Therapeutic interfering particles exploiting viral replication and assembly mechanisms show promising performance: a modelling study

AbstractDefective interfering particles arise spontaneously during a viral infection as mutants lacking essential parts of the viral genome. Their ability to replicate in the presence of the wild-type (WT) virus (at the expense of viable viral particles) is mimicked and exploited by therapeutic interfering particles. We propose a strategy for the design of therapeutic interfering RNAs (tiRNAs) against positive-sense single-stranded RNA viruses that assemble via packaging signal-mediated assembly. These tiRNAs contain both an optimised version of the virus assembly manual that is encoded by multiple dispersed RNA packaging signals and a replication signal for viral polymerase, but lack any protein coding information. We use an intracellular model for hepatitis C viral (HCV) infection that captures key aspects of the competition dynamics between tiRNAs and viral genomes for virally produced capsid protein and polymerase. We show that only a small increase in the assembly and replication efficiency of the tiRNAs compared with WT virus is required in order to achieve a treatment efficacy greater than 99%. This demonstrates that the proposed tiRNA design could be a promising treatment option for RNA viral infections.

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Human Immunodeficiency Virus Types 1 and 2 Differ in the Predominant Mechanism Used for Selection of Genomic RNA for Encapsidation

Journal of Virology ◽

10.1128/jvi.73.4.3023-3031.1999 ◽

1999 ◽

Vol 73 (4) ◽

pp. 3023-3031 ◽

Cited By ~ 67

Author(s):

Jane F. Kaye ◽

Andrew M. L. Lever

Keyword(s):

Human Immunodeficiency Virus ◽

Genomic Rna ◽

Rna Packaging ◽

Packaging Signal ◽

Initiation Mechanism ◽

Gag Protein ◽

Gag Proteins ◽

Viral Genomes ◽

Immunodeficiency Virus

ABSTRACT Retroviral RNA encapsidation is a highly selective process mediated through recognition by the viral Gag proteins of cis-acting RNA packaging signals in genomic RNA. This RNA species is also translated, producing the viral gag gene products. The relationship between these processes is poorly understood. Unlike that of human immunodeficiency virus type 1 (HIV-1), the dominant packaging signal of HIV-2 is upstream of the major splice donor and present in both unspliced and spliced viral RNAs, necessitating additional mechanisms for preferential packaging of unspliced genomic RNA. Encapsidation studies of a series of HIV-2-based vectors showed efficient packaging of viral genomes only if the unspliced, encapsidated RNA expressed full-length Gag protein, including functional nucleocapsid. We propose a novel encapsidation initiation mechanism, providing selectivity, in which unspliced HIV-2 RNA is captured in cis by the Gag protein. This has implications for the use of HIV-2 and other lentiviruses as vectors.

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RNA Structures and Their Role in Selective Genome Packaging

Viruses ◽

10.3390/v13091788 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1788

Author(s):

Liqing Ye ◽

Uddhav B. Ambi ◽

Marco Olguin-Nava ◽

Anne-Sophie Gribling-Burrer ◽

Shazeb Ahmad ◽

...

Keyword(s):

Viral Evolution ◽

Complex Mixture ◽

Rna Structures ◽

Rna Packaging ◽

Genome Packaging ◽

Viral Genomes ◽

Viral Particles ◽

The Impact ◽

Packaging Signals

To generate infectious viral particles, viruses must specifically select their genomic RNA from milieu that contains a complex mixture of cellular or non-genomic viral RNAs. In this review, we focus on the role of viral encoded RNA structures in genome packaging. We first discuss how packaging signals are constructed from local and long-range base pairings within viral genomes, as well as inter-molecular interactions between viral and host RNAs. Then, how genome packaging is regulated by the biophysical properties of RNA. Finally, we examine the impact of RNA packaging signals on viral evolution.

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Identification of the initial nucleocapsid recognition element in the HIV-1 RNA packaging signal

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2008519117 ◽

2020 ◽

Vol 117 (30) ◽

pp. 17737-17746 ◽

Cited By ~ 5

Author(s):

Pengfei Ding ◽

Siarhei Kharytonchyk ◽

Alexis Waller ◽

Ugonna Mbaekwe ◽

Sapna Basappa ◽

...

Keyword(s):

Binding Sites ◽

Virus Assembly ◽

Rna Packaging ◽

Packaging Signal ◽

Nmr Studies ◽

Recognition Element ◽

High Affinity ◽

Molecule Inhibitor ◽

Encapsidation Signal ◽

Hiv 1

Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5ʹ-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. Here, we show that the dimeric 5′-leader contains more than two dozen NC binding sites with affinities ranging from 40 nM to 1.4 μM, and that all high-affinity sites (Kd≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, ΨCES). The four initial binding sites with highest affinity reside near two symmetrically equivalent three-way junction structures. Unlike the other high-affinity sites, which bind NC with exothermic energetics, binding to these sites occurs endothermically due to concomitant unwinding of a weakly base-paired [UUUU]:[GGAG] helical element. Mutations that stabilize base pairing within this element eliminate NC binding to this site and severely impair RNA packaging into virus-like particles. NMR studies reveal that a recently discovered small-molecule inhibitor of HIV-1 RNA packaging that appears to function by stabilizing the structure of the leader binds directly to the [UUUU]:[GGAG] helix. Our findings suggest a sequential NC binding mechanism for Gag-genome assembly and identify a potential RNA Achilles’ heel to which HIV therapeutics may be targeted.

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Coxsackieviruses Undergo Intercellular Transmission as Pools of Sibling Viral Genomes Associated to Membranes

Proceedings ◽

10.3390/proceedings2020050120 ◽

2020 ◽

Vol 50 (1) ◽

pp. 120

Author(s):

Juan-Vicente Bou ◽

Ron Geller ◽

Rafael Sanjuán

Keyword(s):

Genetic Variants ◽

Social Evolution ◽

Viral Population ◽

Multiplicity Of Infection ◽

Defective Interfering Particles ◽

Viral Genomes ◽

Viral Particles ◽

A Cell ◽

Infected Cells ◽

Population Sequence

Some viruses are released from cells as pools of membrane-associated virions. By increasing the multiplicity of infection, this type of collective dispersal could favor viral cooperation, but also the emergence of cheater-like viruses, such as defective interfering particles. To better understand this process, we examined the genetic diversity of membrane-associated coxsackievirus infectious units. We found that infected cells released large membranous structures containing 8–21 infectious particles on average, including vesicles. However, in most cases (62–93%), these structures did not promote the co-transmission of different viral genetic variants present in a cell. Furthermore, collective dispersal had no effect on viral population sequence diversity. Our results indicate that membrane-associated collective infectious units typically contain viral particles derived from the same parental genome. Hence, if cooperation occurred, it should probably involve sibling viral particles rather than different variants. As shown by social evolution theory, cooperation among siblings should be robust against cheater invasion.

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dsRNA-Seq: Identification of Viral Infection by Purifying and Sequencing dsRNA

Viruses ◽

10.3390/v11100943 ◽

2019 ◽

Vol 11 (10) ◽

pp. 943 ◽

Cited By ~ 7

Author(s):

Carolyn J. Decker ◽

Halley R. Steiner ◽

Laura L. Hoon-Hanks ◽

James H. Morrison ◽

Kelsey C. Haist ◽

...

Keyword(s):

Viral Infections ◽

Rna Viruses ◽

Emerging Infectious Diseases ◽

Viral Disease ◽

Viral Genomes ◽

Naturally Occurring ◽

Viral Particles ◽

Rrna Depletion ◽

Disease Agent ◽

Viral Sequences

RNA viruses are a major source of emerging and re-emerging infectious diseases around the world. We developed a method to identify RNA viruses that is based on the fact that RNA viruses produce double-stranded RNA (dsRNA) while replicating. Purifying and sequencing dsRNA from the total RNA isolated from infected tissue allowed us to recover dsRNA virus sequences and replicated sequences from single-stranded RNA (ssRNA) viruses. We refer to this approach as dsRNA-Seq. By assembling dsRNA sequences into contigs we identified full length or partial RNA viral genomes of varying genome types infecting mammalian culture samples, identified a known viral disease agent in laboratory infected mice, and successfully detected naturally occurring RNA viral infections in reptiles. Here, we show that dsRNA-Seq is a preferable method for identifying viruses in organisms that don’t have sequenced genomes and/or commercially available rRNA depletion reagents. In addition, a significant advantage of this method is the ability to identify replicated viral sequences of ssRNA viruses, which is useful for distinguishing infectious viral agents from potential noninfectious viral particles or contaminants.

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Collective Viral Spread Mediated by Virion Aggregates Promotes the Evolution of Defective Interfering Particles

mBio ◽

10.1128/mbio.02156-19 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 4

Author(s):

Iván Andreu-Moreno ◽

Rafael Sanjuán

Keyword(s):

Vesicular Stomatitis Virus ◽

Viral Infections ◽

Cell Types ◽

Viral Fitness ◽

Target Cells ◽

Short Term ◽

Defective Interfering Particles ◽

Viral Genomes ◽

Viral Spread ◽

Vesicular Stomatitis

ABSTRACT A growing number of studies report that viruses can spread in groups in so-called collective infectious units. By increasing the cellular multiplicity of infection, collective dispersal may allow for social-like interactions, such as cooperation or cheating. Yet, little is known about how such interactions evolve. In previous work with vesicular stomatitis virus, we showed that virion aggregation accelerates early infection stages in most cell types, providing a short-term fitness benefit to the virus. Here, we examine the effects of virion aggregation over several infection cycles. Flow cytometry, deep sequencing, infectivity assays, reverse transcription-quantitative PCR, and electron microscopy revealed that virion aggregation rapidly promotes the emergence of defective interfering particles. Therefore, virion aggregation provides immediate fitness benefits to the virus but incurs fitness costs after a few viral generations. This suggests that an optimal strategy for the virus is to undergo virion aggregation only episodically, for instance, during interhost transmission. IMPORTANCE Recent insights have revealed that viruses use a highly diverse set of strategies to release multiple viral genomes into the same target cells, allowing the emergence of beneficial, but also detrimental, interactions among viruses inside infected cells. This has prompted interest among microbial ecologists and evolutionary biologists in studying how collective dispersal impacts the outcome of viral infections. Here, we have used vesicular stomatitis virus as a model system to study the evolutionary implications of collective dissemination mediated by viral aggregates, since this virus can spontaneously aggregate in the presence of saliva. We find that saliva-driven aggregation has a dual effect on viral fitness; whereas aggregation tends to increase infectivity in the very short term, virion aggregates are highly susceptible to invasion by noncooperative defective variants after a few viral generations.

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Conservation of Genetically-Embedded Virus Assembly Instructions: A Novel Route to Antiviral Therapy

Proceedings ◽

10.3390/proceedings2020050087 ◽

2020 ◽

Vol 50 (1) ◽

pp. 87

Author(s):

Reidun Twarock ◽

Peter G. Stockley ◽

Richard J. Bingham ◽

Eric C. Dykeman ◽

Pierre-Philippe Dechant

Keyword(s):

Viral Infections ◽

Core Protein ◽

Virus Assembly ◽

Viral Evolution ◽

Wild Type Virus ◽

Viral Pathogens ◽

Defective Interfering Particles ◽

Novel Approach ◽

Assembly Instructions ◽

Genetic Message

Many single-stranded RNA viruses, including major viral pathogens, present RNA-encoded virus assembly instructions (VAIs) within their genetic message that can be isolated from the genetic code and repurposed for the design of virus-like particles. These VAIs rely on multiple dispersed RNA secondary structure elements with a consensus recognition motif for the capsid (core) protein, called packaging signals (PSs), which collectively promote capsid assembly. In this talk, I will provide evidence for the evolutionary conservation of the PS-encoded assembly instructions among different viruses in a viral family and discuss the implications of this discovery for viral evolution. I will then demonstrate how the VAIs can be exploited for therapy. In particular, defective interfering particles occur spontaneously in viral evolution as mutant strains lacking essential parts of the viral genome. Their ability to replicate in the presence of wild-type virus at the expense of virally produced resources can be mimicked by therapeutic interfering particles (TIPs). I will introduce a novel approach to the design of such TIPs based on synthetic nucleic acid sequences containing the VAIs but otherwise lacking genetic information. Using multiscale models of a viral infection, I will demonstrate the potential of these particles in both the prophylaxis and treatment of RNA viral infections.

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An epitranscriptomic switch at the 5′-UTR controls genome selection during HIV-1 genomic RNA packaging

10.1101/676031 ◽

2019 ◽

Cited By ~ 1

Author(s):

Camila Pereira-Montecinos ◽

Daniela Toro-Ascuy ◽

Cecilia Rojas-Fuentes ◽

Sebastián Riquelme-Barrios ◽

Bárbara Rojas-Araya ◽

...

Keyword(s):

Full Length ◽

Genomic Rna ◽

Rna Packaging ◽

Gag Protein ◽

Rna Molecules ◽

Viral Genomes ◽

Viral Particles ◽

Demethylase Activity ◽

Retroviral Replication ◽

Hiv 1

ABSTRACTDuring retroviral replication, the full-length RNA serves both as mRNA and genomic RNA (gRNA). While the simple retrovirus MLV segregates its full-length RNA into two functional populations, the HIV-1 full-length RNA was proposed to exist as a single population used indistinctly for protein synthesis or packaging. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that HIV-1 full-length RNA packaging is regulated through an epitranscriptomic switch requiring demethylation of two conserved adenosine residues present within the 5′-UTR. As such, while m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation was required for the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus and drives full-length RNA demethylation. Finally, the specific inhibition of the FTO RNA demethylase activity suppressed HIV-1 full-length RNA packaging. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the full-length RNA molecules that will be used as viral genomes.

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Reovirus Low-Density Particles Package Cellular RNA

Viruses ◽

10.3390/v13061096 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1096

Author(s):

Timothy W. Thoner ◽

Xiang Ye ◽

John Karijolich ◽

Kristen M. Ogden

Keyword(s):

Rna Polymerase ◽

Viral Proteins ◽

Viral Rna ◽

Low Density ◽

Rna Packaging ◽

Genome Packaging ◽

Double Stranded Rna ◽

Viral Genomes ◽

Mammalian Orthoreovirus ◽

Insight Into

Packaging of segmented, double-stranded RNA viral genomes requires coordination of viral proteins and RNA segments. For mammalian orthoreovirus (reovirus), evidence suggests either all ten or zero viral RNA segments are simultaneously packaged in a highly coordinated process hypothesized to exclude host RNA. Accordingly, reovirus generates genome-containing virions and “genomeless” top component particles. Whether reovirus virions or top component particles package host RNA is unknown. To gain insight into reovirus packaging potential and mechanisms, we employed next-generation RNA-sequencing to define the RNA content of enriched reovirus particles. Reovirus virions exclusively packaged viral double-stranded RNA. In contrast, reovirus top component particles contained similar proportions but reduced amounts of viral double-stranded RNA and were selectively enriched for numerous host RNA species, especially short, non-polyadenylated transcripts. Host RNA selection was not dependent on RNA abundance in the cell, and specifically enriched host RNAs varied for two reovirus strains and were not selected solely by the viral RNA polymerase. Collectively, these findings indicate that genome packaging into reovirus virions is exquisitely selective, while incorporation of host RNAs into top component particles is differentially selective and may contribute to or result from inefficient viral RNA packaging.

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Aptamer Applications in Emerging Viral Diseases

Pharmaceuticals ◽

10.3390/ph14070622 ◽

2021 ◽

Vol 14 (7) ◽

pp. 622

Author(s):

Arne Krüger ◽

Ana Paula de Jesus Santos ◽

Vanessa de Sá ◽

Henning Ulrich ◽

Carsten Wrenger

Keyword(s):

Virus Assembly ◽

Viral Diseases ◽

Tissue Penetration ◽

Rna Molecules ◽

Viral Particles ◽

Pharmacokinetic Properties ◽

Oligonucleotide Library ◽

In Vivo Diagnosis ◽

Emerging Viral Diseases

Aptamers are single-stranded DNA or RNA molecules which are submitted to a process denominated SELEX. SELEX uses reiterative screening of a random oligonucleotide library to identify high-affinity binders to a chosen target, which may be a peptide, protein, or entire cells or viral particles. Aptamers can rival antibodies in target recognition, and benefit from their non-proteic nature, ease of modification, increased stability, and pharmacokinetic properties. This turns them into ideal candidates for diagnostic as well as therapeutic applications. Here, we review the recent accomplishments in the development of aptamers targeting emerging viral diseases, with emphasis on recent findings of aptamers binding to coronaviruses. We focus on aptamer development for diagnosis, including biosensors, in addition to aptamer modifications for stabilization in body fluids and tissue penetration. Such aptamers are aimed at in vivo diagnosis and treatment, such as quantification of viral load and blocking host cell invasion, virus assembly, or replication, respectively. Although there are currently no in vivo applications of aptamers in combating viral diseases, such strategies are promising for therapy development in the future.

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