- Open Access
Differential unfolded protein response during Chikungunya and Sindbis virus infection: CHIKV nsP4 suppresses eIF2α phosphorylation
© Rathore et al.; licensee BioMed Central Ltd. 2013
Received: 17 October 2012
Accepted: 11 January 2013
Published: 28 January 2013
Chikungunya (CHIKV) and Sindbis (SINV) are arboviruses belonging to the alphavirus genus within the Togaviridae family. They cause frequent epidemics of febrile illness and long-term arthralgic sequelae that affect millions of people each year. Both viruses replicate prodigiously in infected patients and in vitro in mammalian cells, suggesting some level of control over the host cellular translational machinery that senses and appropriately directs the cell’s fate through the unfolded protein response (UPR). The mammalian UPR involves BIP (or GRP78), the master sensor in the endoplasmic reticulum (ER) together with the three downstream effector branches: inositol-requiring ser/thr protein kinase/endonuclease (IRE-1), PKR-like ER resident kinase (PERK) and activating transcription factor 6 (ATF-6). Through careful analysis of CHIKV and SINV infections in cell culture we found that the former selectively activates ATF-6 and IRE-1 branches of UPR and suppresses the PERK pathway. By separately expressing each of the CHIKV proteins as GFP-fusion proteins, we found that non-structural protein 4 (nsP4), which is a RNA-dependent-RNA polymerase, suppresses the serine-51 phosphorylation of eukaryotic translation initiation factor, alpha subunit (eIF2α), which in turn regulates the PERK pathway. This study provides insight into a mechanism by which CHIKV replication responds to overcome the host UPR machinery.
Chikungunya virus (CHIKV) is a member of the alphavirus genus, which contains 26 known arboviruses with a wide host range . During the past 50 years, numerous CHIKV epidemics have been documented in both Africa and Asia . Since, its discovery, CHIKV has spread widely and currently Chikungunya fever has been detected in nearly 40 countries with a potential to affect millions of people worldwide . In general, alphaviruses are divided into viruses that cause human diseases characterized by rash and arthritis, that are primarily found in the “old world” such as CHIKV, O nyong nyong, Sindbis (SINV), Ross River, Barmah Forest and Mayaro virus  and viruses that cause encephalitis, which are primarily found in the “new world”. The first clear association of an alphavirus with arthritic disease was made in 1953 when CHIKV was isolated from the blood of individuals in Tanzania with severe arthritis . SINV was first isolated in 1952, which causes similar disease to CHIKV in humans known as sindbis fever and the symptoms include arthralgia, rash and malaise . These arthritogenic alphaviruses share certain antigenic determinants  and also considerable genome similarity that makes them interesting for comparative responses to the host. In humans, CHIKV infection is characterized by a rapid onset of fever that is cleared in 5–7 days with long lasting immunity . The major pathology associated with CHIKV infection is very high viremia and polyarthritis [8–11]. The mortality rate associated with CHIKV infection has been estimated to be 1:1000 with most deaths occurring in neonates, adults with underlying conditions and the elderly . The persistent detection of viral RNA or antigen in the host has suggested the long-term persistence of these viruses in humans [12, 13]. The alphavirus genome is a single-stranded RNA genome of ~12 kb in size of positive polarity. It encodes two polyproteins of which the first encodes nonstructural proteins (nsPs) 1–4: nsP1 contains methyl transferase and guanyl transferase activities, nsP2 is a helicase/protease, nsP3 is an accessory protein involved in RNA synthesis and nsP4 is the RNA dependent RNA polymerase. The second polypeptide, translated from a subgenomic RNA codes for structural proteins, capsid (C) and the envelope glycoproteins, E1 and E2 that constitute the virion coat [4, 14, 15]. Several studies have shown that alphavirus replication in mammalian cells usually results in severe cytopathicity, mainly caused by dramatic shutdown of host translation machinery [16–20]. However, the mechanism by which CHIKV maintains such a high replication rate in the infected cells is poorly understood.
One host response mechanism that has the potential to limit virus replication is the endoplasmic reticulum (ER) stress response, also known as unfolded protein response (UPR) which, maintains cellular protein homeostasis and prevents the over-accumulation of unfolded proteins in the lumen of the ER during normal and diseased states . ER chaperone immunoglobulin heavy chain binding protein (BIP), also known as glucose regulated protein 78 (GRP78) plays a central role in this process via a three-pronged regulatory pathway involving PKR-like ER kinases (PERK), activating transcription factor 6 (ATF-6) and the ER transmembrane protein kinase/endoribonuclease (IRE-1). Under stress conditions, BIP is sequestered to misfolded or unfolded proteins in the ER whereupon it activates PERK, ATF-6 and IRE-1 . During UPR, PERK activates by self-dimerization and phosphorylation. Activated PERK phosphorylates eIF2α at serine-51 and leads to an inhibition of general protein synthesis. PERK activation also induces the activation of C/EBP homologous protein (CHOP) and growth arrest and DNA damage-inducible protein GADD34 . CHOP is responsible for apoptosis mediated cell death when functions of ER are severely impaired to protect the organism by eliminating the damaged cell  whilst GADD34 and its binding partner protein phosphatase-1 catalytic subunit (PP1c) are involved in eIF2α de-phosphorylation that also modulates cell fate during protein translational stress. The activation of IRE-1 branch of UPR pathway leads to transcription induction of a subset of genes encoding protein degradation and pro-survival enzymes such as components of ER associated degradation (ERAD) including ER degradation-enhancing-α-mannosidase like protein (EDEM) [25–27]. Autoproteolytic activation of ATF-6 stimulates transcription of genes encoding chaperones that assist in the refolding of misfolded proteins . On balance, the UPR pathway in conjunction with ERAD controls the survival vs apoptosis decision of cells stressed by increased protein translation from external stimulus .
To circumvent the host cellular translational response, several viruses [respiratory syncytial virus, simian virus-5, Tula virus, African swine fever virus, herpes simplex virus, cytomegalovirus, dengue virus and hepatitis C virus [30–34] have been shown to regulate UPR machinery. For example, in the case of hepatitis C virus, the virus encoded NS5A phosphoprotein, inhibits PKR activation by direct protein-protein interaction . Likewise, K3L gene product of vaccinia virus also binds to PERK and inhibits its activation . Others such as herpes simplex viruses encode proteins that mimic host factors to regulate the protein synthesis traffic . In light of these various mechanisms by which viruses modulate UPR pathway, we investigated the impact of CHIKV replication on the various components of the UPR machinery and compared it to another representative alphavirus, SINV, in order to reveal differential host responses to these unique but closely related pathogens. Real-time RT-PCR monitoring of transcriptional changes and Western blotting of infected cells were used to reveal the UPR components during both CHIKV and SINV infections. By carefully examining the UPR pathway components and by selectively inducing the ER stress using thapsigargin or tunicamycin treatment, we identified the suppression of eIF2α phosphorylation during CHIKV infection in the early phase of virus replication that does not occur with SINV infection. Subsequently, transfection of individual CHIKV-encoded proteins as GFP-fusion proteins revealed a mechanistic basis for the phenomenon dependent on nsP4.
Materials and methods
Cells and viruses
Mosquito cells Aedes albopictus clone (C6/36) and baby hamster kidney cells (BHK-21) were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco). Human embryonic kidney cells (HEK293) and human lung fibroblast cells (MRC-5) were cultured in DMEM (Gibco) supplemented with 10% FBS. C6/36 cells were grown and maintained in 28°C temperature incubator. BHK-21, MRC-5 and HEK293 cells were grown and maintained at 37°C in a humidified incubator with 5% CO2 atmosphere. CHIKV strain ‘ROSS’ and a laboratory strain of SINV MRM-39 strain (isolated in Australia ) was a generous gift from Dr. Ooi Eng Eong (Duke-NUS GMS). Both the viruses were amplified in C6/36 cells supplemented with 5% FBS at 28°C and titrated by plaque assay as described previously . Low passage number (below passage 5) was used for performing all experiments. Tunicamycin (Sigma) or thapsigargin (sigma) was used to induce UPR stress in the cells.
In vitro virus quantification
Prior to their use, plaque assays were carried out to quantify the number of infectious viral particles for CHIKV and SINV viruses used in the study. Briefly, BHK-21 cells were cultured to approximately 80% confluency in 24-well plates (NUNC). The virus stock was 10-fold serially diluted from 10−1 to 10−12 in RPMI 1640 (Gibco). BHK-21 monolayers were infected with 200μl of each virus dilution. After incubation at 37°C and 5% CO2 atmosphere for 1h with rocking at 15 min intervals, the medium was decanted and 1ml of 1% (w/v) carboxymethyl cellulose in RPMI supplemented with 2% FBS was added to each well. After 72h of incubation at 37°C in 5% CO2, the cells were fixed with 4% paraformaldehyde and stained for 30 min with 200 μl of 1% crystal violet dissolved in 1X-PBS. After thorough rinsing with water, the plates were dried and the plaques were scored visually.
Primer sequences used in the study
Real-time PCR primer sequences: - CHIKV nsP1 (F-TAGAGCAGGAAATTGATCCC, R- CTTTAATCGCCTGGTGGTAT), SINV E1 (F-CACCCCGCACAAAAATGAC, R- AAAAGGGCAAACAGCCAACTC), EDEM (F-TCATCCGAGTTCCAGAAAGCAGTC, R- TTGACATAGAGTGGAGGGTCTCCT), XBP-1 (F-TCACCCCTCCAGAACATCTC, R- ACTGGGTCCAAGTTGTCCAG), CHOP (F-TCTGATTGACCGAATGGTG, R- TCTGGGAAAGGTGGGTAGTG), BIP (F-TAGTGCAAGCTGAAGGCTGA, R- GGGCTGGAGTACAGTGGTGT), GADD34 (F-AACCTCTACTTCTGCCTTGTCT, R- CGCCTCTCCTGAACGATACTC), eIF2αK2 (F-TTTGGACAAAGCTTCCAACC, R- ACTCCCTGCTTCTGACGGTA), 18s (F-TGTTCAAAGCAGGCCCGAG, R-CGGAACTACGACGGTATCTGATC), GAPDH (F- ACAGTCAGCCGCATCTTCTT, R- ACGACCAAATCCGTTGACTC), Actin (F-CAGGGGAACCGCTCATTGCCAATGG, R-TCACCACACACTGTGCCCATCTACGA), XBP-1 splicing (F- AAACAGAGTAGCAGCTCAGACTGC, R- TCCTTCTGGGTAGACCTCTGGGAG).
CHIKV recombination cloning primer sequences: - nsP1 (F- AGATCTCGAGCTCAAGCT TCGATGGATCCTGTGTACGTG, R- TTAACCGTCGACTGCAGATCCTGCACCCGCTCTGTC), nsP2 (F- TCCGGACTCAGATCTCGAGCTATAATAGAGACTCCGAGAGGA, R-GGATCCCGGGCCCGCGGTACCACATCCTGCTCGGGTGGC), nsP3 (F- TCCGGACTCAGATCTCGAGCTGCACCGTCGTACCGGGTA, R- GGATCCCGGGCCCGCGGTACCCCCACCTGCCCTGTCTAG), nsP4 (F- TCCGGACTCAGATCTCGAGCTTATATATTCTCGTCGGAC, R- GGATCCCGGGCCCGCGGTACCCTATTTAGGACCGCCGTA), Capsid (F- TCCGGACTCAGATCTCGAGCTTGCATGAAAATCGAAAATGAC, R- GGATCCCGGGCCCGCGGTACCCCACTCTTCGGCTCCCTC), E2 (F- AGATCTCGAGCTCAAGCTTCGCCATACTTAGCTCACTGT, R- TTATCTAGATCCGGTGGATCCGCAGCATATTAGGCTAAG), E1 (F- AGATCTCGAGCTCAAGCTTCGAGAACAGCTAAAGCGGCC, R- TTATCTAGATCCGGTGGATCCTTAGTGCCTGCTGAACGA).
RNA extraction and real-time RT-PCR analysis
HEK293 cells (1×105) were infected with virus (CHIKV/SINV) at a multiplicity of infection (MOI) of 1. At indicated time intervals, total RNA was isolated using the trizol (Invitrogen) extraction method and 1μg of total RNA was used for cDNA synthesis using ImProm II reverse transcription system (Promega), with oligo dT as primer. cDNA (50 ng) was used for real-time amplification of specific genes using respective primers (Materials and Methods) in Bio-Rad iQ-5 real time thermal cycler. The expression of viral and host gene products was normalized to Actin and GAPDH mRNA expression, followed by normalization to expression levels at uninfected conditions.
XBP-1 splicing assay
The XBP-1 splicing assay was performed essentially as described elsewhere . Briefly, total RNA from the mock or virus (CHIKV/SINV) infected cells was extracted as described above and 1 μg each of the total RNA was used for cDNA synthesis using ImProm II reverse transcription system (Promega), with oligo dT as primer, followed by PCR amplification of XBP-1 spliced genes using XBP-1 splicing specific primers (Materials and Methods). Amplified products were run on 2.5% Agarose gel and visualized under UV ImageQuant.
HEK293 cells (1×105) were infected with MOI of 1 with CHIKV/SINV and total cell lysate was collected in NET lysis buffer (20 mM Tris, 100 mM NaCl & 1 mM EDTA) containing 0.1% Triton X-100 with protease inhibitor cocktail (Roche) at indicated time points post infections. After 30 min on ice, lysates were centrifuged at 13000 rpm for 10 min and supernatants were used to quantitate the amount of total protein by BCA assay (Pierce). Equal amount (2-5 μg each) of protein was loaded on 12% SDS PAGE followed by Western blotting. Blots were blocked overnight with blocking solution [2% Fish gelatin (sigma) in 1X PBS] and were probed using primary antibodies against various proteins: GFP (Abcam), BIP (Abcam), ATF-6 (Abcam), HSP-90 (cell signaling), p58IPK (cell signaling), CHOP (cell signaling), phospho (Thr 980) PERK (cell signaling), eIF2α (cell signaling) and phospho (Ser 51) eIF2α (cell signaling). Anti-GAPDH antibody (cell signaling) and anti-Actin antibody (sigma) were used as the loading control antibodies. All the antibodies used were diluted in blocking solution. After incubating with secondary HRP-conjugated antibodies, blots were developed using ECL detection reagent (GE healthcare) and exposed on Amersham hyper films prior to development or visualized using Image-quant chemiluminiscent machine. Where required, image quantification was done using Image-J software.
Construction of CHIKV-pEGFP clones
Vector pEGFP-C1 (Clontech) was used to clone all the four non-structural (nsP1-4) and three major structural (C, E2 & E1) genes of CHIKV. Briefly, CHIKV RNA was extracted using a viral RNA extraction kit (Qiagen). All the genes were amplified using gene specific primers (Materials and Methods) and superscript III one step RT PCR with platinum Taq kit (Invitrogen) in a thermal cycler (Applied Biosystem). Amplified genes were run on 1% agarose gel and amplicons were gel eluted using QIA-quick gel extraction kit (Qiagen). Individual purified PCR products were then inserted in to the pEGFP-C1 vector using cloneEZ PCR cloning kit (Genscript) as per the manufacturer’s recommendations. For convenience of restriction digestion analysis for screening positive clones, nsP1 was inserted in between HindIII-PstI restriction sites and nsP2-4 and C were cloned using XhoI-KpnI restriction sites. Similarly, E1 and E2 were cloned using HindIII-BamHI restriction sites. All the positive clones were further confirmed by DNA sequencing.
Transfection of plasmids
For transfection of plasmid DNA into HEK293 or MRC-5 cells, cells were seeded to 70% confluency in a 24 well plate (Nunc) and incubated overnight in 37°C incubator supplemented with 5% CO2 atmosphere. One μg of each of the plasmids (GFP vector, GFP-nsP1/2/3/4 or GFP-C/E1/E2) was transfected using jet prime transfection reagent (Polypus BST scientific) as per the manufacturers described protocol. Transfected cells were incubated for 48h for protein expression and then washed once with 1X-PBS (Gibco). Finally, cells were collected in TNET-lysis buffer as described above and then subjected to Western blotting. The transfection efficiencies by fluorescence microscopic visualization for each of the plasmids except GFP-nsp2 were measured to be around ~70% using polyplus jet prime transfection reagent, strictly as per the manufacturer’s protocol. For GFP-nsP2 transfection was done using 2 μg of the plasmid and nearly 60% of transfection efficiency was achieved. No cytotoxicity was observed upon transfection of plasmids till 72h post transfection. However, with GFP-nsP2 some cytotoxicity (less than 20% cell death) was observed after 48h post transfection.
HEK293 cells were seeded on coverslips at a density of 1×105 cells/well in a 12-well plate. Following incubation for overnight at 37°C with 5% CO2, the cells were infected with CHIKV or SINV at an MOI of 1. At indicated time points after infection cells were fixed with ice cold 80% acetone for 10 min followed by overnight incubation with blocking buffer (5% BSA in 1X PBS) at 4°C. The CHIKV RNA was detected using monoclonal dsRNA antibody (J2). The phosphorylated form of ER resident protein eIF2α was detected using antibody against phospho (Ser 51) eIF2α (cell signaling). Secondary antibodies used were anti-mouse alexa 488 and anti-rabbit alexa 594. All the antibodies used were diluted in blocking buffer. The coverslips were mounted on glass slides using prolong gold anti-fade mounting medium (Invitrogen) containing DAPI. Immunofluorescence images were captured using an inverted fluorescence microscope (Olympus IX71, USA) or upright confocal microscope (Zeiss) and image analysis was performed with Image-J software.
Statistical comparison of results were performed using unpaired Student’s t test on the GraphPad Prism 5.0 software with p<0.005 considered statistically significant.
Growth kinetics of CHIKV and SINV in vitro
The ATF-6 signaling branch of UPR pathway during CHIKV and SINV infection
The IRE-1 signaling branch of UPR pathway during CHIKV and SINV infection
The PERK signaling branch of UPR pathway during CHIKV and SINV infection
CHIKV infection suppress phosphorylation (ser 51) of eIF2α
CHIKV protein nsP4 suppresses phosphorylation (Ser 51) of eIF2α
Virus infection in mammalian cells consists of a series of events from entry to maturation and egress of virus. Remarkably, as intracellular parasites, viruses rely on the utilization of cellular machinery and resources to complete their life cycle. In this complex process, RNA viruses synthesize dsRNA intermediates and produce viral proteins within host cells. Consequently, viral replication elicits cellular responses, such as ER stress and the interferon response, as a first line of defense against the invading pathogen. To overcome this natural resistance, viruses have evolved various mechanisms to subvert host responses that limit or inhibit viral replication. Recently, several groups [44, 57–59] have reported the impact of CHIKV or SINV replication on host cellular interferon and apoptotic machinery. In this study we specifically examined the cellular UPR signaling during CHIKV and SINV infections and show that the gene/protein expression responses in the pathway are differentially modulated although the two viruses are considered to be closely related to each other. We explored in more detail the mechanistic basis for CHIKV modulation of the UPR pathway.
The stimulation of transcription and translation of BIP (the master regulator of UPR) has been observed for several viruses [33, 60]. Not surprisingly the massive replication of CHIKV resulted in the induction of ER resident chaperones, such as BIP and HSP-90, which presumably assists in the folding of unfolded proteins in order to relieve the UPR stress within the cell. SINV infection, on the other hand, did not show significant induction in the expression of BIP and HSP-90, suggesting the possible early buildup of ER stress, which may contribute to the apoptosis and early cell death that was observed . However SINV infection caused a more pronounced IRE-1 mediated splicing of XBP-1 gene that resulted in transcriptional induction of XBP-1 and EDEM, a pro-survival gene-product. Although the induction of XBP-1 and EDEM was less prominent during CHIKV infection in comparison to SINV infection, the present data is consistent with the recently reported role of IRE-1 signaling in delaying caspase-induced cell death . In the PERK branch of UPR pathway, the phosphorylation of PERK was observed in both CHIKV and SINV infected cells but intriguingly the kinetics of the concomitant phosphorylation of eIF2α showed marked difference between the two. At the early time points following CHIKV infection although increased PERK phosphorylation could be detected from 12 h post infection, the phosphorylation of eIF2α was not detected until 48h post infection whereas in SINV infected cells the eIF2α phosphorylation could be detected from 3 h post infection. This discrepancy was addressed by treating CHIKV infected cells with thapsigargin or tunicamycin, the well known strong inducers of PERK and eIF2α phosphorylation. This clearly demonstrated that eIF2α phosphorylation in the cell was suppressed at the early stages of CHIKV infection (3-24 h) even with thapsigargin or tunicamycin treatment so as to allow high and sustained viral protein production without building up the ER stress. At 48 h post CHIKV infection the eIF2α phosphorylation was quite prominent and comparable to the level observed at the same time point in SINV infected cells. However at this time point GADD34, a negative regulator of PERK, which mediates the de-phosphorylation of phospho-eIF2α and p58IPK, a chaperone, which suppresses the PERK mediated phosphorylation of eIF2α were also induced, suggesting that even when the cell tries to overcome its control by CHIKV infection, negative loop transcripts like GADD34 and p58IPK are activated in order to rescue viral protein synthesis. To further explore the importance of GADD34 in mediating CHIKV induced suppression of eIF2α-phosphorylation we used a specific GADD34 inhibitor ‘salubrinal’. Interestingly salubrinal treatment during CHIKV infection lead to an increased phosphorylation of eIF2α suggesting the involvement of GADD34 in suppression of eIF2α-phosphorylation. Salubrinal treatment during SINV infection however did not show any significant change in the phosphorylation of eIF2α over untreated SINV infected cells. Also, interestingly CHOP activity was not detected at both protein and transcription levels throughout the CHIKV infection time course. In stark contrast to CHIKV, SINV infection leads to phosphorylation of PERK and a dramatic increase in the phosphorylation of eIF2α starting from 3h post infection. The enhanced expression of CHOP detected as early as 3h suggests the signature cell death by apoptosis during SINV infection. Although, GADD34 was transcriptionally induced during SINV infection the heightened phosphorylation of eIF2α and further increase in CHOP activity triggers massive cell death, which could be observed starting from 12 h post infection (data not shown). Altogether, our data suggest that the PERK branch of UPR pathway is regulated during CHIKV infection as reflected by the suppression in the phosphorylation of eIF2α during the early stage of infection and the reduced CHOP activity.
A mechanistic basis for the suppression in the phosphorylation of eIF2α during the early stage of CHIKV infection was investigated using EGFP-tagged clones of seven CHIKV proteins and we discovered that the observed phenotype in the PERK pathway (i.e. suppression of the phosphorylation of eIF2α) is mediated by CHIKV nsP4 protein, which contains the RNA-dependent-RNA polymerase activity. An interesting conjunction to our finding is that nsP4 protein of alphavirus is the first non-structural protein to be cleaved from the nsP1-4 polyprotein. and this cleavage as well as its enzymatic activity play a critical role in the synthesis of minus strand viral RNA . Furthermore it is also well known that the alphavirus nsP4 is unstable, short-lived and degrades rapidly in the infected cell . This instability of nsP4 could possibly explain why infected cells recover some degree of eIF2α phosphorylation in the late phase of infection (48 h). Together, we suspect that early suppression of the translation inhibition involving nsP4 could permit the buildup of template RNA for further translation and, thereby, support robust replication.
The question of how CHIKV regulates the host translational machinery to achieve a high level of replication is important to examine in detail particularly in light of seemingly contradictory reports on this topic. White et al. , reported independence of CHIKV induced translational shut-off from the phosphorylation of eIF2α, an intriguing finding since eIF2α phosphorylation has a well established role in the shut-off of the host translational machinery . However, in our detailed time course experiments with HEK293 cells, we did not observe eIF2α phosphorylation until 48 h post infection, which was also consistently not observed in another cell type MRC-5 cells until 48 h. We believe our detailed time course study provides advantage in understanding the complex early events of virus-host interactions in the UPR pathways. That it occurs, mechanistically, is interesting since the actions of transiently stable nsP4 function correlate to viral RNA replication and life cycle. Even at the late phase of infection induction of ER chaperones (BIP, HSP-90) along with pro-survival gene-product (EDEM) could work synergistically with negative regulators of eIF2α phosphorylation (p58IPK, GADD34) to possibly support sustained CHIKV replication. SINV infection, on the contrary, is characterized by uncontrolled UPR as reflected by its failure to induce synthesis of ER chaperones followed by increased phosphorylation of eIF2α and CHOP activity leading to early cell death. Since both CHIKV and SINV infections showed differential activation or modulation of the UPR, further detailed studies on the effects of infection on host cellular UPR machinery is required to better understand their characteristic prolific replication profiles.
In conclusion, we show that the two closely linked viruses CHIKV and SINV from the same family, responds differently to the host cellular UPR machinery. Indeed, CHIKV infection modulates the PERK branch of UPR machinery and that it occurs mechanistically through the involvement of the viral protein nsP4 in direct or indirect conjunction with host factors such as GADD34. The early suppression of UPR provides a mechanism for robust replication. Our observation opens up the possibility to explore in detail the interplay of CHIKV nsP4 protein in establishing the infection and exploit possible avenues to use this in identifying a suitable target for antiviral intervention.
We thank Dr. Ooi Eng Eong for providing the laboratory strains of SINV. We also thank Drs. Shirish Shenolikar, Parmeen Akhtar, Ashley St. John and Nicole Moreland for critical reading of the manuscript and their discussions. This research in the SGV laboratory is supported by a DUKE-NUS Signature Research Program start-up grant funded by the Agency for Science, Technology and Research, Singapore and the Ministry of Health, Singapore.
- Rulli NE, Melton J, Wilmes A, Ewart G, Mahalingam S: The molecular and cellular aspects of arthritis due to alphavirus infections: lesson learned from Ross River virus. Ann N Y Acad Sci 2007, 1102: 96-108. 10.1196/annals.1408.007PubMedView ArticleGoogle Scholar
- Powers AM, Logue CH: Changing patterns of chikungunya virus: re-emergence of a zoonotic arbovirus. J Gen Virol 2007, 88: 2363-2377. 10.1099/vir.0.82858-0PubMedView ArticleGoogle Scholar
- Schwartz O, Albert ML: Biology and pathogenesis of chikungunya virus. Nat Rev Microbiol 2010, 8: 491-500. 10.1038/nrmicro2368PubMedView ArticleGoogle Scholar
- Strauss JH, Strauss EG: The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 1994, 58: 491-562.PubMedPubMed CentralGoogle Scholar
- Ross RW: The Newala epidemic. III. The virus: isolation, pathogenic properties and relationship to the epidemic. J Hyg (Lond) 1956, 54: 177-191. 10.1017/S0022172400044442View ArticleGoogle Scholar
- Taylor RM, Hurlbut HS, Work TH, Kingston JR, Frothingham TE: Sindbis virus: a newly recognized arthropodtransmitted virus. Am J Trop Med Hyg 1955, 4: 844-862.PubMedGoogle Scholar
- Brighton SW, Prozesky OW, de la Harpe AL: Chikungunya virus infection. A retrospective study of 107 cases. S Afr Med J 1983, 63: 313-315.PubMedGoogle Scholar
- Borgherini G, Poubeau P, Jossaume A, Gouix A, Cotte L, Michault A, Arvin-Berod C, Paganin F: Persistent arthralgia associated with chikungunya virus: a study of 88 adult patients on reunion island. Clin Infect Dis 2008, 47: 469-475. 10.1086/590003PubMedView ArticleGoogle Scholar
- Chhabra M, Mittal V, Bhattacharya D, Rana U, Lal S: Chikungunya fever: a re-emerging viral infection. Indian J Med Microbiol 2008, 26: 5-12. 10.4103/0255-0857.38850PubMedView ArticleGoogle Scholar
- Labadie K, Larcher T, Joubert C, Mannioui A, Delache B, Brochard P, Guigand L, Dubreil L, Lebon P, Verrier B, de Lamballerie X, Suhrbier A, Cherel Y, Le Grand R, Roques P: Chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages. J Clin Invest 2010, 120: 894-906. 10.1172/JCI40104PubMedPubMed CentralView ArticleGoogle Scholar
- Mavalankar D, Shastri P, Raman P: Chikungunya epidemic in India: a major public-health disaster. Lancet Infect Dis 2007, 7: 306-307. 10.1016/S1473-3099(07)70091-9PubMedView ArticleGoogle Scholar
- Hoarau JJ, Jaffar Bandjee MC, Krejbich Trotot P, Das T, Li-Pat-Yuen G, Dassa B, Denizot M, Guichard E, Ribera A, Henni T, Tallet F, Moiton MP, Gauzere BA, Bruniquet S, Jaffar Bandjee Z, Morbidelli P, Martigny G, Jolivet M, Gay F, Grandadam M, Tolou H, Vieillard V, Debre P, Autran B, Gasque P: Persistent chronic inflammation and infection by Chikungunya arthritogenic alphavirus in spite of a robust host immune response. J Immunol 2010, 184: 5914-5927. 10.4049/jimmunol.0900255PubMedView ArticleGoogle Scholar
- Levine B, Hardwick JM, Griffin DE: Persistence of alphaviruses in vertebrate hosts. Trends Microbiol 1994, 2: 25-28. 10.1016/0966-842X(94)90341-7PubMedView ArticleGoogle Scholar
- Li L, Jose J, Xiang Y, Kuhn RJ, Rossmann MG: Structural changes of envelope proteins during alphavirus fusion. Nature 2010, 468: 705-708. 10.1038/nature09546PubMedPubMed CentralView ArticleGoogle Scholar
- Voss JE, Vaney MC, Duquerroy S, Vonrhein C, Girard-Blanc C, Crublet E, Thompson A, Bricogne G, Rey FA: Glycoprotein organization of Chikungunya virus particles revealed by X-ray crystallography. Nature 2010, 468: 709-712. 10.1038/nature09555PubMedView ArticleGoogle Scholar
- Bourai M, Lucas-Hourani M, Gad HH, Drosten C, Jacob Y, Tafforeau L, Cassonnet P, Jones LM, Judith D, Couderc T, Lecuit M, Andre P, Kummerer BM, Lotteau V, Despres P, Tangy F, Vidalain PO: Mapping of Chikungunya Virus Interactions with Host Proteins Identified nsP2 as a Higly Connected Viral Component. J Virol 2012, 86: 3121-3134. 10.1128/JVI.06390-11PubMedPubMed CentralView ArticleGoogle Scholar
- Garmashova N, Gorchakov R, Volkova E, Paessler S, Frolova E, Frolov I: The Old World and New World alphaviruses use different virus-specific proteins for induction of transcriptional shutoff. J Virol 2007, 81: 2472-2484. 10.1128/JVI.02073-06PubMedPubMed CentralView ArticleGoogle Scholar
- Sawicki DL, Perri S, Polo JM, Sawicki SG: Role for nsP2 proteins in the cessation of alphavirus minus-strand synthesis by host cells. J Virol 2006, 80: 360-371. 10.1128/JVI.80.1.360-371.2006PubMedPubMed CentralView ArticleGoogle Scholar
- Toribio R, Ventoso I: Inhibition of host translation by virus infection in vivo. Proc Natl Acad Sci U S A 2010, 107: 9837-9842. 10.1073/pnas.1004110107PubMedPubMed CentralView ArticleGoogle Scholar
- van Steeg H, Kasperaitis M, Voorma HO, Benne R: Infection of neuroblastoma cells by Semliki Forest virus. The interference of viral capsid protein with the binding of host messenger RNAs into initiation complexes is the cause of the shut-off of host protein synthesis. Eur J Biochem 1984, 138: 473-478. 10.1111/j.1432-1033.1984.tb07940.xPubMedView ArticleGoogle Scholar
- Tardif KD, Waris G, Siddiqui A: Hepatitis C virus, ER stress, and oxidative stress. Trends Microbiol 2005, 13: 159-163. 10.1016/j.tim.2005.02.004PubMedView ArticleGoogle Scholar
- Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D: Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol 2000, 2: 326-332. 10.1038/35014014PubMedView ArticleGoogle Scholar
- Novoa I, Zhang Y, Zeng H, Jungreis R, Harding HP, Ron D: Stress-induced gene expression requires programmed recovery from translational repression. EMBO J 2003, 22: 1180-1187. 10.1093/emboj/cdg112PubMedPubMed CentralView ArticleGoogle Scholar
- Oyadomari S, Araki E, Mori M: Endoplasmic reticulum stress-mediated apoptosis in pancreatic beta-cells. Apoptosis 2002, 7: 335-345. 10.1023/A:1016175429877PubMedView ArticleGoogle Scholar
- Kopito RR: ER quality control: the cytoplasmic connection. Cell 1997, 88: 427-430. 10.1016/S0092-8674(00)81881-4PubMedView ArticleGoogle Scholar
- Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, Walter P: Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 2000, 101: 249-258. 10.1016/S0092-8674(00)80835-1PubMedView ArticleGoogle Scholar
- Yoshida H, Matsui T, Hosokawa N, Kaufman RJ, Nagata K, Mori K: A time-dependent phase shift in the mammalian unfolded protein response. Dev Cell 2003, 4: 265-271. 10.1016/S1534-5807(03)00022-4PubMedView ArticleGoogle Scholar
- Kozutsumi Y, Segal M, Normington K, Gething MJ, Sambrook J: The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature 1988, 332: 462-464. 10.1038/332462a0PubMedView ArticleGoogle Scholar
- Harding HP, Calfon M, Urano F, Novoa I, Ron D: Transcriptional and translational control in the Mammalian unfolded protein response. Annu Rev Cell Dev Biol 2002, 18: 575-599. 10.1146/annurev.cellbio.18.011402.160624PubMedView ArticleGoogle Scholar
- Bitko V, Barik S: An endoplasmic reticulum-specific stress-activated caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells by respiratory syncytial virus. J Cell Biochem 2001, 80: 441-454. 10.1002/1097-4644(20010301)80:3<441::AID-JCB170>3.0.CO;2-CPubMedView ArticleGoogle Scholar
- Isler JA, Skalet AH, Alwine JC: Human cytomegalovirus infection activates and regulates the unfolded protein response. J Virol 2005, 79: 6890-6899. 10.1128/JVI.79.11.6890-6899.2005PubMedPubMed CentralView ArticleGoogle Scholar
- Netherton CL, Parsley JC, Wileman T: African swine fever virus inhibits induction of the stress-induced proapoptotic transcription factor CHOP/GADD153. J Virol 2004, 78: 10825-10828. 10.1128/JVI.78.19.10825-10828.2004PubMedPubMed CentralView ArticleGoogle Scholar
- Paradkar PN, Ooi EE, Hanson BJ, Gubler DJ, Vasudevan SG: Unfolded protein response (UPR) gene expression during antibody-dependent enhanced infection of cultured monocytes correlates with dengue disease severity. Biosci Rep 2011, 31: 221-230. 10.1042/BSR20100078PubMedView ArticleGoogle Scholar
- Pena J, Harris E: Dengue virus modulates the unfolded protein response in a time-dependent manner. J Biol Chem 2011, 286: 14226-14236. 10.1074/jbc.M111.222703PubMedPubMed CentralView ArticleGoogle Scholar
- Gale MJ Jr, Korth MJ, Tang NM, Tan SL, Hopkins DA, Dever TE, Polyak SJ, Gretch DR, Katze MG: Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology 1997, 230: 217-227. 10.1006/viro.1997.8493PubMedView ArticleGoogle Scholar
- Sood R, Porter AC, Ma K, Quilliam LA, Wek RC: Pancreatic eukaryotic initiation factor-2alpha kinase (PEK) homologues in humans, Drosophila melanogaster and Caenorhabditis elegans that mediate translational control in response to endoplasmic reticulum stress. Biochem J 2000, 346 Pt 2: 281-293.PubMedView ArticleGoogle Scholar
- Cheng G, Feng Z, He B: Herpes simplex virus 1 infection activates the endoplasmic reticulum resident kinase PERK and mediates eIF-2alpha dephosphorylation by the gamma(1)34.5 protein. J Virol 2005, 79: 1379-1388. 10.1128/JVI.79.3.1379-1388.2005PubMedPubMed CentralView ArticleGoogle Scholar
- Doherty RL, Carley JG, Mackerras MJ, Marks EN: Studies of arthropod-borne virus infections in Queensland. III. Isolation and characterization of virus strains from wild-caught mosquitoes in North Queensland. Aust J Exp Biol Med Sci 1963, 41: 17-39. 10.1038/icb.1963.2PubMedView ArticleGoogle Scholar
- Rathore AP, Paradkar PN, Watanabe S, Tan KH, Sung C, Connolly JE, Low J, Ooi EE, Vasudevan SG: Celgosivir treatment misfolds dengue virus NS1 protein, induces cellular pro-survival genes and protects against lethal challenge mouse model. Antiviral Res 2011, 92: 453-460. 10.1016/j.antiviral.2011.10.002PubMedView ArticleGoogle Scholar
- Umareddy I, Pluquet O, Wang QY, Vasudevan SG, Chevet E, Gu F: Dengue virus serotype infection specifies the activation of the unfolded protein response. Virol J 2007, 4: 91. 10.1186/1743-422X-4-91PubMedPubMed CentralView ArticleGoogle Scholar
- Solignat M, Gay B, Higgs S, Briant L, Devaux C: Replication cycle of chikungunya: a re-emerging arbovirus. Virology 2009, 393: 183-197. 10.1016/j.virol.2009.07.024PubMedPubMed CentralView ArticleGoogle Scholar
- Sourisseau M, Schilte C, Casartelli N, Trouillet C, Guivel-Benhassine F, Rudnicka D, Sol-Foulon N, Le Roux K, Prevost MC, Fsihi H, Frenkiel MP, Blanchet F, Afonso PV, Ceccaldi PE, Ozden S, Gessain A, Schuffenecker I, Verhasselt B, Zamborlini A, Saib A, Rey FA, Arenzana-Seisdedos F, Despres P, Michault A, Albert ML, Schwartz O: Characterization of reemerging chikungunya virus. PLoS Pathog 2007, 3: e89. 10.1371/journal.ppat.0030089PubMedPubMed CentralView ArticleGoogle Scholar
- Dhanushkodi NR, Mohankumar V, Raju R: Sindbis virus induced phosphorylation of IRF3 in human embryonic kidney cells is not dependent on mTOR. Innate Immun 2012, 18: 325-332. 10.1177/1753425911406944PubMedView ArticleGoogle Scholar
- Krejbich-Trotot P, Gay B, Li-Pat-Yuen G, Hoarau JJ, Jaffar-Bandjee MC, Briant L, Gasque P, Denizot M: Chikungunya triggers an autophagic process which promotes viral replication. Virol J 2011, 8: 432. 10.1186/1743-422X-8-432PubMedPubMed CentralView ArticleGoogle Scholar
- Davis JL, Hodge HM, Campbell WE Jr: Growth of chikungunya virus in baby hamster kidney cell (BHK-21-clone 13) suspension cultures. Appl Microbiol 1971, 21: 338-341.PubMedPubMed CentralGoogle Scholar
- Frolov I, Akhrymuk M, Akhrymuk I, Atasheva S, Frolova EI: Early events in alphavirus replication determine the outcome of infection. J Virol 2012, 86: 5055-5066. 10.1128/JVI.07223-11PubMedPubMed CentralView ArticleGoogle Scholar
- Kurkela S, Manni T, Myllynen J, Vaheri A, Vapalahti O: Clinical and laboratory manifestations of Sindbis virus infection: prospective study, Finland, 2002–2003. J Infect Dis 2005, 191: 1820-1829. 10.1086/430007PubMedView ArticleGoogle Scholar
- van Huizen R, Martindale JL, Gorospe M, Holbrook NJ: P58IPK, a novel endoplasmic reticulum stress-inducible protein and potential negative regulator of eIF2alpha signaling. J Biol Chem 2003, 278: 15558-15564. 10.1074/jbc.M212074200PubMedView ArticleGoogle Scholar
- Wang Y, Shen J, Arenzana N, Tirasophon W, Kaufman RJ, Prywes R: Activation of ATF6 and an ATF6 DNA binding site by the endoplasmic reticulum stress response. J Biol Chem 2000, 275: 27013-27020.PubMedGoogle Scholar
- Plongthongkum N, Kullawong N, Panyim S, Tirasophon W: Ire1 regulated XBP1 mRNA splicing is essential for the unfolded protein response (UPR) in Drosophila melanogaster. Biochem Biophys Res Commun 2007, 354: 789-794. 10.1016/j.bbrc.2007.01.056PubMedView ArticleGoogle Scholar
- Tardif KD, Mori K, Kaufman RJ, Siddiqui A: Hepatitis C virus suppresses the IRE1-XBP1 pathway of the unfolded protein response. J Biol Chem 2004, 279: 17158-17164. 10.1074/jbc.M312144200PubMedView ArticleGoogle Scholar
- Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K: XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 2001, 107: 881-891. 10.1016/S0092-8674(01)00611-0PubMedView ArticleGoogle Scholar
- Lin JH, Li H, Yasumura D, Cohen HR, Zhang C, Panning B, Shokat KM, Lavail MM, Walter P: IRE1 signaling affects cell fate during the unfolded protein response. Science 2007, 318: 944-949. 10.1126/science.1146361PubMedPubMed CentralView ArticleGoogle Scholar
- Molinari M, Calanca V, Galli C, Lucca P, Paganetti P: Role of EDEM in the release of misfolded glycoproteins from the calnexin cycle. Science 2003, 299: 1397-1400. 10.1126/science.1079474PubMedView ArticleGoogle Scholar
- Boyce M, Bryant KF, Jousse C, Long K, Harding HP, Scheuner D, Kaufman RJ, Ma D, Coen DM, Ron D, Yuan J: A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress. Science 2005, 307: 935-939. 10.1126/science.1101902PubMedView ArticleGoogle Scholar
- Oliver BL, Cronin CG, Zhang-Benoit Y, Goldring MB, Tanzer ML: Divergent stress responses to IL-1beta, nitric oxide, and tunicamycin by chondrocytes. J Cell Physiol 2005, 204: 45-50. 10.1002/jcp.20261PubMedView ArticleGoogle Scholar
- Fros JJ, Liu WJ, Prow NA, Geertsema C, Ligtenberg M, Vanlandingham DL, Schnettler E, Vlak JM, Suhrbier A, Khromykh AA, Pijlman GP: Chikungunya virus nonstructural protein 2 inhibits type I/II interferon-stimulated JAK-STAT signaling. J Virol 2010, 84: 10877-10887. 10.1128/JVI.00949-10PubMedPubMed CentralView ArticleGoogle Scholar
- Krejbich-Trotot P, Denizot M, Hoarau JJ, Jaffar-Bandjee MC, Das T, Gasque P: Chikungunya virus mobilizes the apoptotic machinery to invade host cell defenses. FASEB J 2011, 25: 314-325. 10.1096/fj.10-164178PubMedView ArticleGoogle Scholar
- White LK, Sali T, Alvarado D, Gatti E, Pierre P, Streblow D, Defilippis VR: Chikungunya virus induces IPS-1-dependent innate immune activation and protein kinase R-independent translational shutoff. J Virol 2011, 85: 606-620. 10.1128/JVI.00767-10PubMedPubMed CentralView ArticleGoogle Scholar
- Buchkovich NJ, Yu Y, Pierciey FJ Jr, Alwine JC: Human cytomegalovirus induces the endoplasmic reticulum chaperone BiP through increased transcription and activation of translation by using the BiP internal ribosome entry site. J Virol 2010, 84: 11479-11486. 10.1128/JVI.01330-10PubMedPubMed CentralView ArticleGoogle Scholar
- Jan JT, Griffin DE: Induction of apoptosis by Sindbis virus occurs at cell entry and does not require virus replication. J Virol 1999, 73: 10296-10302.PubMedPubMed CentralGoogle Scholar
- Joubert PE, Werneke SW, de la Calle C, Guivel-Benhassine F, Giodini A, Peduto L, Levine B, Schwartz O, Lenschow DJ, Albert ML: Chikungunya virus-induced autophagy delays caspase-dependent cell death. J Exp Med 2012, 209: 1029-1047. 10.1084/jem.20110996PubMedPubMed CentralView ArticleGoogle Scholar
- de Groot RJ, Rumenapf T, Kuhn RJ, Strauss EG, Strauss JH: Sindbis virus RNA polymerase is degraded by the N-end rule pathway. Proc Natl Acad Sci U S A 1991, 88: 8967-8971. 10.1073/pnas.88.20.8967PubMedPubMed CentralView ArticleGoogle Scholar
- Sarre TF: The phosphorylation of eukaryotic initiation factor 2: a principle of translational control in mammalian cells. Biosystems 1989, 22: 311-325. 10.1016/0303-2647(89)90053-1PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.