Our results presented here imply that RVFV NSs promotes the degradation of two different substrates, PKR and p62, in order to ensure efficient viral translation and the suppression of the host antiviral response, respectively. Le May et al. (27
) have shown that NSs is able to interfere with TFIIH assembly by sequestering its p44 subunit. Prompted by the observation that nuclear extracts of RVFV-infected cells contain reduced amounts of the TFIIH subunit p62 in comparison to the amounts in uninfected cells (27
) and by the recent discovery that NSs is able to promote the proteasomal degradation of PKR (14
), we identified p62 as a second substrate of NSs-mediated degradation.
Infection of cells with the RVFV MP-12 vaccine strain resulted in a complete reduction of p62 protein levels at 6 h.p.i. and beyond. Transfection of cells with in vitro-synthesized RNA for NSs was equally able to downregulate p62, indicating that this effect was specific to NSs and not a general consequence of RVFV infection. NSs did not affect p62 transcription or alter the mRNA stability of p62, as p62 mRNA levels remained unchanged at 8 h.p.i. in MP-12-infected cells. This is further corroborated by the finding that pharmacological inhibition of transcription by treatment of cells with actinomycin D for 8 h only marginally decreased p62 levels. This finding also suggests that the transcriptional suppression observed in RVFV-infected cells is indeed a consequence and not the cause of p62 downregulation. To determine whether NSs downregulated p62 by inhibiting p62 protein neosynthesis or, rather, by turnover, we treated cells with cycloheximide for 8 h to inhibit translation. Pharmacological inhibition of protein neosynthesis for 8 h did not alter the p62 levels, indicating that NSs promoted p62 downregulation via a posttranslational mechanism.
Because the selective degradation of most cellular proteins is carried out by the ubiquitin-proteasome system (17
), we investigated whether inhibitors of proteasomal (MG132 and lactacystin) or lysosomal (chloroquine) degradation were able to stabilize p62 in the presence of NSs. Interestingly, MG132 was able to inhibit p62 degradation, while treatment with the more specific proteasomal inhibitor lactacystin only resulted in a partial stabilization of p62. Conversely, treatment of cells with chloroquine had no effect on the degradation of p62. Treatment of cells with either a proteasomal or lysosomal inhibitor greatly reduced the amount of NSs transcribed from transfected RNA. We believe this might be caused by activation of the unfolded protein response (UPR) by pharmacological inhibition of protein degradation (34
). Activation of the UPR in turn results in translational arrest (55
) and, ultimately, in reduced NSs levels in comparison to the levels in untreated cells. Since treatment of cells with proteasomal inhibitors stabilizes p62 and reduces NSs levels at the same time, it is unclear whether this stabilization of p62 is mediated directly by inhibition of the proteasome rather than being due to the loss of NSs. However, as the amount of NSs is equally reduced in chloroquine-treated cells, although this inhibitor does not stabilize p62 levels, these results suggest involvement of the proteasome rather than the lysosome in the degradation of p62.
Recent findings demonstrate extensive cross talk between the pathways of lysosomal and proteasomal degradation; most notably, proteasomal substrates have been shown to be targeted to the lysosome via selective autophagy as a compensatory mechanism during proteasomal inhibition (25
). We therefore speculate that by treating cells with MG132, which also has partial activity against lysosomal proteases (26
), this compensatory mechanism is also affected, which ultimately results in a better stabilization of p62 than can be achieved by inhibition of the proteasome alone. Unfortunately, concurrent treatment of cells with both lactacystin and chloroquine resulted in massive cell death (unpublished results), so the effect of MG132 could not be recapitulated by combining these two inhibitors.
Both NSs (52
) and the proteasome (37
) are present in cytoplasmic as well as nuclear compartments, and the nuclear localization of NSs has previously been shown to be required for efficient transcriptional suppression (2
). The localization of p62, on the other hand, is predominantly nuclear, and pharmacological inhibition of nuclear export has no effect on the NSs-mediated degradation of p62. Furthermore, after MP-12 infection and in the presence of proteasomal inhibitors, p62 remained localized in the nucleus. This suggests that p62 does not need to be exported into the cytoplasm to be degraded but, rather, is degraded directly inside the nucleus.
Proteins are canonically targeted for proteasomal degradation by covalent attachment of a chain of ubiquitin moieties (17
), and it will be important to determine whether NSs is able to promote the polyubiquitination of p62. Interestingly, two distinct ubiquitin ligases (NEDD4 and the elongin/Rbx1/Cul5 complex) have recently been identified as being responsible for the polyubiquitination of the large subunit of RNA polymerase II (16
). The elongin/Rbx1/Cul5 complex is also recruited by the adenoviral E4orf6 protein (40
) and HIV-1 Vif (53
). Furthermore, although it displays no sequence similarity to the RVFV NSs protein, the NSs protein of Bunyamwera virus has been shown to interact with MED8 (29
), which also serves as a substrate recognition factor of the elongin/Rbx1/Cul2 E3 complex (5
). We hypothesize that NSs functions by recruiting a cellular ubiquitin ligase to promote the ubiquitination and subsequent degradation of p62, and it will be important to characterize the complex containing NSs and p62 for a more detailed understanding of NSs-mediated p62 degradation.
The question remains as to whether and, if so, why RVFV employs two redundant mechanisms to induce transcriptional suppression in its host cell, i.e., (i) sequestration of p44 by NSs and (ii) NSs-mediated p62 degradation. Le May et al. suggested that the sequestration of p44 can only prevent the assembly of new TFIIH complexes and leaves preexisting ones unaffected (27
). Furthermore, NSs only interacts with free p44 and not with p44 which has been assembled into the TFIIH complex (27
). If NSs acted only through the sequestration of p44, the onset of transcriptional suppression would be delayed until existing TFIIH had been turned over naturally. By promoting the degradation of p62, on the other hand, NSs might be able to disrupt and inactivate fully assembled complexes. Indeed, partial transcriptional suppression can be observed as early as 4 h.p.i. (). Furthermore, substoichiometric amounts of NSs would be sufficient to completely inactivate all existing and newly synthesized TFIIH, which might be important since the synthesis of large amounts of NSs occurs only after secondary transcription of the ambisense S segment and relatively late during viral replication (21