Flavivirus RNA-Dependent RNA Polymerase Interacts with Genome UTRs and Viral Proteins to Facilitate Flavivirus RNA Replication

Flaviviruses, most of which are emerging and re-emerging human pathogens and significant public health concerns worldwide, are positive-sense RNA viruses. Flavivirus replication occurs on the ER and is regulated by many mechanisms and factors. NS5, which consists of a C-terminal RdRp domain and an N-terminal methyltransferase domain, plays a pivotal role in genome replication and capping. The C-terminal RdRp domain acts as the polymerase for RNA synthesis and cooperates with diverse viral proteins to facilitate productive RNA proliferation within the replication complex. Here, we provide an overview of the current knowledge of the functions and characteristics of the RdRp, including the subcellular localization of NS5, as well as the network of interactions formed between the RdRp and genome UTRs, NS3, and the methyltransferase domain. We posit that a detailed understanding of RdRp functions may provide a target for antiviral drug discovery and therapeutics.

Download Full-text

Viruses and viral proteins

IUCrJ ◽

10.1107/s205225251402003x ◽

2014 ◽

Vol 1 (6) ◽

pp. 492-504 ◽

Cited By ~ 15

Author(s):

Nuria Verdaguer ◽

Diego Ferrero ◽

Mathur R. N. Murthy

Keyword(s):

Viral Entry ◽

Current Knowledge ◽

Viral Proteins ◽

Cell Receptor ◽

Genome Replication ◽

Human Pathogens ◽

X Ray Crystallography ◽

Powerful Approach ◽

Virus World ◽

Structural Aspects

For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes.

Download Full-text

The arterivirus replicase is the only viral protein required for genome replication and subgenomic mRNA transcription

Journal of General Virology ◽

10.1099/0022-1317-81-10-2491 ◽

2000 ◽

Vol 81 (10) ◽

pp. 2491-2496 ◽

Cited By ~ 87

Author(s):

Richard Molenkamp ◽

Hans van Tol ◽

Babette C. D. Rozier ◽

Yvonne van der Meer ◽

Willy J. M. Spaan ◽

...

Keyword(s):

Rna Synthesis ◽

Point Mutations ◽

Equine Arteritis Virus ◽

Viral Rna ◽

Structural Proteins ◽

Genome Replication ◽

Mrna Transcription ◽

Replication Complex ◽

N Protein ◽

Wild Type Phenotype

Equine arteritis virus (EAV) (Arteriviridae) encodes several structural proteins. Whether any of these also function in viral RNA synthesis is unknown. For the related mouse hepatitis coronavirus (MHV), it has been suggested that the nucleocapsid protein (N) is involved in viral RNA synthesis. As described for MHV, we established that the EAV N protein colocalizes with the viral replication complex, suggesting a role in RNA synthesis. Using an infectious cDNA clone, point mutations and deletions were engineered in the EAV genome to disrupt the expression of each of the structural genes. All structural proteins, including N, were found to be dispensable for genome replication and subgenomic mRNA transcription. We also constructed a mutant in which translation of the intraleader ORF was disrupted. This mutant had a wild-type phenotype, indicating that, at least in cell culture, the product of this ORF does not play a role in the EAV replication cycle.

Download Full-text

Conformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity

10.1101/2021.06.24.449696 ◽

2021 ◽

Author(s):

Tomas Kouba ◽

Dominik Vogel ◽

Sigurdur R. Thorkelsson ◽

Emmanuelle R. J. Quemin ◽

Harry M. Williams ◽

...

Keyword(s):

Conformational Changes ◽

Active Site ◽

Rna Synthesis ◽

Antiviral Drug ◽

Structural Data ◽

Lassa Virus ◽

Genome Replication ◽

L Protein ◽

Active Site Cavity

Lassa virus, which causes annual outbreaks in West Africa with increasing case numbers in recent years, is recognized by the WHO R&D blueprint as a significant threat for public health with high epidemic potential and no effective countermeasures. The viral large (L) protein, which contains the RNA-dependent RNA polymerase, is a key player for transcription of viral mRNA and genome replication. Here we present nine cryo-EM structures of Lassa virus L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved genomic 3' and 5' promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and elongation states, the endonuclease is inhibited by the binding of two distinct L protein peptides in the active site, respectively, whereas in the pre-initiation state, the endonuclease is uninhibited. In the stalled, early elongation state, a template-product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide. In this configuration, the full C-terminal region of the L protein, including the putative cap-binding domain, is highly ordered. The structural data are complemented by in vitro and cell-based studies testing a broad range of L protein mutants to probe functional relevance. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated and will underpin antiviral drug development targeting the arenavirus L protein.

Download Full-text

Disruption of the Golgi Apparatus and Contribution of the Endoplasmic Reticulum to the SARS-CoV-2 Replication Complex

Viruses ◽

10.3390/v13091798 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1798

Author(s):

Ted Hackstadt ◽

Abhilash I. Chiramel ◽

Forrest H. Hoyt ◽

Brandi N. Williamson ◽

Cheryl A. Dooley ◽

...

Keyword(s):

Electron Microscopy ◽

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Rna Synthesis ◽

Light And Electron Microscopy ◽

Brefeldin A ◽

Viral Proteins ◽

Protein Markers ◽

Replication Complex ◽

Golgi Fragmentation

A variety of immunolabeling procedures for both light and electron microscopy were used to examine the cellular origins of the host membranes supporting the SARS-CoV-2 replication complex. The endoplasmic reticulum has long been implicated as a source of membrane for the coronavirus replication organelle. Using dsRNA as a marker for sites of viral RNA synthesis, we provide additional evidence supporting ER as a prominent source of membrane. In addition, we observed a rapid fragmentation of the Golgi apparatus which is visible by 6 h and complete by 12 h post-infection. Golgi derived lipid appears to be incorporated into the replication organelle although protein markers are dispersed throughout the infected cell. The mechanism of Golgi disruption is undefined, but chemical disruption of the Golgi apparatus by brefeldin A is inhibitory to viral replication. A search for an individual SARS-CoV-2 protein responsible for this activity identified at least five viral proteins, M, S, E, Orf6, and nsp3, that induced Golgi fragmentation when expressed in eukaryotic cells. Each of these proteins, as well as nsp4, also caused visible changes to ER structure as shown by correlative light and electron microscopy (CLEM). Collectively, these results imply that specific disruption of the Golgi apparatus is a critical component of coronavirus replication.

Download Full-text

How Do Flaviviruses Hijack Host Cell Functions by Phase Separation?

Viruses ◽

10.3390/v13081479 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1479

Author(s):

Akatsuki Saito ◽

Maya Shofa ◽

Hirotaka Ode ◽

Maho Yumiya ◽

Junki Hirano ◽

...

Keyword(s):

Phase Separation ◽

Host Cell ◽

Intrinsically Disordered Proteins ◽

Current Knowledge ◽

Antiviral Drug ◽

Viral Proteins ◽

Disordered Proteins ◽

Cell Organelles ◽

Intrinsically Disordered ◽

Cell Functions

Viral proteins interact with different sets of host cell components throughout the viral life cycle and are known to localize to the intracellular membraneless organelles (MLOs) of the host cell, where formation/dissolution is regulated by phase separation of intrinsically disordered proteins and regions (IDPs/IDRs). Viral proteins are rich in IDRs, implying that viruses utilize IDRs to regulate phase separation of the host cell organelles and augment replication by commandeering the functions of the organelles and/or sneaking into the organelles to evade the host immune response. This review aims to integrate current knowledge of the structural properties and intracellular localizations of viral IDPs to understand viral strategies in the host cell. First, the properties of viral IDRs are reviewed and similarities and differences with those of eukaryotes are described. The higher IDR content in viruses with smaller genomes suggests that IDRs are essential characteristics of viral proteins. Then, the interactions of the IDRs of flaviviruses with the MLOs of the host cell are investigated with emphasis on the viral proteins localized in the nucleoli and stress granules. Finally, the possible roles of viral IDRs in regulation of the phase separation of organelles and future possibilities for antiviral drug development are discussed.

Download Full-text

ACBD3 is an essential pan-enterovirus host factor that mediates the interaction between viral 3A protein and cellular protein PI4KB

10.1101/375550 ◽

2018 ◽

Author(s):

Heyrhyoung Lyoo ◽

Hilde M. van der Schaar ◽

Cristina M. Dorobantu ◽

Jeroen R.P.M. Strating ◽

Frank J.M. van Kuppeveld

Keyword(s):

Virus Replication ◽

Coenzyme A ◽

Rna Viruses ◽

Antiviral Drug ◽

Host Cells ◽

Genome Replication ◽

Human Pathogens ◽

Positive Strand Rna ◽

Acyl Coenzyme A

AbstractThe 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 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 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 acids 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.ImportanceAs all other RNA viruses, enteroviruses reorganize host cellular membranes for efficient genome replication. A host lipid kinase, PI4KB, plays an important role on 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 essential domains for enterovirus replication were identified. Our findings open up a possibility for targeting ACBD3 or its interaction with virus as a novel strategy for a broad-spectrum antiviral drug.

Download Full-text

Open Reading Frame 1a-Encoded Subunits of the Arterivirus Replicase Induce Endoplasmic Reticulum-Derived Double-Membrane Vesicles Which Carry the Viral Replication Complex

Journal of Virology ◽

10.1128/jvi.73.3.2016-2026.1999 ◽

1999 ◽

Vol 73 (3) ◽

pp. 2016-2026 ◽

Cited By ~ 220

Author(s):

Ketil W. Pedersen ◽

Yvonne van der Meer ◽

Norbert Roos ◽

Eric J. Snijder

Keyword(s):

Endoplasmic Reticulum ◽

Rna Synthesis ◽

Membrane Vesicles ◽

Equine Arteritis Virus ◽

Open Reading Frame ◽

Genome Replication ◽

Electron Microscopic ◽

Double Membrane ◽

Reading Frame ◽

Replication Complex

ABSTRACT The replicase of equine arteritis virus (EAV; familyArteriviridae, order Nidovirales) is expressed in the form of two polyproteins (the open reading frame 1a [ORF1a] and ORF1ab proteins). Three viral proteases cleave these precursors into 12 nonstructural proteins, which direct both genome replication and subgenomic mRNA transcription. Immunofluorescence assays showed that most EAV replicase subunits localize to membranes in the perinuclear region of the infected cell. Using replicase-specific antibodies and cryoimmunoelectron microscopy, unusual double-membrane vesicles (DMVs) were identified as the probable site of EAV RNA synthesis. These DMVs were previously observed in cells infected with different arteriviruses but were never implicated in viral RNA synthesis. Extensive electron microscopic analysis showed that they appear to be derived from paired endoplasmic reticulum membranes and that they are most likely formed by protrusion and detachment of vesicular structures with a double membrane. Interestingly, very similar membrane rearrangements were observed upon expression of ORF1a-encoded replicase subunits nsp2 to nsp7 from an alphavirus-based expression vector. Apparently, the formation of a membrane-bound scaffold for the replication complex is a distinct step in the arterivirus life cycle, which is directed by the ORF1a protein and does not depend on other viral proteins and/or EAV-specific RNA synthesis.

Download Full-text

The enzymatic activity of the nsp14 exoribonuclease is critical for replication of Middle East respiratory syndrome-coronavirus

10.1101/2020.06.19.162529 ◽

2020 ◽

Cited By ~ 1

Author(s):

Natacha S. Ogando ◽

Jessika C. Zevenhoven-Dobbe ◽

Clara C. Posthuma ◽

Eric J. Snijder

Keyword(s):

Middle East ◽

Active Site ◽

Rna Synthesis ◽

Reverse Genetics ◽

Antiviral Drug ◽

Middle East Respiratory Syndrome ◽

Genome Replication ◽

Additional Function ◽

Knockout Mutants

AbstractCoronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains an N-terminal 3’-to-5’ exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase) domain. While the latter presumably operates during viral mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN-knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously found to have a crippled but viable hypermutation phenotype. Remarkably, using an identical reverse genetics approach, an extensive mutagenesis study revealed the corresponding ExoN-knockout mutants of another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), to be non-viable. This is in agreement with observations previously made for alpha- and gammacoronaviruses. Only a single MERS-CoV ExoN active site mutant could be recovered, likely because the introduced D191E substitution is highly conservative in nature. For 11 other MERS-CoV ExoN active site mutants, not a trace of RNA synthesis could be detected, unless – in some cases – reversion had first occurred. Subsequently, we expressed and purified recombinant MERS-CoV nsp14 and established in vitro assays for both its ExoN and N7-MTase activities. All ExoN knockout mutations that were lethal when tested via reverse genetics were found to severely decrease ExoN activity, while not affecting N7-MTase activity. Our study thus reveals an additional function for MERS-CoV nsp14 ExoN, which apparently is critical for primary viral RNA synthesis, thus differentiating it from the proofreading activity thought to boost long-term replication fidelity in MHV and SARS-CoV.ImportanceThe bifunctional nsp14 subunit of the coronavirus replicase contains 3’-to-5’ exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) domains. For the betacoronaviruses MHV and SARS-CoV, the ExoN domain was reported to promote the fidelity of genome replication, presumably by mediating some form of proofreading. For these viruses, ExoN knockout mutants are alive while displaying an increased mutation frequency. Strikingly, we now established that the equivalent knockout mutants of MERS-CoV ExoN are non-viable and completely deficient in RNA synthesis, thus revealing an additional and more critical function of ExoN in coronavirus replication. Both enzymatic activities of (recombinant) MERS-CoV nsp14 were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.

Download Full-text

Flavivirus NS1 and Its Potential in Vaccine Development

Vaccines ◽

10.3390/vaccines9060622 ◽

2021 ◽

Vol 9 (6) ◽

pp. 622

Author(s):

Kassandra L. Carpio ◽

Alan D. T. Barrett

Keyword(s):

Vaccine Development ◽

Virus Assembly ◽

Ns1 Protein ◽

Human Pathogens ◽

Unusual Structure ◽

Replication Complex ◽

Virus Challenge ◽

Humoral Immune ◽

Tick Borne Encephalitis ◽

Flavivirus Infection

The Flavivirus genus contains many important human pathogens, including dengue, Japanese encephalitis (JE), tick-borne encephalitis (TBE), West Nile (WN), yellow fever (YF) and Zika (ZIK) viruses. While there are effective vaccines for a few flavivirus diseases (JE, TBE and YF), the majority do not have vaccines, including WN and ZIK. The flavivirus nonstructural 1 (NS1) protein has an unusual structure–function because it is glycosylated and forms different structures to facilitate different roles intracellularly and extracellularly, including roles in the replication complex, assisting in virus assembly, and complement antagonism. It also plays a role in protective immunity through antibody-mediated cellular cytotoxicity, and anti-NS1 antibodies elicit passive protection in animal models against a virus challenge. Historically, NS1 has been used as a diagnostic marker for the flavivirus infection due to its complement fixing properties and specificity. Its role in disease pathogenesis, and the strong humoral immune response resulting from infection, makes NS1 an excellent target for inclusion in candidate flavivirus vaccines.

Download Full-text

Chikungunya and Zika Viruses: Co-Circulation and the Interplay between Viral Proteins and Host Factors

Pathogens ◽

10.3390/pathogens10040448 ◽

2021 ◽

Vol 10 (4) ◽

pp. 448

Author(s):

Sineewanlaya Wichit ◽

Nuttamonpat Gumpangseth ◽

Rodolphe Hamel ◽

Sakda Yainoy ◽

Siwaret Arikit ◽

...

Keyword(s):

Antiviral Drug ◽

Virus Transmission ◽

Antiviral Agent ◽

Viral Proteins ◽

Targeted Therapeutics ◽

Limited Information ◽

Mosquito Vectors ◽

Host Proteins ◽

Host Interactions ◽

Viral Host Interactions

Chikungunya and Zika viruses, both transmitted by mosquito vectors, have globally re-emerged over for the last 60 years and resulted in crucial social and economic concerns. Presently, there is no specific antiviral agent or vaccine against these debilitating viruses. Understanding viral–host interactions is needed to develop targeted therapeutics. However, there is presently limited information in this area. In this review, we start with the updated virology and replication cycle of each virus. Transmission by similar mosquito vectors, frequent co-circulation, and occurrence of co-infection are summarized. Finally, the targeted host proteins/factors used by the viruses are discussed. There is an urgent need to better understand the virus–host interactions that will facilitate antiviral drug development and thus reduce the global burden of infections caused by arboviruses.

Download Full-text