Staufen STAU1 ja Ebolavirus

Ebola virus (EBOV) genome and mRNAs contain long, structured regions that could hijack host RNA-binding proteins to facilitate infection. We performed RNA affinity chromatography coupled with mass spectrometry to identify host proteins that bind to EBOV RNAs and identified four high-confidence proviral host factors, including Staufen1 (STAU1), which specifically binds both 3′ and 5′ extracistronic regions of the EBOV genome. We confirmed that EBOV infection rate and production of infectious particles were significantly reduced in STAU1-depleted cells. STAU1 was recruited to sites of EBOV

RNA synthesis upon infection and enhanced viral RNA synthesis. Furthermore, STAU1 interacts with EBOV nucleoprotein (NP), virion protein 30 (VP30), and VP35; the latter two bridge the viral polymerase to the NP-coated genome, forming the viral ribonucleoprotein (RNP) complex.STAU1 plays a critical role in EBOV replication by coordinating interactions between the viral genome and RNA synthesis machinery.

LÄHDE:  https://mbio.asm.org/content/9/5/e01771-18#sec-9

DISCUSSION

Our data provide evidence that STAU1 is the first cellular factor reported to associate with three essential components of the EBOV RNA synthesis machinery: NP, VP35, and VP30. The NP-VP35 complex serves as a critical backbone for the viral polymerase L to recognize NP-encapsidated RNA genomes, which is the prerequisite for both EBOV transcription and genome replication. Similarly, VP30 is also believed to bridge interactions between L and NP, and it is critical for transcription of viral mRNA (13, 57). Our observations further imply an association of STAU1 with the EBOV RNA genome-NP-VP35 complex, suggesting that regions of the EBOV genome are available for interaction with trans-acting factors in the presence of NP. Related to our finding that STAU1 preferentially binds to 3′ and 5′ extracistronic regions of the EBOV genome in vitro, we propose a model in which STAU1 is involved in the early formation of EBOV RNA synthesis machinery on the 3′ extracistronic region of the EBOV genome, and likewise, in the termination and release of EBOV RNA synthesis machinery on the 5′ extracistronic region (see Fig. S8 in the supplemental material).

Previous efforts have been made to understand EBOV host factor biology by focusing on protein-protein interactions between viral and host proteins (58–62). To provide more insights into how host factors regulate EBOV replication through cis-acting elements in EBOV RNA, we performed a RAC-MS screen and discovered 14 host RBPs specifically enriched for the selected EBOV noncoding RNAs. Among these RBPs, ILF2, ILF3, HNRNPL, and STAU1 stand out as strong EBOV candidate host factors because all three siRNAs tested caused a significant reduction of EBOV infection. While HNRNPL preferentially associated with the 5′ UTR of EBOV NP mRNA, ILF2, ILF3, and STAU1 selectively interacted with the EBOV trailer. It is noteworthy that ILF3 (also known as DRBP76/NF90) was previously identified as a VP35 interactor as well as an EBOV polymerase suppressor (61). Although this previous study did not observe a dramatic effect of reduced ILF3 expression on EBOV infection in 293T cells, the data we present here showed that depletion of ILF3 in HuH-7 cells significantly impaired EBOV infection. This discrepancy could be explained by cell line differences. Nevertheless, the identification of ILF2 (or NF45) in our screen, which is physically and functionally linked to ILF3 (44), highlights the important role that ILF3 perhaps has in EBOV infection.

We focused on STAU1 as a novel EBOV host factor that promotes efficient virus infection. STAU1 is a protein that binds to stable RNA secondary structure (63), a tubulin-binding protein that interacts with cytoskeleton (50), a ribosome-associated protein (64, 65) that accumulates in stress granules induced by certain types of stress (66), and a key mediator of mRNA decay (67, 68). These biochemical characteristics enable STAU1 to fulfill different biological functions: from controlling the localization to enhancing the translation of its target RNAs. Therefore, elucidation of a functional domain(s) of STAU1 that is responsible for EBOV host factor activity may allow generation of targeted inhibitors antagonizing the STAU1 proviral effect.

Previous studies of several RNA viruses have revealed that STAU1 binding to viral RNA regulates infection. For instance, STAU1 facilitates translation of hepatitis C virus RNA through binding to the internal ribosome entry site (46). STAU1 plays a role in the production of viral particles for both influenza A virus (47) and human immunodeficiency virus type 1 (48). Our RAC-MS results indicated that STAU1 preferentially associates with EBOV genomic RNA and not with the 5′ UTR of two viral mRNAs. Therefore, it is unlikely that STAU1 promotes EBOV mRNA translation; however, we tested only the 5′ UTRs of NP and VP24 transcripts, so we cannot eliminate the possibility that STAU1 may bind to other UTRs and participate in their translation. Another caveat is that the EBOV genomic RNA used in the RAC experiment was naked, while during infection, it is likely to be mostly encapsidated by NP and therefore relatively inaccessible (69). Nevertheless, the facts that STAU1 is recruited to sites of viral RNA synthesis and forms a complex in cells with NP, VP35, and minigenome RNA suggest interactions between STAU1 and EBOV genomic RNA in infected cells.

Several other stress granule (SG) markers (for instance, the initiation factors eIF3 and eIF4G) were previously found to localize on discrete, compact granules within EBOV inclusion bodies (30). In contrast to this, STAU1 in EBOV inclusion bodies appeared in a heterogeneous pattern that changed over the course of infection. In some areas near the border of EBOV inclusion bodies, where VP35 was almost absent, STAU1 formed small aggregates that branched out of the inclusion border (Fig. S5C). This distinct distribution of STAU1 suggests that it may function differently than other SG proteins with regard to interactions with EBOV inclusion bodies.

Given the correlation between the level of STAU1 expression and EBOV minigenome activity, it is of interest to pinpoint the molecular event(s) STAU1 regulates during EBOV RNA synthesis. For nonsegmented, negative-sense RNA viruses, the first step of viral RNA synthesis is primary transcription in order to generate sufficient levels of viral proteins. When a supply of NP has been synthesized, genome replication and secondary transcription are allowed to commence, leading to the production of encapsidated antigenomes and progeny genomes as well as naked mRNAs (70). Attempts to distinguish whether STAU1 enhances genome replication or transcription using the minigenome system by strand-specific quantitative PCR (qPCR) were not successful, however, due to residual DNA from transfected plasmids despite DNase treatment of RNA samples. Future studies using other model systems (i.e., replication-deficient minigenome) are required to ascertain whether STAU1 participates in EBOV transcription (71).

Preliminary characterization of STAU1-associated EBOV minigenome RNP (reconstituted by EBOV minigenome RNA, NP, and VP35) revealed another interesting cellular player, PKR. Although PKR is well-known for its antiviral function by sensing viral RNA and phosphorylating the host translation initiation factor eIF2α (72), it has also been implicated in the phosphorylation and regulation of the HIV trans-acting protein Tat, which binds to the transactivation-responsive element (TAR) in the HIV genome, in the context of viral infection (73, 74). Interestingly, no host kinase has yet been reported to regulate phosphorylation of EBOV VP30, even though a growing number of studies indicate a crucial role of dynamic VP30 phosphorylation in EBOV RNA synthesis and RNP assembly (19, 21, 55, 75, 76). Although several reports indicate that EBOV antagonizes PKR activity (53, 54), one can imagine scenarios in which PKR is hijacked by this STAU1-containing viral RNP complex to dynamically control the phosphorylation of EBOV VP30. Further investigation is needed to clarify the role of PKR in EBOV infection.

In conclusion, we identified STAU1 as the first host factor reported to interact with multiple EBOV RNP components, highlighting the significance of this RBP for the EBOV life cycle. These interactions together with redistribution of STAU1 to EBOV inclusion bodies and to NP-induced inclusion bodies link STAU1 to EBOV RNA synthesis, for which we speculate that STAU1 facilitates both the initiation and termination steps. It will be of interest to clarify the exact molecular events occurring during initiation and termination of EBOV RNA synthesis and how STAU1 may participate in these processes. Our study contributes to knowledge of the role of host factors in EBOV RNA synthesis and provides a novel cellular target for the development of possible therapeutic interventions to combat EBOV infection.