scholarly journals Differences in Viral RNA Synthesis but Not Budding or Entry Contribute to the In Vitro Attenuation of Reston Virus Compared to Ebola Virus

2020 ◽  
Vol 8 (8) ◽  
pp. 1215
Author(s):  
Bianca S. Bodmer ◽  
Josephin Greßler ◽  
Marie L. Schmidt ◽  
Julia Holzerland ◽  
Janine Brandt ◽  
...  
Keyword(s):  
Life Cycle ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Severe Disease ◽  
Case Fatality ◽  
Viral Rna ◽  
Pathogenic Potential ◽  
Rnp Complex ◽  
Reston Virus

Most filoviruses cause severe disease in humans. For example, Ebola virus (EBOV) is responsible for the two most extensive outbreaks of filovirus disease to date, with case fatality rates of 66% and 40%, respectively. In contrast, Reston virus (RESTV) is apparently apathogenic in humans, and while transmission of RESTV from domestic pigs to people results in seroconversion, no signs of disease have been reported in such cases. The determinants leading to these differences in pathogenicity are not well understood, but such information is needed in order to better evaluate the risks posed by the repeated spillover of RESTV into the human population and to perform risk assessments for newly emerging filoviruses with unknown pathogenic potential. Interestingly, RESTV and EBOV already show marked differences in their growth in vitro, with RESTV growing slower and reaching lower end titers. In order to understand the basis for this in vitro attenuation of RESTV, we used various life cycle modeling systems mimicking different aspects of the virus life cycle. Our results showed that viral RNA synthesis was markedly slower when using the ribonucleoprotein (RNP) components from RESTV, rather than those for EBOV. In contrast, the kinetics of budding and entry were indistinguishable between these two viruses. These data contribute to our understanding of the molecular basis for filovirus pathogenicity by showing that it is primarily differences in the robustness of RNA synthesis by the viral RNP complex that are responsible for the impaired growth of RESTV in tissue culture.

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 Ebola Virus Inclusion Body Formation and RNA Synthesis Are Controlled by a Novel Domain of Nucleoprotein Interacting with VP35

2020 ◽  
Vol 94 (16) ◽  
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

ABSTRACT Ebola 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. The contents of IBs also must transition prior to further viral maturation, assembly, and release, implying additional steps in IB function. Interestingly, the expression of the viral nucleoprotein (NP) alone is sufficient for the 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. We previously 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 necessary for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for the production of infectious virus-like particles (VLPs), and that defective VLPs with NP-Ct deletions are significantly reduced in viral RNA content. Furthermore, coexpression 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 the 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 that is mediated by a newly identified functional domain of NP, the central domain. IMPORTANCE Inclusion 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 The Integrity of the YxxL Motif of Ebola Virus VP24 Is Important for the Transport of Nucleocapsid-Like Structures and for the Regulation of Viral RNA Synthesis

2020 ◽  
Vol 94 (9) ◽  
Author(s):  
Yuki Takamatsu ◽  
Larissa Kolesnikova ◽  
Martin Schauflinger ◽  
Takeshi Noda ◽  
Stephan Becker
Keyword(s):  
Intracellular Transport ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Case Fatality ◽  
Viral Rna ◽  
Regulatory Function ◽  
Failure To Rescue ◽  
Independent Manner ◽  
Late Domains ◽  
Transcription And Replication

ABSTRACT While it is well appreciated that late domains in the viral matrix proteins are crucial to mediate efficient virus budding, little is known about roles of late domains in the viral nucleocapsid proteins. Here, we characterized the functional relevance of a YxxL motif with potential late-domain function in the Ebola virus nucleocapsid protein VP24. Mutations in the YxxL motif had two opposing effects on the functions of VP24. On the one hand, the mutation affected the regulatory function of VP24 in viral RNA transcription and replication, which correlated with an increased incorporation of minigenomes into released transcription- and replication-competent virus-like particles (trVLPs). Consequently, cells infected with those trVLPs showed higher levels of viral transcription. On the other hand, mutations of the YxxL motif greatly impaired the intracellular transport of nucleocapsid-like structures (NCLSs) composed of the viral proteins NP, VP35, and VP24 and the length of released trVLPs. Attempts to rescue recombinant Ebola virus expressing YxxL-deficient VP24 failed, underlining the importance of this motif for the viral life cycle. IMPORTANCE Ebola virus (EBOV) causes a severe fever with high case fatality rates and, so far, no available specific therapy. Understanding the interplay between viral and host proteins is important to identify new therapeutic approaches. VP24 is one of the essential nucleocapsid components and is necessary to regulate viral RNA synthesis and condense viral nucleocapsids before their transport to the plasma membrane. Our functional analyses of the YxxL motif in VP24 suggested that it serves as an interface between nucleocapsid-like structures (NCLSs) and cellular proteins, promoting intracellular transport of NCLSs in an Alix-independent manner. Moreover, the YxxL motif is necessary for the inhibitory function of VP24 in viral RNA synthesis. A failure to rescue EBOV encoding VP24 with a mutated YxxL motif indicated that the integrity of the YxxL motif is essential for EBOV growth. Thus, this motif might represent a potential target for antiviral interference.

scholarly journals Ebola Virus VP35 Interaction with Dynein LC8 Regulates Viral RNA Synthesis

2015 ◽  
Vol 89 (9) ◽  
pp. 5148-5153 ◽  
Author(s):  
Priya Luthra ◽  
David S. Jordan ◽  
Daisy W. Leung ◽  
Gaya K. Amarasinghe ◽  
Christopher F. Basler
Keyword(s):  
Rna Synthesis ◽  
Ebola Virus ◽  
Mutational Analysis ◽  
Cytoplasmic Dynein ◽  
Viral Rna ◽  
Interferon Production ◽  
Dynein Light Chain ◽  
High Affinity ◽  
Functional Consequences ◽  
Oligomerization Domain

Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.

Studies of two temperature-sensitive mutants of Mengo virus

1979 ◽  
Vol 57 (6) ◽  
pp. 902-913 ◽  
Author(s):  
Patrick W. K. Lee ◽  
John S. Colter
Keyword(s):  
Rna Synthesis ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Supporting Evidence ◽  
Structural Polypeptide ◽  
Infected Cells ◽  
Mengo Virus ◽  
In Vitro Stability

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA−ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 °C) to the nonpermissive (39 °C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA− phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 °C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant.Subviral (53 S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 °C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.

scholarly journals Spatial Perturbations within an RNA Promoter Specifically Recognized by a Viral RNA-Dependent RNA Polymerase (RdRp) Reveal That RdRp Can Adjust Its Promoter Binding Sites

1999 ◽  
Vol 73 (1) ◽  
pp. 198-204 ◽  
Author(s):  
Scott Stevenson Stawicki ◽  
C. Cheng Kao
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Dna And Rna ◽  
In The Wild ◽  
Nucleotide Insertions

ABSTRACT RNA synthesis during viral replication requires specific recognition of RNA promoters by the viral RNA-dependent RNA polymerase (RdRp). Four nucleotides (−17, −14, −13, and −11) within the brome mosaic virus (BMV) subgenomic core promoter are required for RNA synthesis by the BMV RdRp (R. W. Siegel et al., Proc. Natl. Acad. Sci. USA 94:11238–11243, 1997). The spatial requirements for these four nucleotides and the initiation (+1) cytidylate were examined in RNAs containing nucleotide insertions and deletions within the BMV subgenomic core promoter. Spatial perturbations between nucleotides −17 and −11 resulted in decreased RNA synthesis in vitro. However, synthesis was still dependent on the key nucleotides identified in the wild-type core promoter and the initiation cytidylate. In contrast, changes between nucleotides −11 and +1 had a less severe effect on RNA synthesis but resulted in RNA products initiated at alternative locations in addition to the +1 cytidylate. The results suggest a degree of flexibility in the recognition of the subgenomic promoter by the BMV RdRp and are compared with functional regions in other DNA and RNA promoters.

Brome mosaic virus coat protein inhibits viral RNA synthesis in vitro

Virology ◽  
1987 ◽  
Vol 158 (1) ◽  
pp. 15-19 ◽  
Author(s):  
Mamoru Horikoshi ◽  
Masaharu Nakayama ◽  
Naoto Yamaoka ◽  
Iwao Furusawa ◽  
Jiko Shishiyama
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Rna Synthesis ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Virus Coat Protein ◽  
Virus Coat

scholarly journals NUCLEOLAR AND NUCLEAR RNA SYNTHESIS DURING THE CELL LIFE CYCLE IN MONKEY AND PIG KIDNEY CELLS IN VITRO

1967 ◽  
Vol 33 (2) ◽  
pp. 273-279 ◽  
Author(s):  
Jane L. Showacre ◽  
W. G. Cooper ◽  
D. M. Prescott
Keyword(s):  
Life Cycle ◽  
Rna Synthesis ◽  
Kidney Cells ◽  
Pig Kidney ◽  
Rna Labeling ◽  
Cell Life ◽  
Nuclear Rna ◽  
Nucleolar Rna ◽  
Pig Kidney Cells

The incorporation of 5-3H-uridine and 5-3H-cytidine into nucleolar and nonnucleolar RNA in the nucleus of monkey and pig kidney cells was measured in vitro during the cell life cycle. Time-lapse cinematographic records were made of cells during asynchronous exponential proliferation, in order to identify the temporal position of individual cells in relation to the preceding mitosis. Immediately following cinematography, cells were labeled with uridine-3H and cytidine-3H for a short period, fixed, and analyzed by radioautography. Since the data permit correlation of the rate of RNA labeling with the position of a cell within the cycle, curves could be constructed describing the rate of RNA synthesis over the average cell cycle. RNA synthesis was absent in early telophase, and rose very abruptly in rate in late telophase and in very early G1 in both the nucleus and the reconstituting nucleolus. Thereafter, through the G1 and S periods the rate of nuclear RNA synthesis rose gradually. When we used a 10-min pulse, there was no detectable change in the rate for nucleolar RNA labeling in monkey kidney cells during G1 or S. When we used a 30-min labeling time, the rate of nucleolar RNA labeling rose gradually in pig kidney cells. With increasing time after mitosis, the data became more variable, which may, in part, be related to the variation in generation times for individual cells.

scholarly journals SYNCRIP, a Member of the Heterogeneous Nuclear Ribonucleoprotein Family, Is Involved in Mouse Hepatitis Virus RNA Synthesis

2004 ◽  
Vol 78 (23) ◽  
pp. 13153-13162 ◽  
Author(s):  
Keum S. Choi ◽  
Akihiro Mizutani ◽  
Michael M. C. Lai
Keyword(s):  
Mammalian Cells ◽  
Rna Synthesis ◽  
Rna Binding ◽  
Hepatitis Virus ◽  
Affinity Purification ◽  
Mouse Hepatitis Virus ◽  
Viral Rna ◽  
Negative Strand

ABSTRACT Several cellular proteins, including several heterogeneous nuclear ribonucleoproteins (hnRNPs), have been shown to function as regulatory factors for mouse hepatitis virus (MHV) RNA synthesis as a result of their binding to the 5′ and 3′ untranslated regions (UTRs) of the viral RNA. Here, we identified another cellular protein, p70, which has been shown by UV cross-linking to bind both the positive- and negative-strand UTRs of MHV RNA specifically. We purified p70 with a a one-step RNA affinity purification procedure with the biotin-labeled 5′-UTR. Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry identified it as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a member of the hnRNP family and localizes largely in the cytoplasm. The p70 was cross-linked to the MHV positive- or negative-strand UTR in vitro and in vivo. The bacterially expressed SYNCRIP was also able to bind to the 5′-UTR of both strands. The SYNCRIP-binding site was mapped to the leader sequence of the 5′-UTR, requiring the UCUAA repeat sequence. To investigate the functional significance of SYNCRIP in MHV replication, we expressed a full-length or a C-terminally truncated form of SYNCRIP in mammalian cells expressing the MHV receptor. The overexpression of either form of SYNCRIP inhibited syncytium formation induced by MHV infection. Furthermore, downregulation of the endogenous SYNCRIP with a specific short interfering RNA delayed MHV RNA synthesis; in contrast, overexpression or downregulation of SYNCRIP did not affect MHV translation. These results suggest that SYNCRIP may be directly involved in MHV RNA replication as a positive regulator. This study identified an additional cellular hnRNP as an MHV RNA-binding protein potentially involved in viral RNA synthesis.

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