infected cells. In chapter 2 the changes in host protein abundance following CHIKV infec- tion were determined, while chapter 3 identified proteins of which the phosphorylation status changed during infection. This chapter summarizes these findings, places them in a broader context and discusses the reproducibility of proteomics studies.
Changes in protein abundance and phosphorylation status during ChiKv infection
As described in chapter 2, the changes in protein abundance in CHIKV-infected cells during the course of infection were rather limited, especially prior to the onset of host translational shut-off. Not only the number of proteins showing a significantly changed abundance was limited, but also the extent of change was quite small (<2 fold) for most proteins. Despite the lack of stronger effects, this study yielded interesting results. The majority of the proteins for which a significant change was observed showed a decreasing abundance after CHIKV infection (figure 1, chapter 2). For many proteins this decrease was probably the result of their normal turn-over, i.e. natural degradation in the absence of translation due to the virus-induced host shut-off that starts around 8 h p.i. However, certain proteins are most likely specifically targeted by the virus for degradation. The best example is the RNA polymerase II (POLR2) complex of which most subunits were progressively degraded during CHIKV infection (Table 2, chapter 2). This is in line with a previous study that found that the Rpb1 subunit, which is the catalytic subunit of the POLR2 complex, is targeted for degradation by nsP2 [63]. The same could be true for Rnd3, DDX56 and Plk1, three of the proteins chosen for follow-up research. CHIKV replication was reduced in cells overexpressing these proteins, suggesting that their presence is not beneficial for the virus (figure 3,chapter 2). The decreasing abun- dance of these proteins might be explained by the viral manipulation of the intracellular environment to create optimal conditions for replication, or could even be part of a specific strategy to evade antiviral responses.
Based on the results in chapter 2, one could argue that a proteomics study studying only changes in protein abundance may not be the best approach to identify host fac- tors that respond to viral infection in the case of viruses that kill their host cell relatively quickly. During the relatively short course of the infection, the time frame for induction of changes in protein abundance is simply not large enough, especially when the virus induces a transcriptional and/or translational host shut-off. For viruses that cause a per- sistent infection, meaning that cells can be monitored over the course of several days, this strategy seems a better option [202].
The low number of changes observed during CHIKV infection suggested that the cel- lular response to infection during the first cycle of replication does not affect protein abundance. The much more abundant and larger changes that were observed at the
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level of phosphorylation status, described in chapter 3, proved this assumption to be correct. Several phosphorylation sites showed large (>4 fold) increases or decreases in phosphorylation during CHIKV infection, especially at 8 and 12 h p.i. Phosphoryla- tion on one site, T56 on eEF2, even increased >50 fold at 8 and 12 h p.i., and already showed a significant increase in phosphorylation at 2 h p.i. Therefore, the role of eEF2 in the infection with CHIKV and other viruses was studied in more detail. At this point it remains unknown what the trigger is for the induction of eEF2 phosphorylation dur- ing alphavirus infection. However, it does not appear to rely on pathogen-associated molecular pattern (PAMP) recognition (figure 5, chapter 3) or energy sensing pathways (figure 4, chapter 3), although additional studies are required to completely exclude their involvement. The presence of the viral structural proteins is not required and merely the presence of a small amount of viral (uncapped) RNA is not enough to induce phosphorylation (figure 6, chapter 3). Perhaps the RNA structures in the viral UTRs are sensed. The reduction of available eEF2 appears to restrict alphavirus replication since siRNA-mediated knockdown of eEF2 reduced translation of viral proteins, although this was a minor effect.
The induction of eEF2 phosphorylation slows down translation, which is unfavorable for a replicating virus, and this might be a general antiviral response. However, while this strong induction of eEF2 phosphorylation in response to infection was also observed with two other alphaviruses and a picornavirus (figure 2b and C, chapter 3), and was previously reported to occur during infection with rift valley fever virus (RVFV), a –RNA virus from the bunyavirus family [235], two other viruses, equine arteritis virus (EAV), a +RNA virus from the arterivirus family and human adenovirus, a dsDNA virus, did not induce a measurable increase in eEF2 phosphorylation (figure 2d, chapter 3). It will be interesting to determine whether viruses from other families also induce eEF2 phosphorylation as this may help to determine what the trigger is. The large increase in phosphorylation of eEF2 residue T56 upon alphavirus infection is induced early, as a 1.6-fold increase could already be observed as early as 2 h p.i. in CHIKV-infected MRC-5 cells in the proteomics analysis. This relatively small increase proved to be relevant since phosphorylation levels strongly increased as infection progressed. The western blot analysis was not equally sensitive as phosphorylated eEF2 could only be detected at 6 h p.i. in these cells (figure 2A, chapter 3). This is an important caveat, since western blot analysis is often used to confirm mass spectrometry findings. However, in case of a phos- phorylation site with a low occupancy at the start of the experiment, a relatively small increase in phosphorylation may be hard to detect with phospho-specific antibodies. It proved helpful to analyze different time points as the 2 h p.i. mass spectrometry analysis indicated that the induction of eEF2 phosphorylation started several hours prior to what we would have been able to determine from the WB analysis.
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