scholarly journals Biological Roles and Functional Mechanisms of Arenavirus Z Protein in Viral Replication

2012 ◽  
Vol 86 (18) ◽  
pp. 9794-9801 ◽  
Author(s):  
Jialong Wang ◽  
Shamika Danzy ◽  
Naveen Kumar ◽  
Hinh Ly ◽  
Yuying Liang
Keyword(s):  
Viral Replication ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Hemorrhagic Fever ◽  
Ring Domain ◽  
Viral Rna ◽  
Small Ring ◽  
Protein Z ◽  
Virus Growth ◽  
Z Protein

Arenaviruses can cause severe hemorrhagic fever diseases in humans, with limited prophylactic or therapeutic measures. A small RING-domain viral protein Z has been shown to mediate the formation of virus-like particles and to inhibit viral RNA synthesis, although its biological roles in an infectious viral life cycle have not been directly addressed. By taking advantage of the available reverse genetics system for a model arenavirus, Pichinde virus (PICV), we provide the direct evidence for the essential biological roles of the Z protein's conserved residues, including the G2 myristylation site, the conserved C and H residues of RING domain, and the poorly characterized C-terminal L79 and P80 residues. Dicodon substitutions within the late (L) domain (PSAPPYEP) of the PICV Z protein, although producing viable mutant viruses, have significantly reduced virus growth, a finding suggestive of an important role for the intact L domain in viral replication. Further structure-function analyses of both PICV and Lassa fever virus Z proteins suggest that arenavirus Z proteins have similar molecular mechanisms in mediating their multiple functions, with some interesting variations, such as the role of the G2 residue in blocking viral RNA synthesis. In summary, our studies have characterized the biological roles of the Z protein in an infectious arenavirus system and have shed important light on the distinct functions of its domains in virus budding and viral RNA regulation, the knowledge of which may lead to the development of novel antiviral drugs.

scholarly journals Tacaribe Virus Z Protein Interacts with the L Polymerase Protein To Inhibit Viral RNA Synthesis

2003 ◽  
Vol 77 (19) ◽  
pp. 10383-10393 ◽  
Author(s):  
Rodrigo Jácamo ◽  
Nora López ◽  
Maximiliano Wilda ◽  
María T. Franze-Fernández
Keyword(s):  
Inhibitory Activity ◽  
Rna Synthesis ◽  
Ring Finger ◽  
Direct Interaction ◽  
Viral Rna ◽  
Reverse Genetic System ◽  
Small Ring ◽  
Protein Z ◽  
Glycoprotein Precursor ◽  
Tacaribe Virus

ABSTRACT Tacaribe virus (TV) is the prototype of the New World group of arenaviruses. The TV genome encodes four proteins, the nucleoprotein (N), the glycoprotein precursor, the polymerase (L), and a small RING finger protein (Z). Using a reverse genetic system, we recently demonstrated that TV N and L are sufficient to drive transcription and full-cycle RNA replication mediated by TV-like RNAs and that Z is a powerful inhibitor of these processes (N. López, R. Jácamo, and M. T. Franze-Fernández, J. Virol. 65:12241-12251, 2001). In the present study we investigated whether Z might interact with either of the proteins, N and L, required for RNA synthesis. To that end, we used coimmunoprecipitation with monospecific antibodies against the viral proteins and coimmunoprecipitation with serum against glutathione S-transferase (GST) and binding to glutathione-Sepharose beads when Z was expressed as a fusion protein with GST. We demonstrated that Z interacted with L but not with N and that Z inhibitory activity was dependent on its ability to bind to L. We also evaluated the contribution of different Z regions to its binding ability and functional activity. We found that integrity of the RING structure is essential for Z binding to L and for Z inhibitory activity. Mutants with deletions at the N and C termini of Z showed that amino acids within the C-terminal region and immediately adjacent to the RING domain N terminus contribute to efficient Z-L interaction and are required for inhibitory activity. The data presented here provide the first evidence of an interaction between Z and L, suggesting that Z interferes with viral RNA synthesis by direct interaction with L. In addition, coimmunoprecipitation studies revealed a previously unreported interaction between N and L.

EV71 2A Protease inhibits P-body formation to promote viral RNA synthesis

2021 ◽  
Author(s):  
Shanshan Fan ◽  
Zihang Xu ◽  
Pengfei Liu ◽  
Yali Qin ◽  
Mingzhou Chen
Keyword(s):  
Viral Replication ◽  
Rna Synthesis ◽  
Enterovirus 71 ◽  
Viral Rna ◽  
Processing Bodies ◽  
Body Formation ◽  
P Bodies ◽  
Body Components ◽  
2A Protease ◽  
Enterovirus 71 Infection

Several viruses were proved to inhibit the formation of RNA processing bodies (P-bodies); however, knowledge regarding whether enterovirus blocks P-body formation remains unclear, and the detailed molecular mechanisms and functions of picornavirus regulation of P-bodies are limited. Here we show the crucial role of 2A protease in inhibiting P-bodies to promote viral replication during enterovirus 71 infection. Moreover, we found that the activity of 2A protease is essential to inhibit P-body formation, which was proved by the result that infection of EV71-2A C110S , the 2A protease activity-inactivated recombinant virus, failed to block the formation of P-bodies. Furthermore, we showed DDX6, a scaffolding protein of P-bodies, interacted with viral RNA to facilitate viral replication rather than viral translation, by using a Renilla luciferase mRNA reporter system and capturing the nascent RNA assay. Altogether, our data firstly demonstrate that the 2A protease of enterovirus inhibits P-body formation to facilitate viral RNA synthesis by recruiting the P-body components to viral RNA. IMPORTANCE Processing bodies (P-bodies) are constitutively present in eukaryotic cells and play an important role in the mRNA cycle, including regulating gene expression and mRNA degradation. P-bodies are the structure that viruses to manipulate to facilitate their survival. Here, we show that the 2A protease alone was efficient to block P-body formation during enterovirus 71 infection and its activity was essential. When the assembly of P-bodies was blocked by 2A, DDX6 and 4E-T which were required for P-body formation bound to viral RNA to facilitate viral RNA synthesis. We propose a model revealing that EV71 manipulates P-body formation to generate an environment that is conducive to viral replication by facilitating viral RNA synthesis: 2A protease blocked P-body assembly to make it possible for virus to take advantage of P-body components.

scholarly journals Zinc Finger-Containing Cellular Transcription Corepressor ZBTB25 Promotes Influenza Virus RNA Transcription and Is a Target for Zinc Ejector Drugs

2017 ◽  
Vol 91 (20) ◽  
Author(s):  
Shu-Chuan Chen ◽  
King-Song Jeng ◽  
Michael M. C. Lai
Keyword(s):  
Viral Replication ◽  
Zinc Finger ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Interferon Production ◽  
Zinc Finger Domain ◽  
Rna Transcription ◽  
Viral Rdrp ◽  
Cellular Factors ◽  
Transcription And Replication

ABSTRACT Influenza A virus (IAV) replication relies on an intricate interaction between virus and host cells. How the cellular proteins are usurped for IAV replication remains largely obscure. The aim of this study was to search for novel and potential cellular factors that participate in IAV replication. ZBTB25, a transcription repressor of a variety of cellular genes, was identified by an RNA interference (RNAi) genomic library screen. Depletion of ZBTB25 significantly reduced IAV production. Conversely, overexpression of ZBTB25 enhanced it. ZBTB25 interacted with the viral RNA-dependent RNA polymerase (RdRp) protein and modulated its transcription activity. In addition, ZBTB25 also functioned as a viral RNA (vRNA)-binding protein, binding preferentially to the U-rich sequence within the 5′ untranslated region (UTR) of vRNA. Both protein-protein and protein-RNA interactions involving ZBTB25 facilitated viral RNA transcription and replication. In addition, ZBTB25 suppressed interferon production, further enhancing viral replication. ZBTB25-associated functions required an intact zinc finger domain and posttranslational SUMO-1 modification of ZBTB25. Furthermore, treatment with disulfiram (a zinc ejector) of ZBTB25-overexpressing cells showed significantly reduced IAV production as a result of reduced RNA synthesis. Our findings indicate that IAV usurps ZBTB25 for IAV RNA synthesis and serves as a novel and potential therapeutic antiviral target. IMPORTANCE IAV-induced seasonal influenza causes severe illness and death in high-risk populations. However, IAV has developed resistance to current antiviral drugs due to its high mutation rate. Therefore, development of drugs targeting cellular factors required for IAV replication is an attractive alternative for IAV therapy. Here, we discovered a cellular protein, ZBTB25, that enhances viral RdRp activity by binding to both viral RdRp and viral RNA to stimulate viral RNA synthesis. A unique feature of ZBTB25 in the regulation of viral replication is its dual transcription functions, namely, promoting viral RNA transcription through binding to the U-rich region of vRNA and suppressing cellular interferon production. ZBTB25 contains a zinc finger domain that is required for RNA-inhibitory activity by chelating zinc ions. Disulfiram treatment disrupts the zinc finger functions, effectively repressing IAV replication. Based on our findings, we demonstrate that ZBTB25 regulates IAV RNA transcription and replication and serves as a promising antiviral target for IAV treatment.

scholarly journals UGGT1 enhances enterovirus 71 pathogenicity by promoting viral RNA synthesis and viral replication

2017 ◽  
Vol 13 (5) ◽  
pp. e1006375 ◽  
Author(s):  
Peng-Nien Huang ◽  
Jia-Rong Jheng ◽  
Jamie J. Arnold ◽  
Jen-Ren Wang ◽  
Craig E. Cameron ◽  
...  
Keyword(s):  
Viral Replication ◽  
Rna Synthesis ◽  
Enterovirus 71 ◽  
Viral Rna

Characterization of the interaction domains between the phosphoprotein and the nucleoprotein of human Metapneumovirus

2021 ◽  
Author(s):  
Hortense Decool ◽  
Benjamin Bardiaux ◽  
Luis Checa Ruano ◽  
Olivier Sperandio ◽  
Jenna Fix ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Viral Replication ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Structural Model ◽  
Cytoplasmic Inclusion ◽  
Human Metapneumovirus ◽  
Human Populations ◽  
Functional Assay ◽  
Interaction Domains

Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein (N), forming an RNA-N complex (N Nuc ), which serves as template for genome replication and mRNA transcription by the RNA-dependent RNA polymerase (RdRp). The RdRp is formed by the association of the large polymerase subunit (L), which has RNA polymerase, capping and methyltransferase activities, and the tetrameric phosphoprotein (P). P plays a central role in the RdRp complex by binding to N Nuc and L, allowing the attachment of the L polymerase to the N Nuc template. During infection these proteins concentrate in cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. By analogy to the closely related pneumovirus respiratory syncytial virus (RSV), it is likely that the formation of IBs depends on the interaction between HMPV P and N Nuc , which has not been demonstrated yet. Here, we finely characterized the binding P- N Nuc interaction domains by using recombinant proteins, combined with a functional assay for the polymerase complex activity, and the study of the recruitment of these proteins to IBs by immunofluorescence. We show that the last 6 C-terminal residues of HMPV P are necessary and sufficient for binding to N Nuc , that P binds to the N-terminal domain of N (N NTD ), and identified conserved N residues critical for the interaction. Our results allowed to propose a structural model for the HMPV P-N Nuc interaction. IMPORTANCE Human metapneumovirus (HMPV) is a leading cause of severe respiratory infections in children but also affects human populations of all ages worldwide. Nowadays, no vaccine or efficient antiviral treatments are available for this pneumovirus. A better understanding of the molecular mechanisms involved in viral replication could help the design or discovery of specific antiviral compounds. In this work we have investigated the interaction between two major viral proteins involved in HMPV RNA synthesis, the N and P proteins. We finely characterized their domains of interaction, and identified a pocket on the surface of the N protein, a potential target of choice for the design of compounds interfering with N-P complexes and inhibiting viral replication.

scholarly journals Influenza A Virus Panhandle Structure Is Directly Involved in RIG-I Activation and Interferon Induction

2015 ◽  
Vol 89 (11) ◽  
pp. 6067-6079 ◽  
Author(s):  
GuanQun Liu ◽  
Hong-Su Park ◽  
Hyun-Mi Pyo ◽  
Qiang Liu ◽  
Yan Zhou
Keyword(s):  
Viral Replication ◽  
Influenza A Virus ◽  
Promoter Region ◽  
Influenza A ◽  
Rna Synthesis ◽  
Nucleotide Composition ◽  
Viral Rna ◽  
Viral Transcription ◽  
Wild Type ◽  
Viral Promoter

ABSTRACTRetinoic acid-inducible gene I (RIG-I) is an important innate immune sensor that recognizes viral RNA in the cytoplasm. Its nonself recognition largely depends on the unique RNA structures imposed by viral RNA. The panhandle structure residing in the influenza A virus (IAV) genome, whose primary function is to serve as the viral promoter for transcription and replication, has been proposed to be a RIG-I agonist. However, this has never been proved experimentally. Here, we employed multiple approaches to determine if the IAV panhandle structure is directly involved in RIG-I activation and type I interferon (IFN) induction. First, in porcine alveolar macrophages, we demonstrated that the viral genomic coding region is dispensable for RIG-I-dependent IFN induction. Second, usingin vitro-synthesized hairpin RNA, we showed that the IAV panhandle structure could directly bind to RIG-I and stimulate IFN production. Furthermore, we investigated the contributions of the wobble base pairs, mismatch, and unpaired nucleotides within the wild-type panhandle structure to RIG-I activation. Elimination of these destabilizing elements within the panhandle structure promoted RIG-I activation and IFN induction. Given the function of the panhandle structure as the viral promoter, we further monitored the promoter activity of these panhandle variants and found that viral replication was moderately affected, whereas viral transcription was impaired dramatically. In all, our results indicate that the IAV panhandle promoter region adopts a nucleotide composition that is optimal for balanced viral RNA synthesis and suboptimal for RIG-I activation.IMPORTANCEThe IAV genomic panhandle structure has been proposed to be an RIG-I agonist due to its partial complementarity; however, this has not been experimentally confirmed. Here, we provide direct evidence that the IAV panhandle structure is competent in, and sufficient for, RIG-I activation and IFN induction. By constructing panhandle variants with increased complementarity, we demonstrated that the wild-type panhandle structure could be modified to enhance RIG-I activation and IFN induction. These panhandle variants posed moderate influence on viral replication but dramatic impairment of viral transcription. These results indicate that the IAV panhandle promoter region adopts a nucleotide composition to achieve optimal balance of viral RNA synthesis and suboptimal RIG-I activation. Our results highlight the multifunctional role of the IAV panhandle promoter region in the virus life cycle and offer novel insights into the development of antiviral agents aiming to boost RIG-I signaling or virus attenuation by manipulating this conserved region.

scholarly journals HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

2015 ◽  
Vol 89 (22) ◽  
pp. 11356-11371 ◽  
Author(s):  
Shivaprasad Shwetha ◽  
Anuj Kumar ◽  
Ranajoy Mullick ◽  
Deeptha Vasudevan ◽  
Nilanjan Mukherjee ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Untranslated Region ◽  
Rna Synthesis ◽  
Binding Protein ◽  
Viral Rna ◽  
Host Protein ◽  
Polypyrimidine Tract ◽  
Polypyrimidine Tract Binding Protein

ABSTRACTHuR is a ubiquitous, RNA binding protein that influences the stability and translation of several cellular mRNAs. Here, we report a novel role for HuR, as a regulator of proteins assembling at the 3′ untranslated region (UTR) of viral RNA in the context of hepatitis C virus (HCV) infection. HuR relocalizes from the nucleus to the cytoplasm upon HCV infection, interacts with the viral polymerase (NS5B), and gets redistributed into compartments of viral RNA synthesis. Depletion in HuR levels leads to a significant reduction in viral RNA synthesis. We further demonstrate that the interaction of HuR with the 3′ UTR of the viral RNA affects the interaction of two host proteins, La and polypyrimidine tract binding protein (PTB), at this site. HuR interacts with La and facilitates La binding to the 3′ UTR, enhancing La-mediated circularization of the HCV genome and thus viral replication. In addition, it competes with PTB for association with the 3′ UTR, which might stimulate viral replication. Results suggest that HuR influences the formation of a cellular/viral ribonucleoprotein complex, which is important for efficient initiation of viral RNA replication. Our study unravels a novel strategy of regulation of HCV replication through an interplay of host and viral proteins, orchestrated by HuR.IMPORTANCEHepatitis C virus (HCV) is highly dependent on various host factors for efficient replication of the viral RNA. Here, we have shown how a host factor (HuR) migrates from the nucleus to the cytoplasm and gets recruited in the protein complex assembling at the 3′ untranslated region (UTR) of HCV RNA. At the 3′ UTR, it facilitates circularization of the viral genome through interaction with another host factor, La, which is critical for replication. Also, it competes with the host protein PTB, which is a negative regulator of viral replication. Results demonstrate a unique strategy of regulation of HCV replication by a host protein through alteration of its subcellular localization and interacting partners. The study has advanced our knowledge of the molecular mechanism of HCV replication and unraveled the complex interplay between the host factors and viral RNA that could be targeted for therapeutic interventions.

scholarly journals Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

2016 ◽  
Vol 90 (7) ◽  
pp. 3676-3683 ◽  
Author(s):  
James R. Short ◽  
Jeffrey A. Speir ◽  
Radhika Gopal ◽  
Logan M. Pankratz ◽  
Jason Lanman ◽  
...  
Keyword(s):  
Host Defense ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Rna Viruses ◽  
Rna Replication ◽  
Viral Rna ◽  
Flock House Virus ◽  
Double Stranded Rna ◽  
Content Type ◽  
Positive Sense

ABSTRACTViruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infectsDrosophilacells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ∼50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infectedDrosophilacells. These preparations can be programmed to synthesize both single- and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis.IMPORTANCEIt is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infectedDrosophilacells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.

scholarly journals Characterization of the Arenavirus RING Finger Z Protein Regions Required for Z-Mediated Inhibition of Viral RNA Synthesis

2002 ◽  
Vol 76 (13) ◽  
pp. 6678-6688 ◽  
Author(s):  
Tatjana I. Cornu ◽  
Juan Carlos de la Torre
Keyword(s):  
Inhibitory Activity ◽  
Rna Synthesis ◽  
Structural Integrity ◽  
Ring Finger ◽  
Surface Glycoprotein ◽  
Reverse Genetic System ◽  
Ring Finger Protein ◽  
Protein Z ◽  
Finger Protein ◽  
Z Protein

ABSTRACT The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an enveloped virus with a bisegmented negative-strand RNA genome whose proteomic capability is limited to four polypeptides, namely, nucleoprotein; surface glycoprotein (GP), which is proteolytically processed into GP1 and GP2; polymerase (L); and a small (11-kDa) RING finger protein (Z). Using a reverse genetic system based on the ARM strain of LCMV, we have previously shown that Z has a strong inhibitory activity on LCMV minigenome transcription and RNA replication (T. I. Cornu and J. C. de la Torre, J. Virol. 75:9415-9426, 2001). In the present study, we have identified regions and specific amino acid residues within Z which contribute to its inhibitory activity on RNA synthesis mediated by the LCMV polymerase. Z proteins from different LCMV strains had similar inhibitory activities on the expression of the LCMV ARM minigenome, whereas the Z protein of the genetically more distantly related Tacaribe virus had an approximately 10-fold lower inhibitory activity on ARM minigenome expression. Results from the use of chimera proteins between Z and Xenopus Neuralized, a nonviral RING finger protein, indicated that the structural integrity of the Z RING domain (RD) was required but not sufficient for the inhibitory activity of Z. Serial deletion mutants of the N and C termini of Z showed that the N terminus (residues 1 through 16) and C terminus (residues 79 through 90) do not contribute to the Z inhibitory activity. A highly conserved tryptophan (W) residue located at position 36 in ARM-Z, next to the second conserved cysteine (C) residue of the Z RD, also contributed to the Z inhibitory activity.

Sign in / Sign up

Export Citation Format

Share Document