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19 pages, 2035 KiB

Open AccessArticle

An Aedes aegypti-Derived Ago2 Knockout Cell Line to Investigate Arbovirus Infections

by Christina Scherer, Jack Knowles, Vattipally B. Sreenu, Anthony C. Fredericks, Janina Fuss, Kevin Maringer, Ana Fernandez-Sesma, Andres Merits, Margus Varjak, Alain Kohl and Esther Schnettler

Viruses 2021, 13(6), 1066; https://doi.org/10.3390/v13061066 - 3 Jun 2021

Cited by 12 | Viewed by 5053

Abstract

Mosquitoes are known as important vectors of many arthropod-borne (arbo)viruses causing disease in humans. These include dengue (DENV) and Zika (ZIKV) viruses. The exogenous small interfering (si)RNA (exo-siRNA) pathway is believed to be the main antiviral defense in arthropods, including mosquitoes. During infection, [...] Read more.

Mosquitoes are known as important vectors of many arthropod-borne (arbo)viruses causing disease in humans. These include dengue (DENV) and Zika (ZIKV) viruses. The exogenous small interfering (si)RNA (exo-siRNA) pathway is believed to be the main antiviral defense in arthropods, including mosquitoes. During infection, double-stranded RNAs that form during viral replication and infection are cleaved by the enzyme Dicer 2 (Dcr2) into virus-specific 21 nt vsiRNAs, which are subsequently loaded into Argonaute 2 (Ago2). Ago2 then targets and subsequently cleaves complementary RNA sequences, resulting in degradation of the target viral RNA. Although various studies using silencing approaches have supported the antiviral activity of the exo-siRNA pathway in mosquitoes, and despite strong similarities between the siRNA pathway in the Drosophila melanogaster model and mosquitoes, important questions remain unanswered. The antiviral activity of Ago2 against different arboviruses has been previously demonstrated. However, silencing of Ago2 had no effect on ZIKV replication, whereas Dcr2 knockout enhanced its replication. These findings raise the question as to the role of Ago2 and Dcr2 in the control of arboviruses from different viral families in mosquitoes. Using a newly established Ago2 knockout cell line, alongside the previously reported Dcr2 knockout cell line, we investigated the impact these proteins have on the modulation of different arboviral infections. Infection of Ago2 knockout cell line with alpha- and bunyaviruses resulted in an increase of viral replication, but not in the case of ZIKV. Analysis of small RNA sequencing data in the Ago2 knockout cells revealed a lack of methylated siRNAs from different sources, such as acute and persistently infecting viruses-, TE- and transcriptome-derived RNAs. The results confirmed the importance of the exo-siRNA pathway in the defense against arboviruses, but highlights variability in its response to different viruses and the impact the siRNA pathway proteins have in controlling viral replication. Moreover, this established Ago2 knockout cell line can be used for functional Ago2 studies, as well as research on the interplay between the RNAi pathways. Full article

(This article belongs to the Special Issue RNA-Binding Proteins as Cellular Targets to Mediate the Virus Lifecycle)

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17 pages, 19824 KiB

Open AccessArticle

The HIV 5′ Gag Region Displays a Specific Nucleotide Bias Regulating Viral Splicing and Infectivity

by Bastian Grewe, Carolin Vogt, Theresa Horstkötter, Bettina Tippler, Han Xiao, Bianca Müller, Klaus Überla, Ralf Wagner, Benedikt Asbach and Jens Bohne

Viruses 2021, 13(6), 997; https://doi.org/10.3390/v13060997 - 27 May 2021

Cited by 1 | Viewed by 3309

Abstract

Alternative splicing and the expression of intron-containing mRNAs is one hallmark of HIV gene expression. To facilitate the otherwise hampered nuclear export of non-fully processed mRNAs, HIV encodes the Rev protein, which recognizes its intronic response element and fuels the HIV RNAs into [...] Read more.

Alternative splicing and the expression of intron-containing mRNAs is one hallmark of HIV gene expression. To facilitate the otherwise hampered nuclear export of non-fully processed mRNAs, HIV encodes the Rev protein, which recognizes its intronic response element and fuels the HIV RNAs into the CRM-1-dependent nuclear protein export pathway. Both alternative splicing and Rev-dependency are regulated by the primary HIV RNA sequence. Here, we show that these processes are extremely sensitive to sequence alterations in the 5’coding region of the HIV genomic RNA. Increasing the GC content by insertion of either GFP or silent mutations activates a cryptic splice donor site in gag, entirely deregulates the viral splicing pattern, and lowers infectivity. Interestingly, an adaptation of the inserted GFP sequence toward an HIV-like nucleotide bias reversed these phenotypes completely. Of note, the adaptation yielded completely different primary sequences although encoding the same amino acids. Thus, the phenotypes solely depend on the nucleotide composition of the two GFP versions. This is a strong indication of an HIV-specific mRNP code in the 5′ gag region wherein the primary RNA sequence bias creates motifs for RNA-binding proteins and controls the fate of the HIV-RNA in terms of viral gene expression and infectivity. Full article

(This article belongs to the Special Issue RNA-Binding Proteins as Cellular Targets to Mediate the Virus Lifecycle)

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Review

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17 pages, 1269 KiB

Open AccessReview

Pro-Viral and Anti-Viral Roles of the RNA-Binding Protein G3BP1

by Aravinth Kumar Jayabalan, Diane E. Griffin and Anthony K. L. Leung

Viruses 2023, 15(2), 449; https://doi.org/10.3390/v15020449 - 6 Feb 2023

Cited by 12 | Viewed by 4101

Abstract

Viruses depend on host cellular resources to replicate. Interaction between viral and host proteins is essential for the pathogens to ward off immune responses as well as for virus propagation within the infected cells. While different viruses employ unique strategies to interact with [...] Read more.

Viruses depend on host cellular resources to replicate. Interaction between viral and host proteins is essential for the pathogens to ward off immune responses as well as for virus propagation within the infected cells. While different viruses employ unique strategies to interact with diverse sets of host proteins, the multifunctional RNA-binding protein G3BP1 is one of the common targets for many viruses. G3BP1 controls several key cellular processes, including mRNA stability, translation, and immune responses. G3BP1 also serves as the central hub for the protein–protein and protein–RNA interactions within a class of biomolecular condensates called stress granules (SGs) during stress conditions, including viral infection. Increasing evidence suggests that viruses utilize distinct strategies to modulate G3BP1 function—either by degradation, sequestration, or redistribution—and control the viral life cycle positively and negatively. In this review, we summarize the pro-viral and anti-viral roles of G3BP1 during infection among different viral families. Full article

(This article belongs to the Special Issue RNA-Binding Proteins as Cellular Targets to Mediate the Virus Lifecycle)

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38 pages, 2342 KiB

Open AccessReview

RNA-Binding Proteins as Regulators of Internal Initiation of Viral mRNA Translation

by Brenda López-Ulloa, Yazmín Fuentes, Magdalena S. Pizarro-Ortega and Marcelo López-Lastra

Viruses 2022, 14(2), 188; https://doi.org/10.3390/v14020188 - 19 Jan 2022

Cited by 15 | Viewed by 5213

Abstract

Viruses are obligate intracellular parasites that depend on the host’s protein synthesis machinery for translating their mRNAs. The viral mRNA (vRNA) competes with the host mRNA to recruit the translational machinery, including ribosomes, tRNAs, and the limited eukaryotic translation initiation factor (eIFs) pool. [...] Read more.

Viruses are obligate intracellular parasites that depend on the host’s protein synthesis machinery for translating their mRNAs. The viral mRNA (vRNA) competes with the host mRNA to recruit the translational machinery, including ribosomes, tRNAs, and the limited eukaryotic translation initiation factor (eIFs) pool. Many viruses utilize non-canonical strategies such as targeting host eIFs and RNA elements known as internal ribosome entry sites (IRESs) to reprogram cellular gene expression, ensuring preferential translation of vRNAs. In this review, we discuss vRNA IRES-mediated translation initiation, highlighting the role of RNA-binding proteins (RBPs), other than the canonical translation initiation factors, in regulating their activity. Full article

(This article belongs to the Special Issue RNA-Binding Proteins as Cellular Targets to Mediate the Virus Lifecycle)

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25 pages, 3957 KiB

Open AccessReview

RNA Binding Proteins as Pioneer Determinants of Infection: Protective, Proviral, or Both?

by Samantha Lisy, Katherine Rothamel and Manuel Ascano

Viruses 2021, 13(11), 2172; https://doi.org/10.3390/v13112172 - 28 Oct 2021

Cited by 12 | Viewed by 4098

Abstract

As the first intracellular host factors that directly interact with the genomes of RNA viruses, RNA binding proteins (RBPs) have a profound impact on the outcome of an infection. Recent discoveries brought about by new methodologies have led to an unprecedented ability to [...] Read more.

As the first intracellular host factors that directly interact with the genomes of RNA viruses, RNA binding proteins (RBPs) have a profound impact on the outcome of an infection. Recent discoveries brought about by new methodologies have led to an unprecedented ability to peer into the earliest events between viral RNA and the RBPs that act upon them. These discoveries have sparked a re-evaluation of current paradigms surrounding RBPs and post-transcriptional gene regulation. Here, we highlight questions that have bloomed from the implementation of these novel approaches. Canonical RBPs can impact the fates of both cellular and viral RNA during infection, sometimes in conflicting ways. Noncanonical RBPs, some of which were first characterized via interactions with viral RNA, may encompass physiological roles beyond viral pathogenesis. We discuss how these RBPs might discriminate between an RNA of either cellular or viral origin and thus exert either pro- or antiviral effects—which is a particular challenge as viruses contain mechanisms to mimic molecular features of cellular RNA. Full article

(This article belongs to the Special Issue RNA-Binding Proteins as Cellular Targets to Mediate the Virus Lifecycle)

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15 pages, 1281 KiB

Open AccessReview

Does the Zinc Finger Antiviral Protein (ZAP) Shape the Evolution of Herpesvirus Genomes?

by Yao-Tang Lin, Long-Fung Chau, Hannah Coutts, Matin Mahmoudi, Vayalena Drampa, Chen-Hsuin Lee, Alex Brown, David J. Hughes and Finn Grey

Viruses 2021, 13(9), 1857; https://doi.org/10.3390/v13091857 - 17 Sep 2021

Cited by 3 | Viewed by 3155

Abstract

An evolutionary arms race occurs between viruses and hosts. Hosts have developed an array of antiviral mechanisms aimed at inhibiting replication and spread of viruses, reducing their fitness, and ultimately minimising pathogenic effects. In turn, viruses have evolved sophisticated counter-measures that mediate evasion [...] Read more.

An evolutionary arms race occurs between viruses and hosts. Hosts have developed an array of antiviral mechanisms aimed at inhibiting replication and spread of viruses, reducing their fitness, and ultimately minimising pathogenic effects. In turn, viruses have evolved sophisticated counter-measures that mediate evasion of host defence mechanisms. A key aspect of host defences is the ability to differentiate between self and non-self. Previous studies have demonstrated significant suppression of CpG and UpA dinucleotide frequencies in the coding regions of RNA and small DNA viruses. Artificially increasing these dinucleotide frequencies results in a substantial attenuation of virus replication, suggesting dinucleotide bias could facilitate recognition of non-self RNA. The interferon-inducible gene, zinc finger antiviral protein (ZAP) is the host factor responsible for sensing CpG dinucleotides in viral RNA and restricting RNA viruses through direct binding and degradation of the target RNA. Herpesviruses are large DNA viruses that comprise three subfamilies, alpha, beta and gamma, which display divergent CpG dinucleotide patterns within their genomes. ZAP has recently been shown to act as a host restriction factor against human cytomegalovirus (HCMV), a beta-herpesvirus, which in turn evades ZAP detection by suppressing CpG levels in the major immediate-early transcript IE1, one of the first genes expressed by the virus. While suppression of CpG dinucleotides allows evasion of ZAP targeting, synonymous changes in nucleotide composition that cause genome biases, such as low GC content, can cause inefficient gene expression, especially in unspliced transcripts. To maintain compact genomes, the majority of herpesvirus transcripts are unspliced. Here we discuss how the conflicting pressures of ZAP evasion, the need to maintain compact genomes through the use of unspliced transcripts and maintaining efficient gene expression may have shaped the evolution of herpesvirus genomes, leading to characteristic CpG dinucleotide patterns. Full article

(This article belongs to the Special Issue RNA-Binding Proteins as Cellular Targets to Mediate the Virus Lifecycle)

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25 pages, 1564 KiB

Open AccessReview

From A to m⁶A: The Emerging Viral Epitranscriptome

by Belinda Baquero-Perez, Daryl Geers and Juana Díez

Viruses 2021, 13(6), 1049; https://doi.org/10.3390/v13061049 - 1 Jun 2021

Cited by 34 | Viewed by 7038

Abstract

There are over 100 different chemical RNA modifications, collectively known as the epitranscriptome. N⁶-methyladenosine (m⁶A) is the most commonly found internal RNA modification in cellular mRNAs where it plays important roles in the regulation of the mRNA structure, stability, [...] Read more.

There are over 100 different chemical RNA modifications, collectively known as the epitranscriptome. N⁶-methyladenosine (m⁶A) is the most commonly found internal RNA modification in cellular mRNAs where it plays important roles in the regulation of the mRNA structure, stability, translation and nuclear export. This modification is also found in viral RNA genomes and in viral mRNAs derived from both RNA and DNA viruses. A growing body of evidence indicates that m⁶A modifications play important roles in regulating viral replication by interacting with the cellular m⁶A machinery. In this review, we will exhaustively detail the current knowledge on m⁶A modification, with an emphasis on its function in virus biology. Full article

(This article belongs to the Special Issue RNA-Binding Proteins as Cellular Targets to Mediate the Virus Lifecycle)

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19 pages, 2164 KiB

Open AccessReview

RNA-Binding Proteins at the Host-Pathogen Interface Targeting Viral Regulatory Elements

by Azman Embarc-Buh, Rosario Francisco-Velilla and Encarnacion Martinez-Salas

Viruses 2021, 13(6), 952; https://doi.org/10.3390/v13060952 - 21 May 2021

Cited by 12 | Viewed by 3469

Abstract

Viral RNAs contain the information needed to synthesize their own proteins, to replicate, and to spread to susceptible cells. However, due to their reduced coding capacity RNA viruses rely on host cells to complete their multiplication cycle. This is largely achieved by the [...] Read more.

Viral RNAs contain the information needed to synthesize their own proteins, to replicate, and to spread to susceptible cells. However, due to their reduced coding capacity RNA viruses rely on host cells to complete their multiplication cycle. This is largely achieved by the concerted action of regulatory structural elements on viral RNAs and a subset of host proteins, whose dedicated function across all stages of the infection steps is critical to complete the viral cycle. Importantly, not only the RNA sequence but also the RNA architecture imposed by the presence of specific structural domains mediates the interaction with host RNA-binding proteins (RBPs), ultimately affecting virus multiplication and spreading. In marked difference with other biological systems, the genome of positive strand RNA viruses is also the mRNA. Here we focus on distinct types of positive strand RNA viruses that differ in the regulatory elements used to promote translation of the viral RNA, as well as in the mechanisms used to evade the series of events connected to antiviral response, including translation shutoff induced in infected cells, assembly of stress granules, and trafficking stress. Full article

(This article belongs to the Special Issue RNA-Binding Proteins as Cellular Targets to Mediate the Virus Lifecycle)

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11 pages, 2886 KiB

Open AccessReview

RNA Helicase A Regulates the Replication of RNA Viruses

by Rui-Zhu Shi, Yuan-Qing Pan and Li Xing

Viruses 2021, 13(3), 361; https://doi.org/10.3390/v13030361 - 25 Feb 2021

Cited by 7 | Viewed by 2384

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue RNA-Binding Proteins as Cellular Targets to Mediate the Virus Lifecycle)

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