Reovirus µ2 protein modulates host cell alternative splicing by reducing protein levels of U5 snRNP core components.

Mammalian orthoreovirus (MRV) is a double-stranded RNA virus from the Reoviridae family presenting a promising activity as an oncolytic virus. Recent studies have underlined MRV's ability to alter cellular alternative splicing (AS) during infection, with a limited understanding of the mechanisms at play. In this study, we investigated how MRV modulates AS. Using a combination of cell biology and reverse genetics experiments, we demonstrated that the M1 gene segment, encoding the µ2 protein, is the primary determinant of MRV's ability to alter AS, and that the amino acid at position 208 in µ2 is critical to induce these changes. Moreover, we showed that the expression of µ2 by itself is sufficient to trigger AS changes, and its ability to enter the nucleus is not required for all these changes. Moreover, we identified core components of the U5 snRNP (i.e. EFTUD2, PRPF8, and SNRNP200) as interactors of µ2 that are required for MRV modulation of AS. Finally, these U5 snRNP components are reduced at the protein level by both MRV infection and µ2 expression. Our findings identify the reduction of U5 snRNP components levels as a new mechanism by which viruses alter cellular AS.

[Emerging reoviruses: the next pandemic?] / Les réovirus émergents : vers une prochaine pandémie?

As the world is experiencing the pandemic of SARS-CoV-2 responsible for COVID-19, one can wonder if members of other family of viruses could possibly emerge. Can such viruses establish a worldwide distribution with consequences similar to SARS-CoV-2? One such threat is the possible emergence of pathogenic reoviruses, especially by zoonotic transmission. Reoviruses are ubiquitous viruses exhibiting a worldwide distribution and various strains or isolates are found in many mammalian species and other vertebrates. When initially discovered, these viruses were named respiratory enteric orphan viruses (hence the acronym "reo") in order to reflect the fact that they could not be clearly associated with any given disease. However, this is not necessarily the case for all of these viruses, as clearly shown for some of these in animals. Significantly, there have been numerous reports of zoonotic transmission, especially from bats to humans. In this manuscript, pertinent properties of reoviruses will be first briefly presented followed by a review of available evidence for zoonotic transmission of pathogenic reoviruses to humans. Future work that appears to be needed for preparedness to the possible emergence of these viruses will then be briefly discussed.

Emerging reoviruses: the next pandemic?

As the world is experiencing the pandemic of SARS-CoV-2 responsible for COVID-19, one can wonder if members of other family of viruses could possibly emerge. Can such viruses establish a worldwide distribution with consequences similar to SARS-CoV-2? One such threat is the possible emergence of pathogenic reoviruses, especially by zoonotic transmission. Reoviruses are ubiquitous viruses exhibiting a worldwide distribution and various strains or isolates are found in many mammalian species and other vertebrates. When initially discovered, these viruses were named respiratory enteric orphan viruses (hence the acronym "reo") in order to reflect the fact that they could not be clearly associated with any given disease. However, this is not necessarily the case for all of these viruses, as clearly shown for some of these in animals. Significantly, there have been numerous reports of zoonotic transmission, especially from bats to humans. In this manuscript, pertinent properties of reoviruses will be first briefly presented followed by a review of available evidence for zoonotic transmission of pathogenic reoviruses to humans. Future work that appears to be needed for preparedness to the possible emergence of these viruses will then be briefly discussed.

Reovirus µ2 Protein Impairs Translation to Reduce U5 snRNP Protein Levels.

Mammalian orthoreovirus (MRV) is a double-stranded RNA virus from the Reoviridae family that infects a large range of mammals, including humans. Recently, studies have shown that MRV alters cellular alternative splicing (AS) during viral infection. The structural protein µ2 appears to be the main determinant of these AS modifications by decreasing the levels of U5 core components EFTUD2, PRPF8, and SNRNP200 during infection. In the present study, we investigated the mechanism by which µ2 exerts this effect on the U5 components. Our results revealed that µ2 has no impact on steady-state mRNA levels, RNA export, and protein stability of these U5 snRNP proteins. However, polysome profiling and metabolic labeling of newly synthesized proteins revealed that µ2 exerts an inhibitory effect on global translation. Moreover, we showed that µ2 mutants unable to accumulate in the nucleus retain most of the ability to reduce PRPF8 protein levels, indicating that the effect of µ2 on U5 snRNP components mainly occurs in the cytoplasm. Finally, co-expression experiments demonstrated that µ2 suppresses the expression of U5 snRNP proteins in a dose-dependent manner, and that the expression of specific U5 snRNP core components have different sensitivities to µ2's presence. Altogether, these results suggest a novel mechanism by which the µ2 protein reduces the levels of U5 core components through translation inhibition, allowing this viral protein to alter cellular AS during infection.

[Viral persistence of mammalian reovirus in cell culture: a model of virus-cell coevolution]. / Infection persistante en culture cellulaire par le réovirus de mammifère : un modèle de coévolution virus-cellule.

Although mammalian reovirus exhibits only limited pathogenicity in humans, it has been, and still remains, instrumental in studies of viral replication and pathogenesis. Generally considered as cytolytic, this virus can sometimes establish long-term persistent infections in tissue culture. In fact, in this context, it constitutes one widely used model to demonstrate coevolution between virus and host cells. Initially limited to the murine L929 fibroblasts model, further studies in different cell types appeared in the last few years. Establishment of viral persistence could also become a preferred approach to isolate new viruses that are better adapted to their applications in virotherapy, for example as oncolytic agents against human or animal cancers. A better understanding of the persistence phenomenon, especially of viral genes involved, is thus essential. The development of new tools, such as reverse genetics, appears very promising to achieve these objectives. Actually, this last approach allows us to establish the biological significance of mutations found on viruses selected during viral persistence.

Viral persistence of mammalian reovirus in cell culture: a model of virus-cell coevolution.

Although mammalian reovirus exhibits only limited pathogenicity in humans, it has been, and still remains, instrumental in studies of viral replication and pathogenesis. Generally considered as cytolytic, this virus can sometimes establish long-term persistent infections in tissue culture. In fact, in this context, it constitutes one widely used model to demonstrate coevolution between virus and host cells. Initially limited to the murine L929 fibroblasts model, further studies in different cell types appeared in the last few years. Establishment of viral persistence could also become a preferred approach to isolate new viruses that are better adapted to their applications in virotherapy, for example as oncolytic agents against human or animal cancers. A better understanding of the persistence phenomenon, especially of viral genes involved, is thus essential. The development of new tools, such as reverse genetics, appears very promising to achieve these objectives. Actually, this last approach allows us to establish the biological significance of mutations found on viruses selected during viral persistence.

Human T-cell leukemia virus type 3 (HTLV-3) and HTLV-4 antisense-transcript-encoded proteins interact and transactivate Jun family-dependent transcription via their atypical bZIP motif.

Human T-cell leukemia virus types 3 and 4 (HTLV-3 and HTLV-4) are recently isolated retroviruses. We have previously characterized HTLV-3- and HTLV-4-encoded antisense genes, termed APH-3 and APH-4, respectively, which, in contrast to HBZ, the HTLV-1 homologue, do not contain a typical bZIP domain (M. Larocque É Halin, S. Landry, S. J. Marriott, W. M. Switzer, and B. Barbeau, J. Virol. 85:12673-12685, 2011, doi:10.1128/JVI.05296-11). As HBZ differentially modulates the transactivation potential of various Jun family members, the effect of APH-3 and APH-4 on JunD-, c-Jun-, and JunB-mediated transcriptional activation was investigated. We first showed that APH-3 and APH-4 upregulated the transactivation potential of all tested Jun family members. Using an human telomerase catalytic subunit (hTERT) promoter construct, our results also highlighted that, unlike HBZ, which solely modulates hTERT expression via JunD, both APH-3 and APH-4 acted positively on the transactivation of the hTERT promoter mediated by tested Jun factors. Coimmunoprecipitation experiments demonstrated that these Jun proteins interacted with APH-3 and APH-4. Although no activation domain was identified for APH proteins, the activation domain of c-Jun was very important in the observed upregulation of its activation potential. We further showed that APH-3 and APH-4 required their putative bZIP-like domains and corresponding leucine residues for interaction and modulation of the transactivation potential of Jun factors. Our results demonstrate that HTLV-encoded antisense proteins behave differently, and that the bZIP-like domains of both APH-3 and APH-4 have retained their interaction potential for Jun members. These studies are important in assessing the differences between HBZ and other antisense proteins, which might further contribute to determining the role of HBZ in HTLV-1-associated diseases. IMPORTANCE HBZ, the antisense transcript-encoded protein from HTLV-1, is now well recognized as a potential factor for adult T-cell leukemia/lymphoma development. In order to better appreciate the mechanism of action of HBZ, comparison to antisense proteins from other HTLV viruses is important. Little is known in relation to the seemingly nonpathogenic HTLV-3 and HTLV-4 viruses, and studies of their antisense proteins are limited to our previously reported study (M. Larocque É Halin, S. Landry, S. J. Marriott, W. M. Switzer, and B. Barbeau, J. Virol. 85:12673-12685, 2011, doi:10.1128/JVI.05296-11). Here, we demonstrate that Jun transcription factors are differently affected by APH-3 and APH-4 compared to HBZ. These intriguing findings suggest that these proteins act differently on viral replication but also on cellular gene expression, and that highlighting their differences of action might lead to important information allowing us to understand the link between HTLV-1 HBZ and ATL in infected individuals.

Further characterization and determination of the single amino acid change in the tsI138 reovirus thermosensitive mutant.

Many temperature-sensitive mutants have been isolated in early studies of mammalian reovirus. However, the biological properties and nature of the genetic alterations remain incompletely explored for most of these mutants. The mutation harbored by the tsI138 mutant was already assigned to the L3 gene encoding the λ1 protein. In the present study, this mutant was further studied as a possible tool to establish the role of the putative λ1 enzymatic activities in viral multiplication. It was observed that synthesis of viral proteins is only marginally reduced, while it was difficult to recover viral particles at the nonpermissive temperature. A single nucleotide substitution resulting in an amino acid change was found; the position of this amino acid is consistent with a probable defect in assembly of the inner capsid at the nonpermissive temperature.

The µ2 and λ1 Proteins of Mammalian Reovirus Modulate Early Events Leading to Induction of the Interferon Signaling Network.

It has been previously shown that amino acid polymorphisms in reovirus proteins µ2 and λ1 are associated with differing levels of interferon induction. In the present study, viruses carrying these polymorphisms in either or both proteins, were further studied. The two viral determinants exert a synergistic effect on the control of ß-interferon induction at the protein and mRNA level, with a concomitant increase in RIG-I. In contrast, levels of phospho-Stat1 and interferon-stimulated genes are increased in singly substituted viruses but with no further increase when both substitutions were present. This suggests that the viral determinants are acting during initial events of viral recognition. Accordingly, difference between viruses was reduced when infection was performed with partially uncoated virions (ISVPs) and transfection of RNA recovered from early-infected cells recapitulates the differences between viruses harboring the different polymorphisms. Altogether, the data are consistent with a redundant or complementary role of µ2 and λ1, affecting either early disassembly or the nature of the viral RNA in the incoming viral particle. Proteins involved in viral RNA synthesis are thus involved in this likely critical aspect of the ability of different reovirus variants to infect various cell types, and to discriminate between parental and transformed/cancer cells.

U5 snRNP Core Proteins Are Key Components of the Defense Response against Viral Infection through Their Roles in Programmed Cell Death and Interferon Induction.

The spliceosome is a massive ribonucleoprotein structure composed of five small nuclear ribonucleoprotein (snRNP) complexes that catalyze the removal of introns from pre-mature RNA during constitutive and alternative splicing. EFTUD2, PRPF8, and SNRNP200 are core components of the U5 snRNP, which is crucial for spliceosome function as it coordinates and performs the last steps of the splicing reaction. Several studies have demonstrated U5 snRNP proteins as targeted during viral infection, with a limited understanding of their involvement in virus-host interactions. In the present study, we deciphered the respective impact of EFTUD2, PRPF8, and SNRNP200 on viral replication using mammalian reovirus as a model. Using a combination of RNA silencing, real-time cell analysis, cell death and viral replication assays, we discovered distinct and partially overlapping novel roles for EFTUD2, PRPF8, and SNRNP200 in cell survival, apoptosis, necroptosis, and the induction of the interferon response pathway. For instance, we demonstrated that EFTUD2 and SNRNP200 are required for both apoptosis and necroptosis, whereas EFTUD2 and PRPF8 are required for optimal interferon response against viral infection. Moreover, we demonstrated that EFTUD2 restricts viral replication, both in a single cycle and multiple cycles of viral replication. Altogether, these results establish U5 snRNP core components as key elements of the cellular antiviral response.

A newly identified interaction between nucleolar NPM1/B23 and the HTLV-I basic leucine zipper factor in HTLV-1 infected cells.

Human T-cell leukemia virus type 1 is the causative agent of HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T-cell leukemia-lymphoma (ATL). The HTLV-1 basic leucine zipper factor (HBZ) has been associated to the cancer-inducing properties of this virus, although the exact mechanism is unknown. In this study, we identified nucleophosmin (NPM1/B23) as a new interaction partner of HBZ. We show that sHBZ and the less abundant uHBZ isoform interact with nucleolar NPM1/B23 in infected cells and HTLV-1 positive patient cells, unlike equivalent antisense proteins of related non-leukemogenic HTLV-2, -3 and-4 viruses. We further demonstrate that sHBZ association to NPM1/B23 is sensitive to RNase. Interestingly, sHBZ was shown to interact with its own RNA. Through siRNA and overexpression experiments, we further provide evidence that NPM1/B23 acts negatively on viral gene expression with potential impact on cell transformation. Our results hence provide a new insight over HBZ-binding partners in relation to cellular localization and potential function on cell proliferation and should lead to a better understanding of the link between HBZ and ATL development.

[Reverse genetics in reovirus study: advances, difficulties and perspectives]. / La génétique inverse dans l'étude des réovirus : progrès, obstacles et développements futurs.

In "classical" genetics, examination of a phenotype leads to the study of the gene(s) involved in its obtention. Reverse genetics is a powerful experimental approach in which, on the contrary, the genetic material is modified and used to reconstruct a complete organism in order to study the result of these modifications. This approach is especially well adapted to the study of viruses, considering their relative simplicity and small size of their genomes; the main obstacle remains to recover infectious viruses from cloned viral genomes. Over the years, this exploit has been achieved with representatives of almost all families of mammalian viruses. Until recently, the Reoviridae, viruses with segmented double-stranded RNA genome, were an exception. In this review, the progress accomplished toward the development of such an approach for the Orthoreovirus will thus be discussed. Reverse genetics could have a major impact for the optimization of novel virus strains for their use in therapy as oncolytic viruses and for the development of vaccines in the case of Rotavirus and Orbivirus. However, current works stress the limitations of the approach, the need for careful analysis of the results obtained, as well as the necessity to develop more efficient and polyvalent systems.

Identification of the nuclear and nucleolar localization signals of the Feline immunodeficiency virus Rev protein.

Lentivirus genomes code for a regulatory protein essential for virus replication termed Rev. The Rev protein binds to partially spliced and unspliced viral RNAs and mediates their nuclear export. Therefore, Rev possesses functional domains that enable its shuttling between the cytoplasm and the nucleus. The Feline immunodeficiency virus (FIV), a lentivirus, can lead to an immunodeficiency syndrome after a long incubation period, similar to that associated with the human immunodeficiency virus type 1 (HIV-1). The FIV Rev functional domains have been predicted only by homology with those of HIV-1 Rev. In the present study, the nuclear and nucleolar localization signals (NLS and NoLS, respectively) of the FIV Rev were examined. A series of FIV Rev deletion mutants fused to the enhanced green fluorescent protein (EGFP) were used to localize the NLS in a region spanning amino acids (aa) 81-100. By using alanine substitution mutants, basic residues present between the amino acids (aa) 84-99 of the FIV Rev protein sequence were identified to form the NLS, whereas those between aa 82-95 were associated with the NoLS function. These results further enhance our understanding of how Rev exerts its role in the replication cycle of lentiviruses.

The Jembrana disease virus Rev protein: Identification of nuclear and novel lentiviral nucleolar localization and nuclear export signals.

The lentiviral Rev protein, which is a regulatory protein essential for virus replication, has been first studied in the human immunodeficiency virus type 1 (HIV-1). The main function of Rev is to mediate the nuclear exportation of viral RNAs. To fulfill its function, Rev shuttles between the cytoplasm and the nucleus. The Jembrana disease virus (JDV), a lentivirus, is the etiologic agent of the Jembrana disease which was first described in Bali cattle in Indonesia in 1964. Despite the high mortality rate associated with JDV, this virus remains poorly studied. Herein the subcellular distribution of JDV Rev, the nuclear and nucleolar localization signals (NLS and NoLS, respectively) and the nuclear export signal (NES) of the protein were examined. JDV Rev fused to the enhanced green fluorescent protein (EGFP) predominantly localized to the cytoplasm and nucleolus of transfected cells, as determined by fluorescence microscopy analyses. Through transfection of a series of deletion mutants of JDV Rev, it was possible to localize the NLS/NoLS region between amino acids (aa) 74 to 105. By substituting basic residues with alanine within this sequence, we demonstrated that the JDV Rev NLS encompasses aa 76 to 86, and is exclusively composed of arginine residues, whereas a bipartite NoLS was observed for the first time in any retroviral Rev/Rev-like proteins. Finally, a NES was identified downstream of the NLS/NoLS and encompasses aa 116 to 128 of the JDV Rev protein. The JDV Rev NES was found to be of the protein kinase A inhibitor (PKI) class instead of the HIV-1 Rev class. It also corresponds to the most optimal consensus sequence of PKI NES and, as such, is novel among lentiviral Rev NES.

How Many Mammalian Reovirus Proteins are involved in the Control of the Interferon Response?

As with most viruses, mammalian reovirus can be recognized and attacked by the host-cell interferon response network. Similarly, many viruses have developed resistance mechanisms to counteract the host-cell response at different points of this response. Reflecting the complexity of the interferon signaling pathways as well as the resulting antiviral response, viruses can-and often have-evolved many determinants to interfere with this innate immune response and allow viral replication. In the last few years, it has been evidenced that mammalian reovirus encodes many different determinants that are involved in regulating the induction of the interferon response or in interfering with the action of interferon-stimulated gene products. In this brief review, we present our current understanding of the different reovirus proteins known to be involved, introduce their postulated modes of action, and raise current questions that may lead to further investigations.

Viral modulation of cellular RNA alternative splicing: A new key player in virus-host interactions?

Upon viral infection, a tug of war is triggered between host cells and viruses to maintain/gain control of vital cellular functions, the result of which will ultimately dictate the fate of the host cell. Among these essential cellular functions, alternative splicing (AS) is an important RNA maturation step that allows exons, or parts of exons, and introns to be retained in mature transcripts, thereby expanding proteome diversity and function. AS is widespread in higher eukaryotes, as it is estimated that nearly all genes in humans are alternatively spliced. Recent evidence has shown that upon infection by numerous viruses, the AS landscape of host-cells is affected. In this review, we summarize recent advances in our understanding of how virus infection impacts the AS of cellular transcripts. We also present various molecular mechanisms allowing viruses to modulate cellular AS. Finally, the functional consequences of these changes in the RNA splicing signatures during virus-host interactions are discussed. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Splicing Regulation/Alternative Splicing.

A single mutation in the mammalian orthoreovirus S1 gene is responsible for increased interferon sensitivity in a virus mutant selected in Vero cells.

In a previous study, a mammalian orthoreovirus mutant was isolated based on its increased ability to infect interferon-defective Vero cells and was referred to as Vero-cells-adapted virus (VeroAV). This virus exhibits reduced ability to resist the antiviral effect of interferon. In the present study, the complete genome sequence of VeroAV was first determined. Reverse genetics was then used to identify a unique mutation on the S1 gene, overlapping the σ1 and σ1 s reading frame, resulting in increased sensitivity to interferon. A virus lacking σ1 s expression consecutive to mutation of its initiation codon was then shown to exhibit a further increase in sensitivity to interferon, supporting the idea that σ1 s is the viral protein responsible. This identification of a new determinant of reovirus sensitivity to interferon gives credentials to the idea that multiple reovirus genes are responsible for the level of interferon induction and susceptibility to the interferon-induced antiviral activities.

Synthesis and Translation of Viral mRNA in Reovirus-Infected Cells: Progress and Remaining Questions.

At the end of my doctoral studies, in 1988, I published a review article on the major steps of transcription and translation during the mammalian reovirus multiplication cycle, a topic that still fascinates me 30 years later. It is in the nature of scientific research to generate further questioning as new knowledge emerges. Our understanding of these fascinating viruses thus remains incomplete but it seemed appropriate at this moment to look back and reflect on our progress and most important questions that still puzzle us. It is also essential of being careful about concepts that seem so well established, but could still be better validated using new approaches. I hope that the few reflections presented here will stimulate discussions and maybe attract new investigators into the field of reovirus research. Many other aspects of the viral multiplication cycle would merit our attention. However, I will essentially limit my discussion to these central aspects of the viral cycle that are transcription of viral genes and their phenotypic expression through the host cell translational machinery. The objective here is not to review every aspect but to put more emphasis on important progress and challenges in the field.

Multiple proteins differing between laboratory stocks of mammalian orthoreoviruses affect both virus sensitivity to interferon and induction of interferon production during infection.

In the course of previous works, it was observed that the virus laboratory stock (T3DS) differs in sequence from the virus encoded by the ten plasmids currently in use in many laboratories (T3DK), and derived from a different original virus stock. Seven proteins are affected by these sequence differences. In the present study, replication of T3DK was shown to be more sensitive to the antiviral effect of interferon. Infection by the T3DK virus was also shown to induce the production of higher amount of ß and α-interferons compared to T3DS. Two proteins, the µ2 and λ2 proteins, were found to be responsible for increased sensitivity to interferon while both µ2 and λ1 are responsible for increased interferon secretion. Altogether this supports the idea that multiple reovirus proteins are involved in the control of induction of interferon and virus sensitivity to the interferon-induced response. While interrelated, interferon induction and sensitivity can be separated by defined gene combinations. While both µ2 and λ2 were previously suspected of a role in the control of the interferon response, other proteins are also likely involved, as first shown here for λ1. This also further stresses that due caution should be exerted when comparing different virus isolates with different genetic background.

Role of envelope processing and gp41 membrane spanning domain in the formation of human immunodeficiency virus type 1 (HIV-1) fusion-competent envelope glycoprotein complex.

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is directed by the envelope (Env) glycoproteins, which are present on the surface of HIV-1 virion or infected cells in the form of trimers consisting of gp120/gp41 complexes. The surface subunit, gp120, initiates the entry process by interacting sequentially with the CD4 receptor and a co-receptor, thereby inducing a conformational change that allows the transmembrane (TM) gp41 subunit to mediate fusion between viral and target cell membranes. Cleavage of Env into its gp120 and gp41 components is necessary for activation of its fusogenic activity. Here, the gp41 TM glycoprotein was altered by either deleting an isoleucine residue (DeltaI642) in a critical region of its ectodomain or by substituting its membrane spanning domain (MSD) by that of the influenza hemagglutinin (HA) glycoprotein (TM-HA) to examine the contribution of these regions to Env functions. Characterization of these mutant forms of gp41 revealed that they both affected the infectivity of pseudotyped virions, however, through distinct defects in Env functions. While deletion of Ile 642 drastically altered processing of Env, replacement of gp41 MSD by that of HA led to a marked fusion defect even though the TM-HA Env was efficiently processed and incorporated into viral particles. Interestingly, both DeltaI642 and TM-HA Env were found to act as trans dominant-negative mutant of viral infectivity, presumably via their ability to form hetero-oligomers with wild type Env. Together, these results support a previously proposed model whereby all three gp120-gp41 monomers must be cleaved for the Env homo-trimer to function and suggest that the gp41 MSD plays a critical role in the formation of fusion-competent Env trimers.