scholarly journals The Ebola Virus Nucleoprotein Recruits the Nuclear RNA Export Factor NXF1 into Inclusion Bodies to Facilitate Viral Protein Expression

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 187 ◽  
Author(s):  
Lisa Wendt ◽  
Janine Brandt ◽  
Bianca S. Bodmer ◽  
Sven Reiche ◽  
Marie Luisa Schmidt ◽  
...  
Keyword(s):  
Life Cycle ◽  
Protein Expression ◽  
Viral Protein ◽  
Inclusion Bodies ◽  
Ebola Virus ◽  
Rna Binding ◽  
Nuclear Rna ◽  
Rna Export ◽  
Nuclear Rna Export

Ebola virus (EBOV) causes severe outbreaks of viral hemorrhagic fever in humans. While virus-host interactions are promising targets for antivirals, there is only limited knowledge regarding the interactions of EBOV with cellular host factors. Recently, we performed a genome-wide siRNA screen that identified the nuclear RNA export factor 1 (NXF1) as an important host factor for the EBOV life cycle. NXF1 is a major component of the nuclear mRNA export pathway that is usurped by many viruses whose life cycles include nuclear stages. However, the role of NXF1 in the life cycle of EBOV, a virus replicating in cytoplasmic inclusion bodies, remains unknown. In order to better understand the role of NXF1 in the EBOV life cycle, we performed a combination of co-immunoprecipitation and double immunofluorescence assays to characterize the interactions of NXF1 with viral proteins and RNAs. Additionally, using siRNA-mediated knockdown of NXF1 together with functional assays, we analyzed the role of NXF1 in individual aspects of the virus life cycle. With this approach we identified the EBOV nucleoprotein (NP) as a viral interaction partner of NXF1. Further studies revealed that NP interacts with the RNA-binding domain of NXF1 and competes with RNA for this interaction. Co-localization studies showed that RNA binding-deficient, but not wildtype NXF1, accumulates in NP-derived inclusion bodies, and knockdown experiments demonstrated that NXF1 is necessary for viral protein expression, but not for viral RNA synthesis. Finally, our results showed that NXF1 interacts with viral mRNAs, but not with viral genomic RNAs. Based on these results we suggest a model whereby NXF1 is recruited into inclusion bodies to promote the export of viral mRNA:NXF1 complexes from these sites. This would represent a novel function for NXF1 in the life cycle of cytoplasmically replicating viruses, and may provide a basis for new therapeutic approaches against EBOV, and possibly other emerging viruses.

Nuclear RNA export: Possible role of an RNA unwindase activity

1990 ◽  
Vol 14 ◽  
pp. 128
Author(s):  
H SCHRODER ◽  
D UGARKOVIC ◽  
M BACHMANN ◽  
W MULLER
Keyword(s):  
Nuclear Rna ◽  
Rna Export ◽  
Nuclear Rna Export

scholarly journals Serine-Arginine Protein Kinase 1 Regulates Ebola Virus Transcription

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuki Takamatsu ◽  
Verena Krähling ◽  
Larissa Kolesnikova ◽  
Sandro Halwe ◽  
Clemens Lier ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Inclusion Bodies ◽  
Ebola Virus ◽  
Viral Transcription ◽  
Recognition Motif ◽  
Virus Transcription ◽  
Regulatory Circuit ◽  
Republic Of The Congo ◽  
Significant Growth Inhibition

ABSTRACT Ebola virus (EBOV) causes a severe and often fatal disease for which no approved vaccines or antivirals are currently available. EBOV VP30 has been described as a viral phosphoprotein, and nonphosphorylated VP30 is essential and sufficient to support secondary transcription in an EBOV-specific minigenome system; however, phosphorylatable serine residues near the N terminus of VP30 are required to support primary viral transcription as well as the reinitiation of VP30-mediated transcription at internal EBOV genes. While the dephosphorylation of VP30 by the cellular phosphatase PP2A was found to be mediated by nucleoprotein, the VP30-specific kinases and the role of phosphorylation remain unknown. Here, we report that serine-arginine protein kinase 1 (SRPK1) and SRPK2 phosphorylate serine 29 of VP30, which is located in an N-terminal R26xxS29 motif. Interaction with VP30 via the R26xxS29 motif recruits SRPK1 into EBOV-induced inclusion bodies, the sites of viral RNA synthesis, and an inhibitor of SRPK1/SRPK2 downregulates primary viral transcription. When the SRPK1 recognition motif of VP30 was mutated in a recombinant EBOV, virus replication was severely impaired. It is presumed that the interplay between SRPK1 and PP2A in the EBOV inclusions provides a comprehensive regulatory circuit to ensure the activity of VP30 in EBOV transcription. Thus, the identification of SRPK1 is an important mosaic stone that completes our picture of the players involved in Ebola virus transcription regulation. IMPORTANCE The largest Ebola virus (EBOV) epidemic in West Africa ever caused more than 28,000 cases and 11,000 deaths, and the current EBOV epidemic in the Democratic Republic of the Congo continues, with more than 3,000 cases to date. Therefore, it is essential to develop antivirals against EBOV. Recently, an inhibitor of the cellular phosphatase PP2A-mediated dephosphorylation of the EBOV transcription factor VP30 has been shown to suppress the spread of Ebola virus. Here, we identified the protein kinase SRPK1 as a VP30-specific kinase that phosphorylates serine 29, the same residue that is dephosphorylated by PP2A. SRPK1-mediated phosphorylation of serine 29 enabled primary viral transcription. Mutation of the SRPK1 recognition motif in VP30 resulted in significant growth inhibition of EBOV. Similarly, elevation of the phosphorylation status of serine 29 by overexpression of SRPK1 inhibited EBOV growth, highlighting the importance of reversible phosphorylation of VP30 as a potential therapeutic target.

scholarly journals Mutational Definition of Functional Domains within the Rev Homolog Encoded by Human Endogenous Retrovirus K

2000 ◽  
Vol 74 (20) ◽  
pp. 9353-9361 ◽  
Author(s):  
Hal P. Bogerd ◽  
Heather L. Wiegand ◽  
Jin Yang ◽  
Bryan R. Cullen
Keyword(s):  
Nuclear Export ◽  
Biological Activities ◽  
Endogenous Retrovirus ◽  
Viral Rna ◽  
Human Endogenous Retrovirus ◽  
Nuclear Rna ◽  
Rna Export ◽  
Nuclear Rna Export ◽  
Hiv 1 ◽  
Rev Protein

ABSTRACT Nuclear export of the incompletely spliced mRNAs encoded by several complex retroviruses, including human immunodeficiency virus type 1 (HIV-1), is dependent on a virally encoded adapter protein, termed Rev in HIV-1, that directly binds both to a cis-acting viral RNA target site and to the cellular Crm1 export factor. Human endogenous retrovirus K, a family of ancient endogenous retroviruses that is not related to the exogenous retrovirus HIV-1, was recently shown to also encode a Crm1-dependent nuclear RNA export factor, termed K-Rev. Although HIV-1 Rev and K-Rev display little sequence identity, they share the ability not only to bind to Crm1 and to RNA but also to form homomultimers and shuttle between nucleus and cytoplasm. We have used mutational analysis to identify sequences in the 105-amino-acid K-Rev protein required for each of these distinct biological activities. While mutations in K-Rev that inactivate any one of these properties also blocked K-Rev-dependent nuclear RNA export, several K-Rev mutants were comparable to wild type when assayed for any of these individual activities yet nevertheless defective for RNA export. Although several nonfunctional K-Rev mutants acted as dominant negative inhibitors of K-Rev-, but not HIV-1 Rev-, dependent RNA export, these were not defined by their inability to bind to Crm1, as is seen with HIV-1 Rev. In total, this analysis suggests a functional architecture for K-Rev that is similar to, but distinct from, that described for HIV-1 Rev and raises the possibility that viral RNA export mediated by the ∼25 million-year-old K-Rev protein may require an additional cellular cofactor that is not required for HIV-1 Rev function.

scholarly journals Pervasive Transcription of a Herpesvirus Genome Generates Functionally Important RNAs

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Susan P. Canny ◽  
Tiffany A. Reese ◽  
L. Steven Johnson ◽  
Xin Zhang ◽  
Amal Kambal ◽  
...  
Keyword(s):  
Life Cycle ◽  
Protein Expression ◽  
Viral Protein ◽  
Viral Life Cycle ◽  
Murine Cytomegalovirus ◽  
Functional Importance ◽  
Viral Protein Expression ◽  
Novel Transcripts ◽  
Herpesvirus Genome ◽  
Pervasive Transcription

ABSTRACTPervasive transcription is observed in a wide range of organisms, including humans, mice, and viruses, but the functional significance of the resulting transcripts remains uncertain. Current genetic approaches are often limited by their emphasis on protein-coding open reading frames (ORFs). We previously identified extensive pervasive transcription from the murine gammaherpesvirus 68 (MHV68) genome outside known ORFs and antisense to known genes (termed expressed genomic regions [EGRs]). Similar antisense transcripts have been identified in many other herpesviruses, including Kaposi’s sarcoma-associated herpesvirus and human and murine cytomegalovirus. Despite their prevalence, whether these RNAs have any functional importance in the viral life cycle is unknown, and one interpretation is that these are merely “noise” generated by functionally unimportant transcriptional events. To determine whether pervasive transcription of a herpesvirus genome generates RNA molecules that are functionally important, we used a strand-specific functional approach to target transcripts from thirteen EGRs in MHV68. We found that targeting transcripts from six EGRs reduced viral protein expression, proving that pervasive transcription can generate functionally important RNAs. We characterized transcripts emanating from EGRs 26 and 27 in detail using several methods, including RNA sequencing, and identified several novel polyadenylated transcripts that were enriched in the nuclei of infected cells. These data provide the first evidence of the functional importance of regions of pervasive transcription emanating from MHV68 EGRs. Therefore, studies utilizing mutation of a herpesvirus genome must account for possible effects on RNAs generated by pervasive transcription.IMPORTANCEThe fact that pervasive transcription produces functionally important RNAs has profound implications for design and interpretation of genetic studies in herpesviruses, since such studies often involve mutating both strands of the genome. This is a common potential problem; for example, a conservative estimate is that there are an additional 73,000 nucleotides transcribed antisense to annotated ORFs from the 119,450-bp MHV68 genome. Recognizing the importance of considering the function of each strand of the viral genome independently, we used strand-specific approaches to identify six regions of the genome encoding transcripts that promoted viral protein expression. For two of these regions, we mapped novel transcripts and determined that targeting transcripts from these regions reduced viral replication and the expression of other viral genes. This is the first description of a function for these RNAs and suggests that novel transcripts emanating from regions of pervasive transcription are critical for the viral life cycle.

scholarly journals The emerging role of RNA-binding proteins in the life cycle ofTrypanosoma brucei

2014 ◽  
Vol 16 (4) ◽  
pp. 482-489 ◽  
Author(s):  
Nikolay G. Kolev ◽  
Elisabetta Ullu ◽  
Christian Tschudi
Keyword(s):  
Life Cycle ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins

scholarly journals Ebola virus inclusion body formation and RNA synthesis are controlled by a novel domain of NP interacting with VP35

2020 ◽  
Author(s):  
Tsuyoshi Miyake ◽  
Charlotte M. Farley ◽  
Benjamin E. Neubauer ◽  
Thomas P. Beddow ◽  
Thomas Hoenen ◽  
...  
Keyword(s):  
Life Cycle ◽  
Inclusion Body ◽  
Inclusion Bodies ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Central Domain ◽  
Body Formation ◽  
Inclusion Body Formation

AbstractEbola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection IBs display dynamic properties regarding their size and location. Also, the contents of IBs must transition prior to further viral maturation, assembly and release, implying additional steps in IB function. Interestingly, expression of the viral nucleoprotein (NP) alone is sufficient for generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30 and the RNA polymerase L. Previously we defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here we show that NP-Ct is absolutely required for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for production of infectious virus-like particles and retention of viral RNA within these particles. Furthermore, co-expression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins only VP35 is able to overcome the defect in IB formation caused by deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself, and which is mediated by a newly identified domain of NP, the “central domain” (CD).ImportanceInclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative sense RNA viruses including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.

scholarly journals 68P Deciphering the interplay between nuclear RNA export factors and long non-coding RNAs in breast cancer metabolism

2020 ◽  
Vol 31 ◽  
pp. S38
Author(s):  
C. Klec ◽  
D. Schwarzenbacher ◽  
B. Gottschalk ◽  
R. Margit ◽  
F. Prinz ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Metabolism ◽  
Nuclear Rna ◽  
Rna Export ◽  
Non Coding Rnas ◽  
Nuclear Rna Export ◽  
Rna Export Factors
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