The transfection of viral RNA into cells circumvents clathrin-mediated endocytosis. Since the transfected replicon RNA was still able to induce strong eEF2 phosphoryla- tion, it is not likely that one of the steps during CHIKV entry is responsible for triggering phosphorylation. Perhaps RNA structures in the viral genome are sensed in the cytosol and this triggers an innate immune response that slows down protein synthesis. Inhibi- tion of CHIKV replication by pretreatment with 5’pppRNA of cells prior to infection also prevented the induction of eEF2 phosphorylation suggesting that the presence of a certain level of e.g. viral RNA or protein is required before phosphorylation is induced. In different cell lines infected with CHIKV, infection does not progress at the same rate. In Vero E6 cells, in which viral proteins could be detected with WB by 4 h p.i., the induction of phosphorylation could also be observed at that time point. In MRC-5 cells, in which infection progresses slower, viral proteins could not be detected until 8 h p.i. while only a small amount of phosphorylated eEF2 could be detected at 6 h p.i. This also suggests that a certain level of e.g. viral RNA or protein has to be reached before the induction of phosphorylation is fully triggered.
It was recently reported that eEF2 is required for the Gag-mediated block of stress granule assembly during HIV-1 infection [264]. Although this paper suggests that eEF2 phosphorylation might decrease during HIV-1 infection, this stress granule connection might be interesting to explore for CHIKV as well since CHIKV also exhibits complex interactions with stress granules and some of their components, such as the G3BPs [65]. An increase of cytosolic Ca2+ early during alphavirus infection could trigger activa- tion of eEF2K, but there is conflicting evidence as to whether alphaviruses induce an increase in cytosolic calcium levels during infection [265, 266].
eEf2 abundance does not change during alphavirus infection
Other groups have reported a decrease in eEF2 abundance during CHIKV infection [111, 112, 119]. However, in our previous proteomics study the abundance of eEF2 did not change [208] and we have not been able to reproduce this finding in our WB analysis at the time points that we studied either. In the other studies, samples were analyzed at much later time points in infection (24 h p.i. or later) than we used and eEF2 might be degraded during this stage of infection. This might not be an eEF2-specific property, however, since alphaviruses induce a host translational shut-off [100]. Two of the studies that quantified a decrease in eEF2 levels used 2D-DIGE and most likely the large increase of phosphorylated eEF2 during CHIKV infection significantly decreased the amount of eEF2 in the spot containing the unmodified form of eEF2, which could explain the observed decrease in abundance in these studies [112, 119].
It has been shown that ribosomes and eEF2 co-localize with aggregation sites of the C protein during SINV infection [267]. eEF2 was also identified as one of the interaction
78 A + RNA virus diptych
partners of SINV nsP3 during infection with a nsP3-GFP expressing SINV mutant [94]. In these studies the phosphorylation status of eEF2 was not determined, but it would be interesting to determine whether these interactions occur with the modified or unmodi- fied form of eEF2.
CoNCLusioN
In our previous proteomics analysis of CHIKV-infected cells, there were only minimal changes in protein abundance and most of the proteins that did show a significantly changed abundance were downregulated [208]. The current study shows that more and larger changes occur on the level of phosphorylation and that both increased phos- phorylation and dephosphorylation can be observed. Our data suggest that, at least for alphaviruses, cellular responses to viral infection may mainly occur on the level of PTMs and it would certainly be interesting to study other PTMs in this context. However, it continues to be important to analyze changes in total protein abundance as well, since a two-fold change in protein abundance can also result in a two-fold change in phosphorylation status, which would falsely indicate that the phosphorylation status is regulated while it is in fact the protein abundance.
Our study revealed that early during infection with several alphaviruses and a picornavirus, eEF2 becomes phosphorylated. The phosphorylation status of sites of other proteins involved in translation also changed significantly during CHIKV infec- tion and possibly also contribute to translation inhibition. The phosphorylation of eEF2 is unfavorable for CHIKV replication as a reduction in the eEF2 levels resulted in a decreased expression of CHIKV proteins. The trigger for eEF2 phosphorylation during CHIKV infection is not yet known, but it does not appear to be mediated through one of the energy-sensing pathways, RIG-I signaling, alphavirus structural proteins or the viral entry process. We hypothesize that the increase in eEF2 phosphorylation is triggered by a special/unique feature of the viral genome (e.g. RNA structure) or by the translation or transcription of (alpha)virus RNA as a novel antiviral/stress response that slows down translation.
ACKNoWLEdGmENTs
The authors are grateful to dr. Sander Piersma for his help with the HAMMOC procedure, to Irina Albulescu for technical assistance in the BSL-3 lab and to Jeroen de Keijzer and dr. George Janssen for helpful discussions.
Chapter 3 79
3
suPPoRTiNG iNfoRmATioNsupporting information tables
supporting information Table s1: Complete list of identified phosphorylation sites, the first sheet con-
tains all sites identified in host proteins, the second sheet contains the sites identified in CHIKV proteins. In case this research has not yet been published this table can be requested from the author.
supporting information Table s3: Go terms that the list with unique phosphoproteins that contained phosphorylation sites that were significantly modulated during ChiKv infection was enriched for. Go_id molecular
function
Term NumberofGenes p-value p-value_fdr p-value_
bonferroni
GO:0044822 poly(A) RNA binding 42 2.81E-24 1.11E-20 1.11E-20
GO:0003723 RNA binding 44 3.76E-23 7.4E-20 1.48E-19
GO:0003779 actin binding 12 1.97E-07 0.000258 0.000775
GO:0003676 nucleic acid binding 39 2.66E-07 0.000262 0.00105
GO:0005515 protein binding 76 5.75E-07 0.000452 0.00226
GO:0008092 cytoskeletal protein binding 15 2.62E-06 0.00172 0.0103
Go_id biological processes
Term NumberofGenes p-value p-value_fdr p-value_
bonferroni
GO:0046907 intracellular transport 23 5.70E-08 7.66E-04 7.66E-04
GO:0051641 cellular localization 28 5.11E-07 2.81E-03 6.88E-03
GO:0051649 establishment of localization in cell 25 6.90E-07 2.81E-03 9.29E-03
GO:0007010 cytoskeleton organization 16 8.84E-07 2.81E-03 1.19E-02
GO:0071840 cellular component organization or
biogenesis
45 1.04E-06 2.81E-03 1.40E-02
GO:0008380 RNA splicing 11 1.44E-06 3.23E-03 1.94E-02
GO:0007167 enzyme linked receptor protein
signaling pathway
17 2.02E-06 3.63E-03 2.71E-02
GO:0033036 macromolecule localization 26 2.16E-06 3.63E-03 2.90E-02
GO:0006996 organelle organization 31 3.49E-06 4.93E-03 4.70E-02
GO:0016043 cellular component organization 43 3.70E-06 4.93E-03 4.98E-02
Go_id Cellular Component
Term NumberofGenes p-value p-value_fdr p-value_
bonferroni
GO:0005925 focal adhesion 27 4.95E-23 3.75E-20 7.84E-20
GO:0005924 cell-substrate adherens junction 27 7.09E-23 3.75E-20 1.12E-19
supporting information Table s2: Lists with phosphorylation sites that were significantly modulated dur-
ing CHIKV infection. Each time point is put in a separate sheet. In case this research has not yet been pub- lished this table can be requested from the author.
80 A + RNA virus diptych
supporting information Table s3 (continued)
Go_id Cellular Component
Term NumberofGenes p-value p-value_fdr p-value_
bonferroni
GO:0030055 cell-substrate junction 27 7.09E-23 3.75E-20 1.12E-19
GO:0005912 adherens junction 27 2.40E-21 9.49E-19 3.80E-18
GO:0070161 anchoring junction 27 6.31E-21 2.00E-18 9.99E-18
GO:0030054 cell junction 31 4.69E-15 1.24E-12 7.42E-12
GO:0015629 actin cytoskeleton 17 1.59E-11 3.60E-09 2.52E-08
GO:0044422 organelle part 68 1.33E-09 2.63E-07 2.11E-06
GO:0044446 intracellular organelle part 67 1.51E-09 2.66E-07 2.39E-06
GO:0043228 non-membrane-bounded organelle 42 2.13E-09 3.07E-07 3.38E-06
GO:0043232 intracellular non-membrane-
bounded organelle
42 2.13E-09 3.07E-07 3.38E-06
GO:0044428 nuclear part 44 9.81E-09 1.29E-06 1.55E-05
GO:0031988 membrane-bounded vesicle 42 1.72E-08 2.09E-06 2.72E-05
GO:0005829 cytosol 38 4.07E-08 4.60E-06 6.44E-05
GO:0031981 nuclear lumen 40 1.32E-07 1.39E-05 2.09E-04
GO:0043230 extracellular organelle 36 2.26E-07 1.89E-05 3.59E-04
GO:0070062 extracellular exosome 36 2.26E-07 1.89E-05 3.59E-04
GO:0065010 extracellular membrane-bounded
organelle
36 2.26E-07 1.89E-05 3.59E-04
GO:0031982 vesicle 40 3.47E-07 2.75E-05 5.50E-04
GO:0005856 cytoskeleton 26 4.92E-07 3.71E-05 7.80E-04
GO:0032991 macromolecular complex 43 5.22E-07 3.76E-05 8.26E-04
GO:0005654 nucleoplasm 35 5.80E-07 3.99E-05 9.18E-04
GO:0005634 nucleus 56 7.48E-07 4.94E-05 1.18E-03
GO:0042641 actomyosin 6 1.31E-06 8.28E-05 2.07E-03
GO:0070013 intracellular organelle lumen 42 1.72E-06 1.05E-04 2.73E-03
GO:0043233 organelle lumen 42 2.54E-06 1.49E-04 4.03E-03
GO:0031974 membrane-enclosed lumen 42 3.55E-06 2.01E-04 5.63E-03
GO:0043227 membrane-bounded organelle 76 3.91E-06 2.14E-04 6.19E-03
GO:0044421 extracellular region part 38 4.85E-06 2.56E-04 7.69E-03
GO:0043234 protein complex 37 7.66E-06 3.92E-04 1.21E-02
GO:0005911 cell-cell junction 10 1.34E-05 6.63E-04 2.12E-02
GO:0016020 membrane 59 2.24E-05 1.08E-03 3.55E-02
GO:0005737 cytoplasm 68 2.55E-05 1.19E-03 4.05E-02
Chapter 3 81