Role of Viral RNA and Co-opted Cellular ESCRT-I and ESCRT-III Factors in Formation of Tombusvirus Spherules Harboring the Tombusvirus Replicase

ABSTRACTPlus-stranded RNA viruses induce membrane deformations in infected cells in order to build viral replication complexes (VRCs).Tomato bushy stunt virus(TBSV) co-opts cellular ESCRT (endosomal sorting complexes required for transport) proteins to induce the formation of vesicle (spherule)-like structures in the peroxisomal membrane with tight openings toward the cytosol. In this study, using a yeast (Saccharomyces cerevisiae)vps23Δbro1Δ double-deletion mutant, we showed that the Vps23p ESCRT-I protein (Tsg101 in mammals) and Bro1p (ALIX) ESCRT-associated protein, both of which bind to the viral p33 replication protein, play partially complementary roles in TBSV replication in cells and in cell extracts. Dual expression of dominant-negative versions ofArabidopsishomologs of Vps23p and Bro1p inhibited tombusvirus replication to greater extent than individual expression inNicotiana benthamianaleaves. We also demonstrated the critical role of Snf7p (CHMP4), Vps20p, and Vps24p ESCRT-III proteins in tombusvirus replication in yeast andin vitro. Electron microscopic imaging ofvps23Δ yeast revealed the lack of tombusvirus-induced spherule-like structures, while crescent-like structures are formed in ESCRT-III deletion yeasts replicating TBSV RNA. In addition, we also showed that the length of the viral RNA affects the sizes of spherules formed inN. benthamianacells. The 4.8-kb genomic RNA is needed for the formation of spherules 66 nm in diameter, while spherules formed during the replication of the ∼600-nucleotide (nt)-long defective interfering RNA in the presence of p33 and p92 replication proteins are 42 nm. We propose that the viral RNA serves as a “measuring string” during VRC assembly and spherule formation.IMPORTANCEPlant positive-strand RNA viruses, similarly to animal positive-strand RNA viruses, replicate in membrane-bound viral replicase complexes in the cytoplasm of infected cells. Identification of cellular and viral factors affecting the formation of the membrane-bound viral replication complex is a major frontier in current virology research. In this study, we dissected the functions of co-opted cellular ESCRT-I (endosomal sorting complexes required for transport I) and ESCRT-III proteins and the viral RNA in tombusvirus replicase complex formation usingin vitro, yeast-based, and plant-based approaches. Electron microscopic imaging revealed the lack of tombusvirus-induced spherule-like structures in ESCRT-I or ESCRT-III deletion yeasts replicating TBSV RNA, demonstrating the requirement for these co-opted cellular factors in tombusvirus replicase formation. The work could be of broad interest in virology and beyond.

Download Full-text

Activation ofTomato Bushy Stunt VirusRNA-Dependent RNA Polymerase by Cellular Heat Shock Protein 70 Is Enhanced by PhospholipidsIn Vitro

Journal of Virology ◽

10.1128/jvi.03711-14 ◽

2015 ◽

Vol 89 (10) ◽

pp. 5714-5723 ◽

Cited By ~ 26

Author(s):

Judit Pogany ◽

Peter D. Nagy

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Inhibitory Effect ◽

Viral Rna ◽

Infected Cells ◽

Membrane Bound ◽

Replicase Complex ◽

Rna Interaction ◽

Positive Strand Rna

ABSTRACTSimilar to other positive-strand RNA viruses, tombusviruses are replicated by the membrane-bound viral replicase complex (VRC). The VRC consists of the p92 virus-coded RNA-dependent RNA polymerase (RdRp), the viral p33 RNA chaperone, and several co-opted host proteins. In order to become a functional RdRp after its translation, the p92 replication protein should be incorporated into the VRC, followed by its activation. We have previously shown in a cell-free yeast extract-based assay that the activation of theTomato bushy stunt virus(TBSV) RdRp requires a soluble host factor(s). In this article, we identify the cellular heat shock protein 70 (Hsp70) as the co-opted host factor required for the activation of an N-terminally truncated recombinant TBSV RdRp. In addition, small-molecule-based blocking of Hsp70 function inhibits RNA synthesis by the tombusvirus RdRpin vitro. Furthermore, we show that neutral phospholipids, namely, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), enhance RdRp activationin vitro. In contrast, phosphatidylglycerol (PG) shows a strong and dominant inhibitory effect onin vitroRdRp activation. We also demonstrate that PE and PC stimulate RdRp-viral plus-strand RNA [(+)RNA] interaction, while PG inhibits the binding of the viral RNA to the RdRp. Based on the stimulatory versus inhibitory roles of various phospholipids in tombusvirus RdRp activation, we propose that the lipid composition of targeted subcellular membranes might be utilized by tombusviruses to regulate new VRC assembly during the course of infection.IMPORTANCEThe virus-coded RNA-dependent RNA polymerase (RdRp), which is responsible for synthesizing the viral RNA progeny in infected cells of several positive-strand RNA viruses, is initially inactive. This strategy is likely to avoid viral RNA synthesis in the cytosol that would rapidly lead to induction of RNA-triggered cellular antiviral responses. During the assembly of the membrane-bound replicase complex, the viral RdRp becomes activated through an incompletely understood process that makes the RdRp capable of RNA synthesis. By using TBSV RdRp, we show that the co-opted cellular Hsp70 chaperone and neutral phospholipids facilitate RdRp activationin vitro. In contrast, phosphatidylglycerol (PG) has a dominant inhibitory effect onin vitroRdRp activation and RdRp-viral RNA interaction, suggesting that the membranous microdomain surrounding the RdRp greatly affects its ability for RNA synthesis. Thus, the activation of the viral RdRp likely depends on multiple host components in infected cells.

Download Full-text

Human Norovirus NS3 Has RNA Helicase and Chaperoning Activities

Journal of Virology ◽

10.1128/jvi.01606-17 ◽

2017 ◽

Vol 92 (5) ◽

Cited By ~ 8

Author(s):

Teng-Feng Li ◽

Myra Hosmillo ◽

Hella Schwanke ◽

Ting Shu ◽

Zhaowei Wang ◽

...

Keyword(s):

Rna Synthesis ◽

Rna Viruses ◽

Rna Helicase ◽

Diverse Group ◽

Rna Helicases ◽

Human Norovirus ◽

Content Type ◽

Rna Remodeling ◽

Positive Strand Rna

ABSTRACTRNA-remodeling proteins, including RNA helicases and chaperones, act to remodel RNA structures and/or protein-RNA interactions and are required for all processes involving RNAs. Although many viruses encode RNA helicases and chaperones, theirin vitroactivities and their roles in infected cells largely remain elusive. Noroviruses are a diverse group of positive-strand RNA viruses in the familyCaliciviridaeand constitute a significant and potentially fatal threat to human health. Here, we report that the protein NS3 encoded by human norovirus has both ATP-dependent RNA helicase activity that unwinds RNA helices and ATP-independent RNA-chaperoning activity that can remodel structured RNAs and facilitate strand annealing. Moreover, NS3 can facilitate viral RNA synthesisin vitroby norovirus polymerase. NS3 may therefore play an important role in norovirus RNA replication. Lastly, we demonstrate that the RNA-remodeling activity of NS3 is inhibited by guanidine hydrochloride, an FDA-approved compound, and, more importantly, that it reduces the replication of the norovirus replicon in cultured human cells. Altogether, these findings are the first to demonstrate the presence of RNA-remodeling activities encoded byCaliciviridaeand highlight the functional significance of NS3 in the noroviral life cycle.IMPORTANCENoroviruses are a diverse group of positive-strand RNA viruses, which annually cause hundreds of millions of human infections and over 200,000 deaths worldwide. For RNA viruses, cellular or virus-encoded RNA helicases and/or chaperones have long been considered to play pivotal roles in viral life cycles. However, neither RNA helicase nor chaperoning activity has been demonstrated to be associated with any norovirus-encoded proteins, and it is also unknown whether norovirus replication requires the participation of any viral or cellular RNA helicases/chaperones. We found that a norovirus protein, NS3, not only has ATP-dependent helicase activity, but also acts as an ATP-independent RNA chaperone. Also, NS3 can facilitatein vitroviral RNA synthesis, suggesting the important role of NS3 in norovirus replication. Moreover, NS3 activities can be inhibited by an FDA-approved compound, which also suppresses norovirus replicon replication in human cells, raising the possibility that NS3 could be a target for antinoroviral drug development.

Download Full-text

Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-Dependent RNA Polymerases

Journal of Virology ◽

10.1128/jvi.01574-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 275-286 ◽

Cited By ~ 63

Author(s):

Grace Campagnola ◽

Seth McDonald ◽

Stéphanie Beaucourt ◽

Marco Vignuzzi ◽

Olve B. Peersen

Keyword(s):

Rna Viruses ◽

Viral Rna ◽

Mutation Rates ◽

Rapid Adaptation ◽

Replication Fidelity ◽

Virus Growth ◽

Polymerase Structure ◽

Positive Strand Rna

ABSTRACTViral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates andin vivofidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects onin vitronucleotide discrimination, and these effects are strongly correlated with elongation rates andin vivomutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity.IMPORTANCEPositive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects onin vitronucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growthin vivo. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.

Download Full-text

Replication Complex of Human Parechovirus 1

Journal of Virology ◽

10.1128/jvi.77.15.8512-8523.2003 ◽

2003 ◽

Vol 77 (15) ◽

pp. 8512-8523 ◽

Cited By ~ 30

Author(s):

Camilla Krogerus ◽

Denise Egger ◽

Olga Samuilova ◽

Timo Hyypiä ◽

Kurt Bienz

Keyword(s):

Viral Protein ◽

Structural Changes ◽

Rna Replication ◽

Viral Rna ◽

Human Parechovirus ◽

Electron Microscopic ◽

Vitro Assay ◽

Replication Complex ◽

Infected Cells

ABSTRACT The parechoviruses differ in many biological properties from other picornaviruses, and their replication strategy is largely unknown. In order to identify the viral RNA replication complex in human parechovirus type 1 (HPEV-1)-infected cells, we located viral protein and RNA in correlation to virus-induced membrane alterations. Structural changes in the infected cells included a disintegrated Golgi apparatus and disorganized, dilated endoplasmic reticulum (ER) which had lost its ribosomes. Viral plus-strand RNA, located by electron microscopic (EM) in situ hybridization, and the viral protein 2C, located by EM immunocytochemistry were found on clusters of small vesicles. Nascent viral RNA, visualized by 5-bromo-UTP incorporation, localized to compartments which were immunocytochemically found to contain the viral protein 2C and the trans-Golgi marker 1,4-galactosyltransferase. Protein 2C was immunodetected additionally on altered ER membranes which displayed a complex network-like structure devoid of cytoskeletal elements and with no apparent involvement in viral RNA replication. This protein also exhibited membrane binding properties in an in vitro assay. Our data suggest that the HPEV-1 replication complex is built up from vesicles carrying a Golgi marker and forming a structure different from that of replication complexes induced by other picornaviruses.

Download Full-text

Membrane Requirements for Uridylylation of the Poliovirus VPg Protein and Viral RNA Synthesis In Vitro

Journal of Virology ◽

10.1128/jvi.77.21.11408-11416.2003 ◽

2003 ◽

Vol 77 (21) ◽

pp. 11408-11416 ◽

Cited By ~ 26

Author(s):

Mark H. Fogg ◽

Natalya L. Teterina ◽

Ellie Ehrenfeld

Keyword(s):

Membrane Trafficking ◽

Rna Synthesis ◽

Rna Replication ◽

Viral Rna ◽

Chain Elongation ◽

Electron Microscopic ◽

Ribonucleoprotein Complexes ◽

Cell Extracts ◽

Infected Cells

ABSTRACT Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation.

Download Full-text

Coronavirus RNA Synthesis Takes Place within Membrane-Bound Sites

Viruses ◽

10.3390/v13122540 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2540

Author(s):

Nicole Doyle ◽

Jennifer Simpson ◽

Philippa C. Hawes ◽

Helena J. Maier

Keyword(s):

Life Cycle ◽

Infectious Bronchitis Virus ◽

Rna Synthesis ◽

Rna Viruses ◽

Viral Rna ◽

Host Cells ◽

Infectious Bronchitis ◽

Welfare Issue ◽

Membrane Bound ◽

Positive Strand Rna

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them.

Download Full-text

Coronavirus RNA synthesis takes place within membrane-bound sites

10.1101/2021.11.04.467246 ◽

2021 ◽

Author(s):

Nicole Doyle ◽

Jennifer Simpson ◽

Philippa C Hawes ◽

Helena J Maier

Keyword(s):

Life Cycle ◽

Infectious Bronchitis Virus ◽

Rna Synthesis ◽

Rna Viruses ◽

Viral Rna ◽

Host Cells ◽

Infectious Bronchitis ◽

Welfare Issue ◽

Membrane Bound ◽

Positive Strand Rna

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Under-standing more about the replication of these viruses is imperative in order to effectively find ways to control them.

Download Full-text

RNA virus replication depends on enrichment of phosphatidylethanolamine at replication sites in subcellular membranes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1418971112 ◽

2015 ◽

Vol 112 (14) ◽

pp. E1782-E1791 ◽

Cited By ~ 58

Author(s):

Kai Xu ◽

Peter D. Nagy

Keyword(s):

Rna Viruses ◽

Rna Virus ◽

Membrane Microdomains ◽

Replication Assay ◽

Replication Sites ◽

Membrane Bound ◽

Surrogate Host ◽

Viral Replicase ◽

Positive Strand Rna

Intracellular membranes are critical for replication of positive-strand RNA viruses. To dissect the roles of various lipids, we have developed an artificial phosphatidylethanolamine (PE) vesicle-based Tomato bushy stunt virus (TBSV) replication assay. We demonstrate that the in vitro assembled viral replicase complexes (VRCs) in artificial PE vesicles can support a complete cycle of replication and asymmetrical RNA synthesis, which is a hallmark of (+)-strand RNA viruses. Vesicles containing ∼85% PE and ∼15% additional phospholipids are the most efficient, suggesting that TBSV replicates within membrane microdomains enriched for PE. Accordingly, lipidomics analyses show increased PE levels in yeast surrogate host and plant leaves replicating TBSV. In addition, efficient redistribution of PE leads to enrichment of PE at viral replication sites. Expression of the tombusvirus p33 replication protein in the absence of other viral compounds is sufficient to promote intracellular redistribution of PE. Increased PE level due to deletion of PE methyltransferase in yeast enhances replication of TBSV and other viruses, suggesting that abundant PE in subcellular membranes has a proviral function. In summary, various (+)RNA viruses might subvert PE to build membrane-bound VRCs for robust replication in PE-enriched membrane microdomains.

Download Full-text

RNA Helicase A Regulates the Replication of RNA Viruses

Viruses ◽

10.3390/v13030361 ◽

2021 ◽

Vol 13 (3) ◽

pp. 361

Author(s):

Rui-Zhu Shi ◽

Yuan-Qing Pan ◽

Li Xing

Keyword(s):

Virus Replication ◽

Rna Binding ◽

Rna Viruses ◽

Rna Helicase ◽

Rna Helicase A ◽

Double Stranded Rna ◽

Binding Domains ◽

N Terminus

The RNA helicase A (RHA) is a member of DExH-box helicases and characterized by two double-stranded RNA binding domains at the N-terminus. RHA unwinds double-stranded RNA in vitro and is involved in RNA metabolisms in the cell. RHA is also hijacked by a variety of RNA viruses to facilitate virus replication. Herein, this review will provide an overview of the role of RHA in the replication of RNA viruses.

Download Full-text

Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

International Journal of Molecular Sciences ◽

10.3390/ijms22010058 ◽

2020 ◽

Vol 22 (1) ◽

pp. 58

Author(s):

Thomas Gremminger ◽

Zhenwei Song ◽

Juan Ji ◽

Avery Foster ◽

Kexin Weng ◽

...

Keyword(s):

Reverse Transcription ◽

Potential Interaction ◽

Viral Rna ◽

Primer Binding Site ◽

Rna Integrity ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Extended Interaction ◽

Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

Download Full-text