BiP overexpression in infected cells. The triggering the XBP1 signaling pathway is beneficial for viral production and flaviviruses take advantage of this cellular response, as it also alleviates virus-induced cytotoxicity. As for as DENV infection is concerned, it was demonstrated that xbp1 splicing is induced by nonstructural protein NS2B and NS3 39.It has also been observed in liver cells infected with DENV2, ATF6 and PERK were not associated with BiP, which results in increased eIF-2? phosphorylation40. Moreover, it has also been reported that DENV can manipulate the sequence of events to activate and suppress UPR to prevent premature apoptosis and prolong the viral life cycle41.
Japanese Encephalitis Virus (JEV) infection triggers the UPR pathway in neuronal cells, which results in apoptotic cell death by vigorous expression of CHOP/GADD153, a death-related transcription factor which is responsible for down-regulation of Bcl-2 and it increases the production of ROS10. JEV induced ER-mediated UPR is also involved in the activation of stress-inducible p38 mitogen-activated protein kinase (p38 MAPK) that could impart stimulation of post-translational CHOP induction37.It has been reported that West Nile Virus (WNV) infection also disrupts ER homeostasis leading to activation of UPR pathways. ATF6 cleavage was observed upon WNV infection, and it also leads to phosphorylation of eIF-2? by PERK activation. The activation of IRE1 pathway corresponds to increasing viral load in the ER 42. One study shows that WNV Modulates the Unfolded Protein Response to progress Replication and Immune Evasion. The exact mechanisms by which WNV can manipulate different aspects of UPR are still to be determined43.
The Hepatitis C Virus (HCV) core processing and folding occurs at the ER, and it is firmly dependent on interaction with the ER membrane 44. Additionally, HCV core expressing liver cells showed escalated calcium release from ER, taken up by mitochondria, resulting in increased levels of ROS production and reduced antioxidant levels. These events ultimately trigger oxidative stress and apoptosis10.
Stress Granules and P-Bodies During Viral Infection
A key aspect of virus-host interactions is manipulation of cellular gene expression by the virus to maintain conditions favorable for productive replication. Eukaryotic genes are post-transcriptionally regulated by evolving mRNP compositions that interfere with splicing, export, subcellular localization and mRNA turnover and regulation of translation. These events are mostly interrelated, e.g., mRNA translation is linked to poly(A) shortening and decay, and the processes share proteins46,47
Translationally silenced mRNPs can organize into two major classes of RNA granules in the cytoplasm, known as stress granules (SGs) and processing bodies (P-bodies, PBs). Stress granules are dynamic structures that quickly form when external stresses are applied to cells, and global translation rates decline, and disperse when translation conditions are restored. Thus, SG is mostly thought to contain stalled 43S and 48S ribosomal preinitiation complexes and are proposed to serve as temporary repositories for these complexes. In this way, largely preassembled translation complexes can be rapidly released to resume gene expression when cellular stress conditions abate. The scenario most often described for SG formation follows when oxidative, nutrient or heat stress activates one of the eIF2? kinases (heme-regulated kinase, HRI; general control non-depressible 2 kinase, GCN2; double-stranded RNA (dsRNA)-activated protein kinase R, PKR; and PKR-like endoplasmic reticulum kinase, PERK), which phosphorylate the alpha subunit of eIF2 and block translation, forcing an accumulation of the stalled 43S and 48S ribosomal preinitiation complexes. Inhibition of the function of eIF4G or eIF4A in translation initiation is also linked to SG formation48and some mechanisms of SG formation can proceed in the absence of eIF2? phosphorylation47 (Fig. 1
P-bodies are constitutively present in cells, and increase in size and number when the translational arrest occurs. P-body constituents include decapping enzymes, exonucleases, deadenylases, RNA binding proteins involved in nonsense-mediated decay and microRNA mediated silencing (Fig. 2). RNA decay occurs both within PBs and outside PBs and proportions of total RNA decay attributed to each compartment are controversial. PBs are proposed to dynamically exchange mRNP cargo with SGs and have been proposed to serve as nucleation sites for SG formation47,49.
Since, SGs and PBs control the mRNA cycle, gene expression, and metabolism, they become another important point of control for viruses to modulate. The degree and type of manipulation taken by viruses are turning out to be as changeable as the replication schemes of the viruses. Virus infection triggers many types of stresses on cells, even during non-lytic infections, and the deviation of cellular homeostasis is sensed in many ways in pathways that feed directly into stress responses. Viruses can induce SGs formation by interacting with translation complexes such as eIF4G or eIF4A50. SG induction upon virus infection is more frequently associated with PKR activation by recognition of viral dsRNA (Figure Figure44). eIF2? is phosphorylated by activated PKR, as described earlier, and promotes mRNP aggregation with the help ofG3BP51.Moreover, it has been demonstrated recently that cytoplasmic virus sensor and interferon responsive genes can gather and colocalize with virus infection induced SGs, forming the so-called antiviral stress granules (avSG; 10,52 (Figure Figure44). Even though PKR had been reported to be a key element for the formation of avSG. It forms a platform that promotes interaction of non-self RNA and ligands with antiviral proteins. Since propagation of virus completely depends on the translational machinery of host cells, therefore SG induction by virus infection often transitory and SG assembly is repressed at some point of virus replication cycle. Most flaviviruses such as DENV, JEV, and WNV block stress granule formation by exploiting multiple SG components, including G3BP1, TIA1, TIAR, and Caprin-110,52.
It was first demonstrated that SG proteins directly interact with WNV RNA and proteins 53. They observed that RNA binding proteins like TIA-1 and, TIAR mostly interacts with minus strand of WNV, which is the site of initiation of genomic RNA synthesis). The Interaction of viral RNA with TIA-1/TIAR might facilitate replication of WNV and, TIAR knockout cells showed reduced WNV replication when compared with control cells. DENV RNA also binds specifically to TIA-1, TIAR, and G3BP;54. Also, the latest study reported that caprin-1 interacts with the 3?UTR of DENV suggesting that this genomic region is a site for SG protein assembly. Both the flavivirus WNV and, DENV inhibited eIF2-? phosphorylation and SG formation thus, prevents the shutdown of host proteins translation, and favors virus RNA translation. Likewise, recruiting of SG proteins to different compartments may allow translation of virus or replication and evades innate immune response as a result of SG assembly10.
Japanese Encephalitis Virus core protein also recruited several SG-associated proteins, including G3BP and USP10, in a way dependent on caprin-1 binding10. These interactions are associated with the repression of SG assembly, resulting in enhanced viral replication. SGs rich in G3BP1/eIF3/eIF4B were also detected upon TBEV infection. Exhaustion of TIA-1 or TIAR resulted in enhanced production of new infectious virus, demonstrating that SGs harboring TIAR and TIA-1 inhibit TBEV replication55.
Hepatitis C Virus also hijacks SG machinery by recruiting PKR-eIF2-? phosphorylation pathway as a strategy for viral escape 56. It has been demonstrated that IFN treatment of HCV-infected cells induced PKR and eIF2-? phosphorylation, thereby inhibiting de novo cellular protein synthesis, which includes antiviral interferon-stimulated gene translation57. Activation of PKR, however, did not inhibit translation of HCV proteins, probably due to IRES-dependent synthesis5610.
It has been reported that HCV exhibited a stress response oscillation as a mechanism to prevent long-lasting translation repression10.It was reported that GADD34(a regulatory subunit of protein phosphatase 1 ) upregulation was involved in reverting eIF2-? phosphorylation, thus reactivating translation during HCV infection in Huh7 cells7.