Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting theCHO2gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes.

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Replication of positive-strand RNA viruses in plants: contact points between plant and virus components

Canadian Journal of Botany ◽

10.1139/b05-121 ◽

2005 ◽

Vol 83 (12) ◽

pp. 1529-1549 ◽

Cited By ~ 25

Author(s):

Hélène Sanfaçon

Keyword(s):

Viral Replication ◽

Rna Viruses ◽

Plant Viruses ◽

Host Proteins ◽

Positive Strand ◽

Viral Genomes ◽

Contact Points ◽

Protein Membrane ◽

Positive Strand Rna

Positive-strand RNA viruses constitute the largest group of plant viruses and have an important impact on world agriculture. These viruses have small genomes that encode a limited number of proteins and depend on their hosts to complete the various steps of their replication cycle. In this review, the contact points between positive-strand RNA plant viruses and their hosts, which are necessary for the translation and replication of the viral genomes, are discussed. Special emphasis is placed on the description of viral replication complexes that are associated with specific membranous compartments derived from plant intracellular membranes and contain viral RNAs and proteins as well as a variety of host proteins. These complexes are assembled via an intricate network of protein–protein, protein–membrane, and protein–RNA interactions. The role of host factors in regulating the assembly, stability, and activity of viral replication complexes are also discussed.

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Co-opted Cellular Sac1 Lipid Phosphatase and PI(4)P Phosphoinositide Are Key Host Factors during the Biogenesis of the Tombusvirus Replication Compartment

Journal of Virology ◽

10.1128/jvi.01979-19 ◽

2020 ◽

Vol 94 (12) ◽

Cited By ~ 3

Author(s):

Zsuzsanna Sasvari ◽

Wenwu Lin ◽

Jun-Ichi Inaba ◽

Kai Xu ◽

Nikolay Kovalev ◽

...

Keyword(s):

Viral Replication ◽

Rna Viruses ◽

Critical Role ◽

Host Factors ◽

Positive Strand ◽

Content Type ◽

Membrane Contact Sites ◽

Viral Replicase ◽

Positive Strand Rna ◽

Replication Compartment

ABSTRACT Positive-strand RNA [(+)RNA] viruses assemble numerous membrane-bound viral replicase complexes (VRCs) with the help of viral replication proteins and co-opted host proteins within large viral replication compartments in the cytosol of infected cells. In this study, we found that deletion or depletion of Sac1 phosphatidylinositol 4-phosphate [PI(4)P] phosphatase reduced tomato bushy stunt virus (TBSV) replication in yeast (Saccharomyces cerevisiae) and plants. We demonstrate a critical role for Sac1 in TBSV replicase assembly in a cell-free replicase reconstitution assay. The effect of Sac1 seems to be direct, based on its interaction with the TBSV p33 replication protein, its copurification with the tombusvirus replicase, and its presence in the virus-induced membrane contact sites and within the TBSV replication compartment. The proviral functions of Sac1 include manipulation of lipid composition, sterol enrichment within the VRCs, and recruitment of additional host factors into VRCs. Depletion of Sac1 inhibited the recruitment of Rab5 GTPase-positive endosomes and enrichment of phosphatidylethanolamine in the viral replication compartment. We propose that Sac1 might be a component of the assembly hub for VRCs, likely in collaboration with the co-opted the syntaxin18-like Ufe1 SNARE protein within the TBSV replication compartments. This work also led to demonstration of the enrichment of PI(4)P phosphoinositide within the replication compartment. Reduction in the PI(4)P level due to chemical inhibition in plant protoplasts; depletion of two PI(4)P kinases, Stt4p and Pik1p; or sequestration of free PI(4)P via expression of a PI(4)P-binding protein in yeast strongly inhibited TBSV replication. Altogether, Sac1 and PI(4)P play important proviral roles during TBSV replication. IMPORTANCE Replication of positive-strand RNA viruses depends on recruitment of host components into viral replication compartments or organelles. Using TBSV, we uncovered the critical roles of Sac1 PI(4)P phosphatase and its substrate, PI(4)P phosphoinositide, in promoting viral replication. Both Sac1 and PI(4)P are recruited to the site of viral replication to facilitate the assembly of the viral replicase complexes, which perform viral RNA replication. We found that Sac1 affects the recruitment of other host factors and enrichment of phosphatidylethanolamine and sterol lipids within the subverted host membranes to promote optimal viral replication. In summary, this work demonstrates the novel functions of Sac1 and PI(4)P in TBSV replication in the model host yeast and in plants.

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Tombusviruses target a major crossroad in the endocytic and recycling pathways via co-opting Rab7 small GTPase

Journal of Virology ◽

10.1128/jvi.01076-21 ◽

2021 ◽

Author(s):

Zhike Feng ◽

Jun-ichi Inaba ◽

Peter D. Nagy

Keyword(s):

Viral Replication ◽

Virus Replication ◽

Rna Viruses ◽

Small Gtpase ◽

Replication Protein ◽

Positive Strand ◽

Sorting Nexin ◽

Retromer Complex ◽

Infected Cells ◽

Positive Strand Rna

Positive-strand RNA viruses induce the biogenesis of unique membranous organelles, called viral replication organelles (VROs), which perform virus replication in infected cells. Tombusviruses have been shown to rewire cellular trafficking and metabolic pathways, remodel host membranes and recruit multiple host factors to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely-related carnation Italian ringspot virus (CIRV) usurp Rab7 small GTPase to facilitate building VROs in the surrogate host yeast and in plants. Depletion of Rab7 small GTPase, which is needed for late endosome and retromer biogenesis, strongly inhibits TBSV and CIRV replication in yeast and in planta. The viral p33 replication protein interacts with Rab7 small GTPase, which results in relocalization of Rab7 into the large VROs. Similar to depletion of Rab7, deletion of either MON1 or CCZ1 heterodymeric GEFs (guanine nucleotide exchange factors) of Rab7, inhibited TBSV repRNA replication in yeast. This suggests that the activated Rab7 has pro-viral functions. We show that the pro-viral function of Rab7 is to facilitate the recruitment of the retromer complex and the endosomal sorting nexin-BAR proteins into VROs. We demonstrate that TBSV p33-driven retargeting Rab7 into VROs results in delivery of several retromer cargos with pro-viral functions. These proteins include lipid enzymes, such as Vps34 PI3K (phosphatidylinositol 3-kinase), PI4Kα-like Stt4 (phosphatidylinositol 4-kinase) and Psd2 phosphatidylserine decarboxylase. In summary, based on these and previous findings, we propose that subversion of Rab7 into VROs allows tombusviruses to reroute endocytic and recycling trafficking to support virus replication. Importance: Replication of positive-strand RNA viruses depends on the biogenesis of viral replication organelles (VROs). However, formation of membranous VROs is not well understood yet. Using tombusviruses and the model host yeast, the authors discovered that the endosomal Rab7 small GTPase is critical for the formation of VROs. Interaction between Rab7 and the TBSV p33 replication protein leads to the recruitment of Rab7 into VROs. TBSV-driven usurping of Rab7 has pro-viral functions through facilitating the delivery of co-opted retromer complex, sorting nexin-BAR proteins and lipid enzymes into VROs to create optimal milieu for virus replication. These results open up the possibility that controlling cellular Rab7 activities in infected cells could be a target for new antiviral strategies.

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Surfeit 4 Contributes to the Replication of Hepatitis C Virus Using Double-Membrane Vesicles

Journal of Virology ◽

10.1128/jvi.00858-19 ◽

2019 ◽

Vol 94 (2) ◽

Author(s):

Lingbao Kong ◽

Haruyo Aoyagi ◽

Zibing Yang ◽

Tao Ouyang ◽

Mami Matsuda ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Replication ◽

Rna Viruses ◽

Membrane Vesicles ◽

Rna Replication ◽

Double Membrane ◽

Positive Strand ◽

Replication Sites ◽

Positive Strand Rna

ABSTRACT A number of positive-strand RNA viruses, such as hepatitis C virus (HCV) and poliovirus, use double-membrane vesicles (DMVs) as replication sites. However, the role of cellular proteins in DMV formation during virus replication is poorly understood. HCV NS4B protein induces the formation of a “membranous web” structure that provides a platform for the assembly of viral replication complexes. Our previous screen of NS4B-associated host membrane proteins by dual-affinity purification, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and small interfering RNA (siRNA) methods revealed that the Surfeit 4 (Surf4) gene, which encodes an integral membrane protein, is involved in the replication of the JFH1 subgenomic replicon. Here, we investigated in detail the effect of Surf4 on HCV replication. Surf4 affects HCV replication in a genotype-independent manner, whereas HCV replication does not alter Surf4 expression. The influence of Surf4 on HCV replication indicates that while Surf4 regulates replication, it has no effect on entry, translation, assembly, or release. Analysis of the underlying mechanism showed that Surf4 is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs and the structural integrity of RNA replication complexes. Surf4 also participates in the replication of poliovirus, which uses DMVs as replication sites, but it has no effect on the replication of dengue virus, which uses invaginated/sphere-type vesicles as replication sites. These findings clearly show that Surf4 is a novel cofactor that is involved in the replication of positive-strand RNA viruses using DMVs as RNA replication sites, which provides valuable clues for DMV formation during positive-strand RNA virus replication. IMPORTANCE Hepatitis C virus (HCV) NS4B protein induces the formation of a membranous web (MW) structure that provides a platform for the assembly of viral replication complexes. The main constituents of the MW are double-membrane vesicles (DMVs). Here, we found that the cellular protein Surf4, which maintains endoplasmic reticulum (ER)-Golgi intermediate compartments and the Golgi compartment, is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs. Moreover, Surf4 participates in the replication of poliovirus, which uses DMVs as replication sites, but has no effect on the replication of dengue virus, which uses invaginated vesicles as replication sites. These results indicate that the cellular protein Surf4 is involved in the replication of positive-strand RNA viruses that use DMVs as RNA replication sites, providing new insights into DMV formation during virus replication and potential targets for the diagnosis and treatment of positive-strand RNA viruses.

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Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis

Journal of Virology ◽

10.1128/jvi.01196-17 ◽

2017 ◽

Vol 92 (1) ◽

Cited By ~ 24

Author(s):

Ina Karen Stoeck ◽

Ji-Young Lee ◽

Keisuke Tabata ◽

Inés Romero-Brey ◽

David Paul ◽

...

Keyword(s):

Viral Replication ◽

Rna Viruses ◽

Rna Replication ◽

Hcv Rna ◽

Lipid Transfer ◽

Lipid Transfer Proteins ◽

Positive Strand ◽

Membrane Contact Sites ◽

Niemann Pick ◽

Positive Strand Rna

ABSTRACT Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell endomembrane system to produce a membranous replication organelle (RO). The underlying mechanisms are far from being elucidated fully. In this report, we provide evidence that HCV RNA replication depends on functional lipid transport along the endosomal-lysosomal pathway that is mediated by several lipid transfer proteins, such as the Niemann-Pick type C1 (NPC1) protein. Pharmacological inhibition of NPC1 function reduced viral replication, impaired the transport of cholesterol to the viral replication organelle, and altered organelle morphology. Besides NPC1, our study reports the importance of additional endosomal and lysosomal lipid transfer proteins required for viral replication, thus contributing to our understanding of how HCV manipulates their function in order to generate a membranous replication organelle. These results might have implications for the biogenesis of replication organelles of other positive-strand RNA viruses.

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ACBD3 Is an Essential Pan-enterovirus Host Factor That Mediates the Interaction between Viral 3A Protein and Cellular Protein PI4KB

mBio ◽

10.1128/mbio.02742-18 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 16

Author(s):

Heyrhyoung Lyoo ◽

Hilde M. van der Schaar ◽

Cristina M. Dorobantu ◽

Huib H. Rabouw ◽

Jeroen R. P. M. Strating ◽

...

Keyword(s):

Virus Replication ◽

Coenzyme A ◽

Rna Viruses ◽

Host Cells ◽

Genome Replication ◽

Human Pathogens ◽

Positive Strand ◽

Positive Strand Rna ◽

Acyl Coenzyme A

ABSTRACT The enterovirus genus of the picornavirus family includes a large number of important human pathogens such as poliovirus, coxsackievirus, enterovirus A71, and rhinoviruses. Like all other positive-strand RNA viruses, genome replication of enteroviruses occurs on rearranged membranous structures called replication organelles (ROs). Phosphatidylinositol 4-kinase IIIβ (PI4KB) is required by all enteroviruses for RO formation. The enteroviral 3A protein recruits PI4KB to ROs, but the exact mechanism remains elusive. Here, we investigated the role of acyl-coenzyme A binding domain containing 3 (ACBD3) in PI4KB recruitment upon enterovirus replication using ACBD3 knockout (ACBD3KO) cells. ACBD3 knockout impaired replication of representative viruses from four enterovirus species and two rhinovirus species. PI4KB recruitment was not observed in the absence of ACBD3. The lack of ACBD3 also affected the localization of individually expressed 3A, causing 3A to localize to the endoplasmic reticulum instead of the Golgi. Reconstitution of wild-type (wt) ACBD3 restored PI4KB recruitment and 3A localization, while an ACBD3 mutant that cannot bind to PI4KB restored 3A localization, but not virus replication. Consistently, reconstitution of a PI4KB mutant that cannot bind ACBD3 failed to restore virus replication in PI4KBKO cells. Finally, by reconstituting ACBD3 mutants lacking specific domains in ACBD3KO cells, we show that acyl-coenzyme A binding (ACB) and charged-amino-acid region (CAR) domains are dispensable for 3A-mediated PI4KB recruitment and efficient enterovirus replication. Altogether, our data provide new insight into the central role of ACBD3 in recruiting PI4KB by enterovirus 3A and reveal the minimal domains of ACBD3 involved in recruiting PI4KB and supporting enterovirus replication. IMPORTANCE Similar to all other positive-strand RNA viruses, enteroviruses reorganize host cellular membranes for efficient genome replication. A host lipid kinase, PI4KB, plays an important role in this membrane rearrangement. The exact mechanism of how enteroviruses recruit PI4KB was unclear. Here, we revealed a role of a Golgi-residing protein, ACBD3, as a mediator of PI4KB recruitment upon enterovirus replication. ACBD3 is responsible for proper localization of enteroviral 3A proteins in host cells, which is important for 3A to recruit PI4KB. By testing ACBD3 and PI4KB mutants that abrogate the ACBD3-PI4KB interaction, we showed that this interaction is crucial for enterovirus replication. The importance of specific domains of ACBD3 was evaluated for the first time, and the domains that are essential for enterovirus replication were identified. Our findings open up a possibility for targeting ACBD3 or its interaction with enteroviruses as a novel strategy for the development of broad-spectrum antienteroviral drugs.

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Host Deadenylation-Dependent mRNA Decapping Factors Are Required for a Key Step in Brome Mosaic Virus RNA Replication

Journal of Virology ◽

10.1128/jvi.80.1.246-251.2006 ◽

2006 ◽

Vol 80 (1) ◽

pp. 246-251 ◽

Cited By ~ 48

Author(s):

Antonio Mas ◽

Isabel Alves-Rodrigues ◽

Amine Noueiry ◽

Paul Ahlquist ◽

Juana Díez

Keyword(s):

Viral Replication ◽

Mosaic Virus ◽

Rna Viruses ◽

Rna Virus ◽

Rna Replication ◽

Brome Mosaic Virus ◽

Virus Rna ◽

Cellular Translation ◽

Cellular Mrnas ◽

Positive Strand Rna

ABSTRACT The genomes of positive-strand RNA [(+)RNA] viruses perform two mutually exclusive functions: they act as mRNAs for the translation of viral proteins and as templates for viral replication. A universal key step in the replication of (+)RNA viruses is the coordinated transition of the RNA genome from the cellular translation machinery to the viral replication complex. While host factors are involved in this step, their nature is largely unknown. By using the ability of the higher eukaryotic (+)RNA virus brome mosaic virus (BMV) to replicate in yeast, we previously showed that the host Lsm1p protein is required for efficient recruitment of BMV RNA from translation to replication. Here we show that in addition to Lsm1p, all tested components of the Lsm1p-7p/Pat1p/Dhh1p decapping activator complex, which functions in deadenylation-dependent decapping of cellular mRNAs, are required for BMV RNA recruitment for RNA replication. In contrast, other proteins of the decapping machinery, such as Edc1p and Edc2p from the deadenylation-dependent decapping pathway and Upf1p, Upf2p, and Upf3p from the deadenylation-independent decapping pathway, had no significant effects. The dependence of BMV RNA recruitment on the Lsm1p-7p/Pat1p/Dhh1p complex was linked exclusively to the 3′ noncoding region of the BMV RNA. Collectively, our results suggest that the Lsm1p-7p/Pat1p/Dhh1p complex that transfers cellular mRNAs from translation to degradation might act as a key regulator in the switch from BMV RNA translation to replication.

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Replication-Coupled Packaging Mechanism in Positive-Strand RNA Viruses: Synchronized Coexpression of Functional Multigenome RNA Components of an Animal and a Plant Virus in Nicotiana benthamiana Cells by Agroinfiltration

Journal of Virology ◽

10.1128/jvi.01540-07 ◽

2007 ◽

Vol 82 (3) ◽

pp. 1484-1495 ◽

Cited By ~ 47

Author(s):

Padmanaban Annamalai ◽

Fady Rofail ◽

Darleen A. DeMason ◽

A. L. N. Rao

Keyword(s):

Nicotiana Benthamiana ◽

Rna Viruses ◽

Rna Virus ◽

Plant Cells ◽

Brome Mosaic Virus ◽

Biologically Active ◽

Flock House Virus ◽

Positive Strand ◽

Chimeric Rnas ◽

Positive Strand Rna

ABSTRACT Flock house virus (FHV), a bipartite RNA virus of insects and a member of the Nodaviridae family, shares viral replication features with the tripartite brome mosaic virus (BMV), an RNA virus that infects plants and is a member of the Bromoviridae family. In BMV and FHV, genome packaging is coupled to replication, a widely conserved mechanism among positive-strand RNA viruses of diverse origin. To unravel the events that modulate the mechanism of replication-coupled packaging, in this study, we have extended the transfer DNA (T-DNA)-based agroinfiltration system to express functional genome components of FHV in plant cells (Nicotiana benthamiana). Replication, intracellular membrane localization, and packaging characteristics in agroinfiltrated plant cells revealed that T-DNA plasmids of FHV were biologically active and faithfully mimicked complete replication and packaging behavior similar to that observed for insect cells. Synchronized coexpression of wild-type BMV and FHV genome components in plant cells resulted in the assembly of virions packaging the respective viral progeny RNA. To further elucidate the link between replication and packaging, coat protein (CP) open reading frames were precisely exchanged between BMV RNA 3 (B3) and FHV RNA 2 (F2), creating chimeric RNAs expressing heterologous CP genes (B3/FCP and F2/BCP). Coinfiltration of each chimera with its corresponding genome counterpart to provide viral replicase (B1+B2+B3/FCP and F1+F2/BCP) resulted in the expected progeny profiles, but virions exhibited a nonspecific packaging phenotype. Complementation with homologous replicase (with respect to CP) failed to enhance packaging specificity. Taken together, we propose that the transcription of CP mRNA from homologous replication and its translation must be synchronized to confer packaging specificity.

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Ultrastructure of the replication sites of positive-strand RNA viruses

Virology ◽

10.1016/j.virol.2015.02.029 ◽

2015 ◽

Vol 479-480 ◽

pp. 418-433 ◽

Cited By ~ 78

Author(s):

Christian Harak ◽

Volker Lohmann

Keyword(s):

Rna Viruses ◽

Positive Strand ◽

Replication Sites ◽

Positive Strand Rna

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Poliovirus CRE-Dependent VPg Uridylylation Is Required for Positive-Strand RNA Synthesis but Not for Negative-Strand RNA Synthesis

Journal of Virology ◽

10.1128/jvi.77.8.4739-4750.2003 ◽

2003 ◽

Vol 77 (8) ◽

pp. 4739-4750 ◽

Cited By ~ 97

Author(s):

Kenneth E. Murray ◽

David J. Barton

Keyword(s):

Rna Structure ◽

Rna Synthesis ◽

Critical Role ◽

Rna Replication ◽

Stem Loop ◽

Positive Strand ◽

Negative Strand ◽

Step Model ◽

Positive Strand Rna

ABSTRACT The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpUOH, the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpUOH, however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpUOH synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpUOH were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpUOH in the asymmetric replication of poliovirus RNA.

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