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.
Subject(s)
Ebolavirus/metabolism , Inclusion Bodies, Viral/metabolism , Nucleoproteins/physiology , Cell Line , Ebolavirus/pathogenicity , Genome, Viral/genetics , HEK293 Cells , Hemorrhagic Fever, Ebola/virology , Humans , Inclusion Bodies/metabolism , Nucleocapsid/metabolism , Nucleocapsid/physiology , Nucleocapsid Proteins/metabolism , Nucleocapsid Proteins/physiology , Nucleoproteins/metabolism , RNA/biosynthesis , RNA, Viral/genetics , Transcription Factors/metabolism , Viral Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Viral Regulatory and Accessory Proteins/physiology , Virion/metabolism , Virus Replication/physiologyABSTRACT
A large subfamily of the type IV secretion systems (T4SSs), termed the conjugation systems, transmit mobile genetic elements (MGEs) among many bacterial species. In the initiating steps of conjugative transfer, DNA transfer and replication (Dtr) proteins assemble at the origin-of-transfer (oriT) sequence as the relaxosome, which nicks the DNA strand destined for transfer and couples the nicked substrate with the VirD4-like substrate receptor. Here, we defined contributions of the Dtr protein TraK, a predicted member of the Ribbon-Helix-Helix (RHH) family of DNA-binding proteins, to transfer of DNA and protein substrates through the pKM101-encoded T4SS. Using a combination of cross-linking/affinity pull-downs and two-hybrid assays, we determined that TraK self-associates as a probable tetramer and also forms heteromeric contacts with pKM101-encoded TraI relaxase, VirD4-like TraJ receptor, and VirB11-like and VirB4-like ATPases, TraG and TraB, respectively. TraK also promotes stable TraJ-TraB complex formation and stimulates binding of TraI with TraB. Finally, TraK is required for or strongly stimulates the transfer of cognate (pKM101, TraI relaxase) and noncognate (RSF1010, MobA relaxase) substrates. We propose that TraK functions not only to nucleate pKM101 relaxosome assembly, but also to activate the TrapKM101 T4SS via interactions with the ATPase energy center positioned at the channel entrance.
Subject(s)
DNA-Binding Proteins/metabolism , Escherichia coli Proteins/metabolism , Nucleoproteins/metabolism , Periplasmic Proteins/metabolism , Type IV Secretion Systems/metabolism , Adenosine Triphosphatases/metabolism , Bacterial Outer Membrane Proteins/metabolism , Bacterial Outer Membrane Proteins/physiology , Bacterial Proteins/metabolism , Conjugation, Genetic/genetics , DNA, Bacterial/metabolism , DNA-Binding Proteins/physiology , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/physiology , Membrane Proteins/metabolism , Nucleoproteins/physiology , Periplasmic Proteins/physiology , Plasmids/geneticsABSTRACT
Ebola virus (EBOV) is a non-segmented, negative-sense RNA virus (NNSV) in the family Filoviridae, and is recognized as one of the most lethal pathogens in the planet. For RNA viruses, cellular or virus-encoded RNA helicases play pivotal roles in viral life cycles by remodelling viral RNA structures and/or unwinding viral dsRNA produced during replication. However, no helicase or helicase-like activity has ever been found to associate with any NNSV-encoded proteins, and it is unknown whether the replication of NNSVs requires the participation of any viral or cellular helicase. Here, we show that despite of containing no conserved NTPase/helicase motifs, EBOV VP35 possesses the NTPase and helicase-like activities that can hydrolyse all types of NTPs and unwind RNA helices in an NTP-dependent manner, respectively. Moreover, guanidine hydrochloride, an FDA-approved compound and inhibitor of certain viral helicases, inhibited the NTPase and helicase-like activities of VP35 as well as the replication/transcription of an EBOV minigenome replicon in cells, highlighting the importance of VP35 helicase-like activity during EBOV life cycle. Together, our findings provide the first demonstration of the NTPase/helicase-like activity encoded by EBOV, and would foster our understanding of EBOV and NNSVs.
Subject(s)
Ebolavirus/genetics , Ebolavirus/metabolism , Hemorrhagic Fever, Ebola/virology , Nucleoproteins/physiology , RNA, Double-Stranded/chemistry , Viral Core Proteins/physiology , Adenosine Triphosphate/chemistry , Amino Acid Motifs , Cells, Cultured , DNA Helicases/metabolism , Humans , Nucleocapsid Proteins , Nucleoproteins/genetics , Nucleoproteins/metabolism , Nucleoside-Triphosphatase/genetics , Protein Binding , RNA Helicases/metabolism , RNA, Viral/metabolism , Viral Core Proteins/genetics , Viral Core Proteins/metabolism , Viral Nonstructural Proteins/metabolism , Virus ReplicationABSTRACT
The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, and the nucleoprotein (NP) interact with the genomic RNA of influenza viruses and form ribonucleoproteins. Especially, the PB2 subunit binds to the host RNA cap [7-methylguanosine triphosphate (m7GTP)] and supports the endonuclease activity of PA to "snatch" the cap from host pre-mRNAs. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is necessary for interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs). In vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m7GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m7GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino acid mutations in the VRG site. Substitutions of 2 or 3 amino acid residues of the VRG site to alanine significantly reduced PB2's binding ability to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication. I will also discuss some novel functions of NP in this review.
Subject(s)
Antiviral Agents , Drug Discovery , Influenza A virus/enzymology , Influenza A virus/genetics , Influenza, Human/drug therapy , RNA, Viral , RNA-Dependent RNA Polymerase/chemistry , Amino Acid Sequence/genetics , Humans , Influenza A virus/physiology , Mutation , Nucleoproteins/chemistry , Nucleoproteins/physiology , RNA-Dependent RNA Polymerase/physiology , Ribonucleoproteins/chemistry , Virus ReplicationABSTRACT
UNLABELLED: Dendritic cells (DCs) are major targets of filovirus infection in vivo Previous studies have shown that the filoviruses Ebola virus (EBOV) and Marburg virus (MARV) suppress DC maturation in vitro Both viruses also encode innate immune evasion functions. The EBOV VP35 (eVP35) and the MARV VP35 (mVP35) proteins each can block RIG-I-like receptor signaling and alpha/beta interferon (IFN-α/ß) production. The EBOV VP24 (eVP24) and MARV VP40 (mVP40) proteins each inhibit the production of IFN-stimulated genes (ISGs) by blocking Jak-STAT signaling; however, this occurs by different mechanisms, with eVP24 blocking nuclear import of tyrosine-phosphorylated STAT1 and mVP40 blocking Jak1 function. MARV VP24 (mVP24) has been demonstrated to modulate host cell antioxidant responses. Previous studies demonstrated that eVP35 is sufficient to strongly impair primary human monocyte-derived DC (MDDC) responses upon stimulation induced through the RIG-I-like receptor pathways. We demonstrate that mVP35, like eVP35, suppresses not only IFN-α/ß production but also proinflammatory responses after stimulation of MDDCs with RIG-I activators. In contrast, eVP24 and mVP40, despite suppressing ISG production upon RIG-I activation, failed to block upregulation of maturation markers or T cell activation. mVP24, although able to stimulate expression of antioxidant response genes, had no measurable impact of DC function. These data are consistent with a model where filoviral VP35 proteins are the major suppressors of DC maturation during filovirus infection, whereas the filoviral VP24 proteins and mVP40 are insufficient to prevent DC maturation. IMPORTANCE: The ability to suppress the function of dendritic cells (DCs) likely contributes to the pathogenesis of disease caused by the filoviruses Ebola virus and Marburg virus. To clarify the basis for this DC suppression, we assessed the effect of filovirus proteins known to antagonize innate immune signaling pathways, including Ebola virus VP35 and VP24 and Marburg virus VP35, VP40, and VP24, on DC maturation and function. The data demonstrate that the VP35s from Ebola virus and Marburg virus are the major suppressors of DC maturation and that the effects on DCs of the remaining innate immune inhibitors are minor.
Subject(s)
Dendritic Cells/physiology , Dendritic Cells/virology , Ebolavirus/chemistry , Marburgvirus/chemistry , RNA Viruses/physiology , Viral Proteins/physiology , Viral Regulatory and Accessory Proteins/physiology , Cell Differentiation , Encephalomyocarditis virus/physiology , Host-Pathogen Interactions , Humans , Interferon-alpha/genetics , Interferon-alpha/immunology , Interferon-beta/genetics , Interferon-beta/immunology , Interferon-gamma/metabolism , Nucleocapsid Proteins , Nucleoproteins/genetics , Nucleoproteins/physiology , Sendai virus/physiology , Transduction, Genetic , Viral Core Proteins/genetics , Viral Core Proteins/physiology , Viral Proteins/geneticsABSTRACT
The influenza A virus (IAV) genome is divided into eight distinct RNA segments believed to be copackaged into virions with nearly perfect efficiency. Here, we describe a mutation in IAV nucleoprotein (NP) that enhances replication and transmission in guinea pigs while selectively reducing neuraminidase (NA) gene segment packaging into virions. We show that incomplete IAV particles lacking gene segments contribute to the propagation of the viral population through multiplicity reactivation under conditions of widespread coinfection, which we demonstrate commonly occurs in the upper respiratory tract of guinea pigs. NP also dramatically altered the functional balance of the viral glycoproteins on particles by selectively decreasing NA expression. Our findings reveal novel functions for NP in selective control of IAV gene packaging and balancing glycoprotein expression and suggest a role for incomplete gene packaging during host adaptation and transmission.
Subject(s)
Influenza A virus/physiology , Influenza, Human/transmission , Neuraminidase/genetics , Nucleoproteins/physiology , Viral Proteins/physiology , Genes, Viral , Humans , Influenza A virus/genetics , Influenza, Human/virology , Virus Assembly , Virus ReplicationABSTRACT
Integration host factor (IHF) is an Escherichia coli protein involved in (i) condensation of the bacterial nucleoid and (ii) regulation of a variety of cellular functions. In its regulatory role, IHF binds to a specific sequence to introduce a strong bend into the DNA; this provides a duplex architecture conducive to the assembly of site-specific nucleoprotein complexes. Alternatively, the protein can bind in a sequence-independent manner that weakly bends and wraps the duplex to promote nucleoid formation. IHF is also required for the development of several viruses, including bacteriophage lambda, where it promotes site-specific assembly of a genome packaging motor required for lytic development. Multiple IHF consensus sequences have been identified within the packaging initiation site (cos), and we here interrogate IHF-cos binding interactions using complementary electrophoretic mobility shift (EMS) and analytical ultracentrifugation (AUC) approaches. IHF recognizes a single consensus sequence within cos (I1) to afford a strongly bent nucleoprotein complex. In contrast, IHF binds weakly but with positive cooperativity to nonspecific DNA to afford an ensemble of complexes with increasing masses and levels of condensation. Global analysis of the EMS and AUC data provides constrained thermodynamic binding constants and nearest neighbor cooperativity factors for binding of IHF to I1 and to nonspecific DNA substrates. At elevated IHF concentrations, the nucleoprotein complexes undergo a transition from a condensed to an extended rodlike conformation; specific binding of IHF to I1 imparts a significant energy barrier to the transition. The results provide insight into how IHF can assemble specific regulatory complexes in the background of extensive nonspecific DNA condensation.
Subject(s)
Bacteriophage lambda/genetics , Bacteriophage lambda/physiology , DNA Packaging/physiology , Integration Host Factors/physiology , Virus Assembly/physiology , DNA, Viral/chemistry , DNA, Viral/physiology , Electrophoretic Mobility Shift Assay , Escherichia coli/genetics , Escherichia coli/virology , Gene Expression Regulation, Bacterial , Genes, Bacterial , Genome, Viral , Integration Host Factors/chemistry , Models, Molecular , Nucleic Acid Conformation , Nucleoproteins/chemistry , Nucleoproteins/physiology , Protein Conformation , ThermodynamicsABSTRACT
Filoviruses are the causative agents of a severe and often fatal hemorrhagic fever with repeated outbreaks in Africa. They are negative sense single stranded enveloped viruses that can cross species barriers from its natural host bats to primates including humans. The small size of the genome poses limits to viral adaption, which may be partially overcome by conformational plasticity. Here we review the different conformational states of the Ebola virus (EBOV) matrix protein VP40 that range from monomers, to dimers, hexamers, and RNA-bound octamers. This conformational plasticity that is required for the viral life cycle poses a unique opportunity for development of VP40 specific drugs. Furthermore, we compare the structure to homologous matrix protein structures from Paramyxoviruses and Bornaviruses and we predict that they do not only share the fold but also the conformational flexibility of EBOV VP40.
Subject(s)
Nucleoproteins , Viral Core Proteins , Ebolavirus/metabolism , Ebolavirus/physiology , Models, Molecular , Nucleoproteins/chemistry , Nucleoproteins/metabolism , Nucleoproteins/physiology , Protein Conformation , Viral Core Proteins/chemistry , Viral Core Proteins/metabolism , Viral Core Proteins/physiology , Virus Assembly , Virus ReleaseABSTRACT
Typical avian influenza A viruses are restricted from replicating efficiently and causing disease in humans. However, an avian virus can become adapted to humans by mutating or recombining with currently circulating human viruses. These viruses have the potential to cause pandemics in an immunologically naïve human population. It is critical that we understand the molecular basis of host-range restriction and how this can be overcome. Here, we review our current understanding of the mechanisms by which influenza viruses adapt to replicate efficiently in a new host. We predominantly focus on the influenza polymerase, which remains one of the least understood host-range barriers.
Subject(s)
Influenza A virus/pathogenicity , Adaptation, Physiological , Animals , Birds , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/physiology , Genes, Viral , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/physiology , Host Specificity/genetics , Host Specificity/immunology , Host Specificity/physiology , Humans , Immunity, Innate , Influenza A virus/genetics , Influenza A virus/physiology , Influenza in Birds/immunology , Influenza in Birds/virology , Influenza, Human/immunology , Influenza, Human/virology , Mutation , Neuraminidase/genetics , Neuraminidase/physiology , Nucleoproteins/genetics , Nucleoproteins/physiology , Viral Proteins/genetics , Viral Proteins/physiologyABSTRACT
Recent biophysical approaches have provided key insights into the enthalpic and entropic forces that compact the nucleoid in the cell. Our biophysical approach combines two complementary, non-invasive and label-free techniques: a precisely timed steerable optical trap and a high throughput microcapillary Coulter counter. We demonstrate the ability of the latter technique to probe the physical properties and size of many purified nucleoids, at the individual nucleoid level. The DNA-binding protein H-NS is central to the organization of the bacterial genome. Our results show that nucleoids purified from the Δhns strain in the stationary phase expand approximately five fold more than the form observed in WT bacteria. This compaction is consistent with the role played by H-NS in regulating the nucleoid structure and the significant organizational changes that occur as the cell adapts to the stationary phase. We also study the permeability to the flow of ions and find that in the experiment nucleoids behave as solid colloids.
Subject(s)
Bacterial Proteins/physiology , DNA-Binding Proteins/physiology , Escherichia coli/physiology , Genome, Bacterial/physiology , Nucleoproteins/physiology , Microfluidics , Optical TweezersABSTRACT
Filoviruses are filamentous lipid-enveloped viruses and include Ebola (EBOV) and Marburg, which are morphologically identical but antigenically distinct. These viruses can be very deadly with outbreaks of EBOV having clinical fatality as high as 90%. In 2012 there were two separate Ebola outbreaks in the Democratic Republic of Congo and Uganda that resulted in 25 and 4 fatalities, respectively. The lack of preventive vaccines and FDA-approved therapeutics has struck fear that the EBOV could become a pandemic threat. The Ebola genome encodes only seven genes, which mediate the entry, replication, and egress of the virus from the host cell. The EBOV matrix protein is VP40, which is found localized under the lipid envelope of the virus where it bridges the viral lipid envelope and nucleocapsid. VP40 is effectively a peripheral protein that mediates the plasma membrane binding and budding of the virus prior to egress. A number of studies have demonstrated specific deletions or mutations of VP40 to abrogate viral egress but to date pharmacological inhibition of VP40 has not been demonstrated. This editorial highlights VP40, which is the most abundantly expressed protein of the virus and discusses VP40 as a potential therapeutic target.
Subject(s)
Nucleoproteins/physiology , Viral Core Proteins/physiology , Animals , Hemorrhagic Fever, Ebola/drug therapy , Humans , Nucleoproteins/chemistry , Viral Core Proteins/chemistryABSTRACT
Ebola, a fatal virus in humans and non-human primates, has no Food and Drug Administration-approved vaccines or therapeutics. The virus from the Filoviridae family causes hemorrhagic fever, which rapidly progresses and in some cases has a fatality rate near 90%. The Ebola genome encodes seven genes, the most abundantly expressed of which is viral protein 40 (VP40), the major Ebola matrix protein that regulates assembly and egress of the virus. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of plasma membrane association by VP40 are not well understood. In this study, we used an array of biophysical experiments and cellular assays along with mutagenesis of VP40 to investigate the role of membrane penetration in VP40 assembly and egress. Here we demonstrate that VP40 is able to penetrate specifically into the plasma membrane through an interface enriched in hydrophobic residues in its C-terminal domain. Mutagenesis of this hydrophobic region consisting of Leu(213), Ile(293), Leu(295), and Val(298) demonstrated that membrane penetration is critical to plasma membrane localization, VP40 oligomerization, and viral particle egress. Taken together, VP40 membrane penetration is an important step in the plasma membrane localization of the matrix protein where oligomerization and budding are defective in the absence of key hydrophobic interactions with the membrane.
Subject(s)
Ebolavirus/metabolism , Gene Expression Regulation, Viral , Nucleoproteins/physiology , Viral Core Proteins/physiology , Viral Matrix Proteins/physiology , Animals , Biophysics/methods , CHO Cells , Cell Membrane/enzymology , Cell Membrane/metabolism , Cricetinae , DNA/genetics , Fatty Acid-Binding Proteins/chemistry , HEK293 Cells , Humans , Hydrophobic and Hydrophilic Interactions , Lipids/chemistry , Models, Molecular , Molecular Conformation , Mutagenesis , Nucleoproteins/chemistry , Protein Structure, Tertiary , Viral Core Proteins/chemistry , Viral Matrix Proteins/metabolismABSTRACT
Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.
Subject(s)
Mitochondria/genetics , Mitochondrial Proteins/biosynthesis , Nucleoproteins/physiology , Protein Biosynthesis , ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/physiology , Cell Line, Tumor , DNA, Mitochondrial/metabolism , HEK293 Cells , Humans , Membrane Proteins/metabolism , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/physiology , Nuclear Proteins/physiology , Prohibitins , RNA/analysis , RNA/isolation & purification , RNA, Messenger/analysis , RNA, Mitochondrial , Repressor Proteins/physiology , Ribosomes/metabolismABSTRACT
BACKGROUND: Double-stranded RNA dependent protein kinase (PKR) is a key regulator of the anti-viral innate immune response in mammalian cells. PKR activity is regulated by a 58 kilo Dalton cellular inhibitor (P58(IPK)), which is present in inactive state as a complex with Hsp40 under normal conditions. In case of influenza A virus (IAV) infection, P58(IPK) is known to dissociate from Hsp40 and inhibit PKR activation. However the influenza virus component responsible for PKR inhibition through P58(IPK) activation was hitherto unknown. PRINCIPAL FINDINGS: Human heat shock 40 protein (Hsp40) was identified as an interacting partner of Influenza A virus nucleoprotein (IAV NP) using a yeast two-hybrid screen. This interaction was confirmed by co-immunoprecipitation studies from mammalian cells transfected with IAV NP expressing plasmid. Further, the IAV NP-Hsp40 interaction was validated in mammalian cells infected with various seasonal and pandemic strains of influenza viruses. Cellular localization studies showed that NP and Hsp40 co-localize primarily in the nucleus. During IAV infection in mammalian cells, expression of NP coincided with the dissociation of P58(IPK) from Hsp40 and decrease PKR phosphorylation. We observed that, plasmid based expression of NP in mammalian cells leads to decrease in PKR phosphorylation. Furthermore, inhibition of NP expression during influenza virus replication led to PKR activation and concomitant increase in eIF2α phosphorylation. Inhibition of NP expression also led to reduced IRF3 phosphorylation, enhanced IFN ß production and concomitant reduction of virus replication. Taken together our data suggest that NP is the viral factor responsible for P58(IPK) activation and subsequent inhibition of PKR-mediated host response during IAV infection. SIGNIFICANCE: Our findings demonstrate a novel role of IAV NP in inhibiting PKR-mediated anti-viral host response and help us understand P58(IPK) mediated inhibition of PKR activity during IAV infection.
Subject(s)
HSP40 Heat-Shock Proteins/metabolism , Influenza A Virus, H5N1 Subtype/metabolism , Nucleoproteins/physiology , eIF-2 Kinase/metabolism , Base Sequence , Cell Line , Cell Nucleus/metabolism , Enzyme Activation , Humans , Influenza A Virus, H5N1 Subtype/physiology , Phosphorylation , RNA, Small Interfering , Virus ReplicationABSTRACT
BACKGROUND: Measles virus nucleoprotein (N) encapsidates the viral RNA, protects it from endonucleases and forms a virus specific template for transcription and replication. It is the most abundant protein during viral infection. Its C-terminal domain is intrinsically disordered imparting it the flexibility to interact with several cellular and viral partners. PRINCIPAL FINDINGS: In this study, we demonstrate that expression of N within mammalian cells resulted in morphological transitions, nuclear condensation, DNA fragmentation and activation of Caspase 3 eventuating into apoptosis. The rapid generation of intracellular reactive oxygen species (ROS) was involved in the mechanism of cell death. Addition of ascorbic acid (AA) or inhibitor of caspase-3 in the extracellular medium partially reversed N induced apoptosis. We also studied the protein profile of cells expressing N protein. MS analysis revealed the differential expression of 25 proteins out of which 11 proteins were up regulated while 14 show signs of down regulation upon N expression. 2DE results were validated by real time and semi quantitative RT-PCR analysis. CONCLUSION: These results show the pro-apoptotic effects of N indicating its possible development as an apoptogenic tool. Our 2DE results present prima facie evidence that the MV nucleoprotein interacts with or causes differential expression of a wide range of cellular factors. At this stage it is not clear as to what the adaptive response of the host cell is and what reflects a strategic modulation exerted by the virus.
Subject(s)
Apoptosis/physiology , Measles virus/metabolism , Nucleoproteins/metabolism , Viral Proteins/metabolism , Caspase 3/metabolism , Cell Line, Tumor , Electrophoresis, Gel, Two-Dimensional , Enzyme Activation , Humans , Mass Spectrometry , Nucleoproteins/physiology , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Viral Proteins/physiologyABSTRACT
The ebolavirus (EBOV) VP35 protein binds to double-stranded RNA (dsRNA), inhibits host alpha/beta interferon (IFN-α/ß) production, and is an essential component of the viral polymerase complex. Structural studies of the VP35 C-terminal IFN inhibitory domain (IID) identified specific structural features, including a central basic patch and a hydrophobic pocket, that are important for dsRNA binding and IFN inhibition. Several other conserved basic residues bordering the central basic patch and a separate cluster of basic residues, called the first basic patch, were also identified. Functional analysis of alanine substitution mutants indicates that basic residues outside the central basic patch are not required for dsRNA binding or for IFN inhibition. However, minigenome assays, which assess viral RNA polymerase complex function, identified these other basic residues to be critical for viral RNA synthesis. Of these, a subset located within the first basic patch is important for VP35-nucleoprotein (NP) interaction, as evidenced by the inability of alanine substitution mutants to coimmunoprecipitate with NP. Therefore, first basic patch residues are likely critical for replication complex formation through interactions with NP. Coimmunoprecipitation studies further demonstrate that the VP35 IID is sufficient to interact with NP and that dsRNA can modulate VP35 IID interactions with NP. Other basic residue mutations that disrupt the VP35 polymerase cofactor function do not affect interaction with NP or with the amino terminus of the viral polymerase. Collectively, these results highlight the importance of conserved basic residues from the EBOV VP35 C-terminal IID and validate the VP35 IID as a potential therapeutic target.
Subject(s)
Ebolavirus/physiology , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/physiology , Amino Acid Substitution , Animals , Cell Line , Chlorocebus aethiops , Ebolavirus/genetics , Ebolavirus/pathogenicity , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/physiology , Humans , Models, Molecular , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Nucleocapsid Proteins , Nucleoproteins/chemistry , Nucleoproteins/genetics , Nucleoproteins/physiology , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , RNA/genetics , RNA/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Static Electricity , Vero Cells , Viral Core Proteins/chemistry , Viral Core Proteins/genetics , Viral Core Proteins/physiology , Viral Regulatory and Accessory Proteins/genetics , Virulence/genetics , Virulence/physiologyABSTRACT
Virulent strains of Newcastle disease virus ([NDV] also known as avian paramyxovirus type 1) can be discriminated from low-virulence strains by the presence of multiple basic amino acid residues at the proteolytic cleavage site of the fusion (F) protein. However, some NDV variants isolated from pigeons (pigeon paramyxovirus type 1 [PPMV-1]) have low levels of virulence, despite the fact that their F protein cleavage sites contain a multibasic amino acid sequence and have the same functionality as that of virulent strains. To determine the molecular basis of this discrepancy, we examined the role of the internal proteins in NDV virulence. Using reverse genetics, the genes encoding the nucleoprotein (NP), phosphoprotein (P), matrix protein (M), and large polymerase protein (L) were exchanged between the nonvirulent PPMV-1 strain AV324 and the highly virulent NDV strain Herts. Recombinant viruses were evaluated for their pathogenicities and replication levels in day-old chickens, and viral genome replication and plaque sizes were examined in cell culture monolayers. We also tested the contributions of the individual NP, P, and L proteins to the activity of the viral replication complex in an in vitro replication assay. The results showed that the replication proteins of Herts are more active than those of AV324 and that the activity of the viral replication complex is directly related to virulence. Although the M protein affected viral replication in vitro, it had only a minor effect on virulence.
Subject(s)
Newcastle disease virus/genetics , Newcastle disease virus/pathogenicity , Virus Replication/genetics , Animals , Base Sequence , Cell Line , Chickens , Columbidae , DNA, Viral/genetics , Genome, Viral , In Vitro Techniques , Newcastle Disease/virology , Newcastle disease virus/isolation & purification , Newcastle disease virus/physiology , Nucleocapsid Proteins , Nucleoproteins/genetics , Nucleoproteins/physiology , Phosphoproteins/genetics , Phosphoproteins/physiology , Quail , Recombination, Genetic , Viral Matrix Proteins/genetics , Viral Matrix Proteins/physiology , Viral Proteins/genetics , Viral Proteins/physiology , Virulence/genetics , Virulence/physiology , Virus Replication/physiologyABSTRACT
The phosphoprotein P of non-segmented negative-sense RNA viruses is an essential component of the replication and transcription complex and acts as a co-factor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. We have obtained the structure of the C-terminal domain of P of Mokola virus (MOKV), a lyssavirus that belongs to the Rhabdoviridae family and mapped at the amino acid level the crucial positions involved in interaction with N and in the formation of the viral replication complex. Comparison of the N-RNA binding domains of P solved to date suggests that the N-RNA binding domains are structurally conserved among paramyxoviruses and rhabdoviruses in spite of low sequence conservation. We also review the numerous other functions of this domain and more generally of the phosphoprotein.
Subject(s)
Lyssavirus/genetics , Nucleoproteins/chemistry , Nucleoproteins/metabolism , Paramyxoviridae/genetics , RNA/metabolism , Rhabdoviridae/genetics , Humans , Lyssavirus/metabolism , Models, Biological , Models, Molecular , Nucleoproteins/genetics , Nucleoproteins/physiology , Paramyxoviridae/metabolism , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphoproteins/physiology , Protein Structure, Tertiary , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Rhabdoviridae/metabolism , Structure-Activity RelationshipABSTRACT
Ebola virus (EBOV) causes severe hemorrhagic fevers in humans and non-human primates. While the role of the EBOV major matrix protein VP40 in morphogenesis is well understood, nothing is known about its contributions to the regulation of viral genome replication and/or transcription. Similarly, while it was reported that the minor matrix protein VP24 impairs viral genome replication, it remains unclear whether it also regulates transcription, since all common experimental systems measure the combined products of replication and transcription. We have developed systems that allow the independent monitoring of viral transcription and replication, based on qRT-PCR and a replication-deficient minigenome. Using these systems we show that VP24 regulates not only viral genome replication, but also transcription. Further, we show for the first time that VP40 is also involved in regulating these processes. These functions are conserved among EBOV species and, in the case of VP40, independent of its budding or RNA-binding functions.
Subject(s)
Ebolavirus/physiology , Gene Expression Regulation, Viral , Nucleoproteins/physiology , RNA, Viral/biosynthesis , Transcription, Genetic , Viral Core Proteins/physiology , Viral Proteins/physiology , Animals , Cell Line , Chlorocebus aethiops , Gene Expression Profiling , Genes, Reporter , Humans , Luciferases/genetics , Luciferases/metabolismABSTRACT
The rabies virus Ni-CE strain causes nonlethal infection in adult mice after intracerebral inoculation, whereas the parental Nishigahara (Ni) strain kills mice. We previously reported that the chimeric CE(NiN) strain with the N gene from the Ni strain in the genetic background of the Ni-CE strain kills adult mice, indicating that the N gene is related to the different pathogenicities of Ni and Ni-CE strains. In the present study, to obtain an insight into the mechanism by which the N gene determines viral pathogenicity, we compared the effects of Ni, Ni-CE, and CE(NiN) infections on host gene expressions using a human neuroblastoma cell line. Microarray analysis of these infected cells revealed that the expression levels of particular genes in Ni- and CE(NiN)-infected cells, including beta interferon (IFN-beta) and chemokine genes (i.e., CXCL10 and CCL5) were lower than those in Ni-CE-infected cells. We also demonstrated that Ni-CE infection activated the interferon regulatory factor 3 (IRF-3)-dependent IFN-beta promoter and induced IRF-3 nuclear translocation more efficiently than did Ni or CE(NiN) infection. Furthermore, we showed that Ni-CE infection, but not Ni or CE(NiN) infection, strongly activates the IRF-3 pathway through activation of RIG-I, which is known as a cellular sensor of virus infection. These findings indicate that the N protein of rabies virus (Ni strain) has a function to evade the activation of RIG-I. To our knowledge, this is the first report that the Mononegavirales N protein functions to evade induction of host IFN and chemokines.