Silvestrol Inhibits Chikungunya Virus Replication

Silvestrol, a natural compound that is isolated from plants of the genus Aglaia, is a specific inhibitor of the RNA helicase eIF4A, which unwinds RNA secondary structures in 5′-untranslated regions (UTRs) of mRNAs and allows translation. Silvestrol has a broad antiviral activity against multiple RNA virus families. Here, we show that silvestrol inhibits the replication of chikungunya virus (CHIKV), a positive single-stranded RNA virus. Silvestrol delayed the protein synthesis of non-structural (nsPs) and structural proteins, resulting in a delayed innate response to CHIKV infection. Interferon-α induced STAT1 phosphorylation was not inhibited nor did eIF2α become phosphorylated 16 h post infection in the presence of silvestrol. In addition, the host protein shut-off induced by CHIKV infection was decreased in silvestrol-treated cells. Silvestrol acts by limiting the amount of nsPs, and thereby reducing CHIKV RNA replication. From our results, we propose that inhibition of the host helicase eIF4A might have potential as a therapeutic strategy to treat CHIKV infections.

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The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

Cells ◽

10.3390/cells10071828 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1828

Author(s):

Jared Kirui ◽

Yara Abidine ◽

Annasara Lenman ◽

Koushikul Islam ◽

Yong-Dae Gwon ◽

...

Keyword(s):

Chikungunya Virus ◽

Molecular Mechanisms ◽

Rna Virus ◽

Ectopic Expression ◽

Point Mutations ◽

Cell Entry ◽

Target Cells ◽

Human Hepatoma Cells ◽

Chikv Infection ◽

Phosphatidylserine Receptor

Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.

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Sensitivity of Alphaviruses to G3BP Deletion Correlates with Efficiency of Replicase Polyprotein Processing

Journal of Virology ◽

10.1128/jvi.01681-19 ◽

2020 ◽

Vol 94 (7) ◽

Cited By ~ 2

Author(s):

Benjamin Götte ◽

Age Utt ◽

Rennos Fragkoudis ◽

Andres Merits ◽

Gerald M. McInerney

Keyword(s):

Chikungunya Virus ◽

Rna Replication ◽

Viral Rna ◽

Host Protein ◽

Regulatory Role ◽

Functional Link ◽

Ongoing Research ◽

Old World ◽

Polyprotein Processing ◽

Viral Replicase

ABSTRACT We present a comprehensive overview of the dependency of several Old World alphaviruses for the host protein G3BP. Based on their replication ability in G3BP-deleted cells, Old World alphaviruses can be categorized into two groups, being either resistant or sensitive to G3BP deletion. We observed that all sensitive viruses have an Arg residue at the P4 position of the cleavage site between the nonstructural protein P1 (nsP1) and nsP2 regions of the replicase precursor polyprotein (1/2 site), while a different residue is found at this site in viruses resistant to G3BP deletion. Swapping this residue between resistant and sensitive viruses also switches the G3BP deletion sensitivity. In the absence of G3BP, chikungunya virus (CHIKV) replication is at the limit of detection. The P4 Arg-to-His substitution partially rescues this defect. The P4 residue of the 1/2 site is known to play a regulatory role during processing at this site, and we found that if processing is blocked, the influence of the P4 residue on the sensitivity to G3BP deletion is abolished. Immunofluorescence experiments with CHIKV replicase with manipulated processing indicate that the synthesis of double-stranded RNA is defective in the absence of G3BP and suggest a role of G3BP during negative-strand RNA synthesis. This study provides a functional link between the host protein G3BP and the P4 residue of the 1/2 site for viral RNA replication of Old World alphaviruses. While this suggests a link between G3BP proteins and viral replicase polyprotein processing, we propose that G3BP proteins do not have a regulatory role during polyprotein processing. IMPORTANCE Old World alphaviruses comprise several medically relevant viruses, including chikungunya virus and Ross River virus. Recurrent outbreaks and the lack of antivirals and vaccines demand ongoing research to fight the emergence of these infectious diseases. In this context, a thorough investigation of virus-host interactions is critical. Here, we highlight the importance of the host protein G3BP for several Old World alphaviruses. Our data strongly suggest that G3BP plays a crucial role for the activity of the viral replicase and, thus, the amplification of the viral RNA genome. To our knowledge, the present work is the first to provide a functional link between the regulation of viral polyprotein processing and RNA replication and a host factor for alphaviruses. Moreover, the results of this study raise several questions about the fundamental regulatory mechanisms that dictate the activity of the viral replicase, thereby paving the way for future studies.

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BHK-21 Cell Clones Differ in Chikungunya Virus Infection and MXRA8 Receptor Expression

Viruses ◽

10.3390/v13060949 ◽

2021 ◽

Vol 13 (6) ◽

pp. 949

Author(s):

Peiqi Yin ◽

Margaret Kielian

Keyword(s):

Cell Lines ◽

Primary Infection ◽

Sindbis Virus ◽

Chikungunya Virus ◽

Mrna Level ◽

Receptor Expression ◽

Chikv Infection ◽

Cell Clones ◽

Clonal Cell Lines ◽

Exogenous Expression

Baby hamster kidney-21 (BHK-21) cells are widely used to propagate and study many animal viruses using infection and transfection techniques. Among various BHK-21 cell clones, the fibroblast-like BHK-21/C-13 line and the epithelial-like BHK-21/WI-2 line are commonly used cell clones for alphavirus research. Here we report that BHK-21/WI-2 cells were significantly less susceptible to primary infection by the alphavirus chikungunya virus (CHIKV) than were BHK-21/C-13 cells. The electroporation efficiency of alphavirus RNA into BHK-21/WI-2 was also lower than that of BHK-21/C-13. The growth of CHIKV was decreased in BHK-21/WI-2 compared to BHK-21/C-13, while primary infection and growth of the alphavirus Sindbis virus (SINV) were equivalent in the two cell lines. Our results suggested that CHIKV entry could be compromised in BHK-21/WI-2. Indeed, we found that the mRNA level of the CHIKV receptor MXRA8 in BHK-21/WI-2 cells was much lower than that in BHK-21/C-13 cells, and exogenous expression of either human MXRA8 or hamster MXRA8 rescued CHIKV infection. Our results affirm the importance of the MXRA8 receptor for CHIKV infection, and document differences in its expression in two clonal cell lines derived from the original BHK-21 cell cultures. Our results also indicate that CHIKV propagation and entry studies in BHK-21 cells will be significantly more efficient in BHK-21/C-13 than in BHK-21/WI-2 cells.

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Insights into Antibody-Mediated Alphavirus Immunity and Vaccine Development Landscape

Microorganisms ◽

10.3390/microorganisms9050899 ◽

2021 ◽

Vol 9 (5) ◽

pp. 899

Author(s):

Anthony Torres-Ruesta ◽

Rhonda Sin-Ling Chee ◽

Lisa F.P. Ng

Keyword(s):

Diagnostic Tests ◽

Inflammatory Arthritis ◽

Chikungunya Virus ◽

Vaccine Development ◽

Antibody Responses ◽

Chikv Infection ◽

Wide Range ◽

Susceptible Populations ◽

Humoral Responses

Alphaviruses are mosquito-borne pathogens distributed worldwide in tropical and temperate areas causing a wide range of symptoms ranging from inflammatory arthritis-like manifestations to the induction of encephalitis in humans. Historically, large outbreaks in susceptible populations have been recorded followed by the development of protective long-lasting antibody responses suggesting a potential advantageous role for a vaccine. Although the current understanding of alphavirus antibody-mediated immunity has been mainly gathered in natural and experimental settings of chikungunya virus (CHIKV) infection, little is known about the humoral responses triggered by other emerging alphaviruses. This knowledge is needed to improve serology-based diagnostic tests and the development of highly effective cross-protective vaccines. Here, we review the role of antibody-mediated immunity upon arthritogenic and neurotropic alphavirus infections, and the current research efforts for the development of vaccines as a tool to control future alphavirus outbreaks.

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Native Structure-Based Peptides as Potential Protein–Protein Interaction Inhibitors of SARS-CoV-2 Spike Protein and Human ACE2 Receptor

Molecules ◽

10.3390/molecules26082157 ◽

2021 ◽

Vol 26 (8) ◽

pp. 2157

Author(s):

Norbert Odolczyk ◽

Ewa Marzec ◽

Maria Winiewska-Szajewska ◽

Jarosław Poznański ◽

Piotr Zielenkiewicz

Keyword(s):

Drug Development ◽

Protein Interactions ◽

Rna Virus ◽

Antiviral Drug ◽

Host Protein ◽

Host Cells ◽

Cell Surface Receptor ◽

Short Peptides ◽

Virus Protein ◽

Positive Strand Rna

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a positive-strand RNA virus that causes severe respiratory syndrome in humans, which is now referred to as coronavirus disease 2019 (COVID-19). Since December 2019, the new pathogen has rapidly spread globally, with over 65 million cases reported to the beginning of December 2020, including over 1.5 million deaths. Unfortunately, currently, there is no specific and effective treatment for COVID-19. As SARS-CoV-2 relies on its spike proteins (S) to bind to a host cell-surface receptor angiotensin-converting enzyme-2(ACE2), and this interaction is proved to be responsible for entering a virus into host cells, it makes an ideal target for antiviral drug development. In this work, we design three very short peptides based on the ACE2 sequence/structure fragments, which may effectively bind to the receptor-binding domain (RBD) of S protein and may, in turn, disrupt the important virus-host protein–protein interactions, blocking early steps of SARS-CoV-2 infection. Two of our peptides bind to virus protein with affinity in nanomolar range, and as very short peptides have great potential for drug development.

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Synthesis and Biological Evaluation of Hydroxylated Monocarbonyl Curcumin Derivatives as Potential Inducers of Neprilysin Activity

Biomedicines ◽

10.3390/biomedicines9080955 ◽

2021 ◽

Vol 9 (8) ◽

pp. 955

Author(s):

Dimitris Matiadis ◽

See-Ting Ng ◽

Eric H.-L. Chen ◽

Georgia Nigianni ◽

Veroniki P. Vidali ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Therapeutic Strategy ◽

Specific Inhibitor ◽

Biological Evaluation ◽

Lead Compound ◽

Cleavage Activity ◽

Curcumin Derivatives ◽

Aβ Clearance ◽

Activity Enhancement ◽

Synthesis And Biological Evaluation

Background: Alzheimer’s disease (AD) involves impairment of Aβ clearance. Neprilysin (NEP) is the most efficient Aβ peptidase. Enhancement of the activity or expression of NEP may provide a prominent therapeutic strategy against AD. Aims: Ten hydroxylated monocarbonyl curcumin derivatives were designed, synthesized and evaluated for their NEP upregulating potential using sensitive fluorescence-based Aβ digestion and inhibition assays. Results: Compound 4 was the most active one, resulting in a 50% increase in Aβ cleavage activity. Cyclohexanone-bearing derivatives exhibited higher activity enhancement compared to their acetone counterparts. Inhibition experiments with the NEP-specific inhibitor thiorphan resulted in dramatic cleavage reduction. Conclusion: The increased Aβ cleavage activity and the ease of synthesis of 4 renders it an extremely attractive lead compound.

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Epidemiological Evidence for Lineage-Specific Differences in the Risk of Inapparent Chikungunya Virus Infection

Journal of Virology ◽

10.1128/jvi.01622-18 ◽

2018 ◽

Vol 93 (4) ◽

Cited By ~ 4

Author(s):

Fausto Bustos Carrillo ◽

Damaris Collado ◽

Nery Sanchez ◽

Sergio Ojeda ◽

Brenda Lopez Mercado ◽

...

Keyword(s):

At Risk ◽

Chikungunya Virus ◽

Literature Search ◽

Systematic Literature Search ◽

Epidemiological Evidence ◽

Chikv Infection ◽

Adjusted Risk ◽

Household Contacts ◽

Literature Reviews ◽

Asymptomatic Infections

ABSTRACTIn late 2013, chikungunya virus (CHIKV) was introduced into the Americas, leading to widespread epidemics. A large epidemic caused by the Asian chikungunya virus (CHIKV) lineage occurred in Managua, Nicaragua, in 2015. Literature reviews commonly state that the proportion of inapparent CHIKV infections ranges from 3 to 28%. This study estimates the ratio of symptomatic to asymptomatic CHIKV infections and identifies risk factors of infection. In October to November 2015, 60 symptomatic CHIKV-infected children were enrolled as index cases and prospectively monitored, alongside 236 household contacts, in an index cluster study. Samples were collected upon enrollment and on day 14 or 35 and tested by real-time reverse transcription-PCR (rRT-PCR), IgM capture enzyme-linked immunosorbent assays (IgM-ELISAs), and inhibition ELISAs to detect pre- and postenrollment CHIKV infections. Of 236 household contacts, 55 (23%) had experienced previous or very recent infections, 41 (17%) had active infections at enrollment, and 21 (9%) experienced incident infections. Vehicle ownership (multivariable-adjusted risk ratio [aRR], 1.58) increased the risk of CHIKV infection, whereas ≥4 municipal trash collections/week (aRR, 0.38) and having externally piped water (aRR, 0.52) protected against CHIKV infection. Among 63 active and incident infections, 31 (49% [95% confidence interval {CI}, 36%, 62%]) were asymptomatic, yielding a ratio of symptomatic to asymptomatic infections of 1:0.97 (95% CI, 1:0.56, 1:1.60). Although our estimate is outside the 3% to 28% range reported previously, Bayesian and simulation analyses, informed by a systematic literature search, suggested that the proportion of inapparent CHIKV infections is lineage dependent and that more inapparent infections are associated with the Asian lineage than the East/Central/South African (ECSA) lineage. Overall, these data substantially improve knowledge regarding chikungunya epidemics.IMPORTANCEChikungunya virus (CHIKV) is an understudied threat to human health. During the 2015 chikungunya epidemic in Managua, Nicaragua, we estimated the ratio of symptomatic to asymptomatic CHIKV infections, which is important for understanding transmission dynamics and the public health impact of CHIKV. This index cluster study identified and monitored persons at risk of infection, enabling capture of asymptomatic infections. We estimated that 31 (49%) of 63 at-risk participants had asymptomatic CHIKV infections, which is significantly outside the 3% to 28% range reported in literature reviews. However, recent seroprevalence studies, including two large pediatric cohort studies in the same setting, had also found percentages of inapparent infections outside the 3% to 28% range. Bayesian and simulation analyses, informed by a systematic literature search, revealed that the percentage of inapparent infections in epidemic settings varies by CHIKV phylogenetic lineage. Our study quantifies and provides the first epidemiological evidence that chikungunya epidemic characteristics are strongly influenced by CHIKV lineage.

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Rheumatic manifestations of Chikungunya virus infection: Prevalence, patterns, and enthesitis

PLoS ONE ◽

10.1371/journal.pone.0249867 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0249867

Author(s):

Saovanee Benjamanukul ◽

Manathip Osiri ◽

Jira Chansaenroj ◽

Chintana Chirathaworn ◽

Yong Poovorawan

Keyword(s):

Chikungunya Virus ◽

Inflammatory Rheumatic Diseases ◽

Infection Prevalence ◽

Chikv Infection ◽

Lateral Epicondyle ◽

Factors Associated ◽

Response Parameter ◽

Rheumatic Manifestations ◽

The Mean ◽

Rheumatic Symptoms

Chikungunya virus (CHIKV) is an arthropod-borne virus transmitted by mosquitoes of the genus Aedes. CHIKV infection causes various rheumatic symptoms, including enthesitis; however, these effects are rarely investigated. The aim of this study was to describe the rheumatic manifestations in CHIKV infection, estimate the prevalence of enthesitis in CHIKV-infected patients, and determine the factors associated with CHIKV-induced enthesitis. We conducted a prospective, observational study in patients with CHIKV infection confirmed by positive RT-PCR or IgM assay from October 2019 to March 2020. Patients with pre-existing inflammatory rheumatic diseases were excluded. A rheumatologist evaluated the demographic and clinical characteristics of the patients, including the number of inflamed joints, enthesitis sites, tendinitis, and tenosynovitis. The Leeds enthesitis index (LEI) and the Maastricht ankylosing spondylitis enthesis score (MASES) were used to evaluate enthesitis sites. Factors associated with enthesitis were determined using logistic regression analysis. One hundred and sixty-four participants diagnosed with CHIKV infection were enrolled. The mean (SD) age of the patients was 48.2 (14) years. The most common pattern of rheumatic manifestations was polyarthritis with or without enthesitis. Enthesitis was observed in 63 patients (38.4%). The most common site of enthesitis was the left lateral epicondyle as assessed by LEI and the posterior superior iliac spine as assessed by MASES. Multivariate analysis indicated that the number of actively inflamed joints and Thai-HAQ score at the initial evaluation were significantly associated with the presence of enthesitis. The main rheumatic manifestations of CHIKV infection were arthritis/arthralgia, with enthesitis as a prominent extraarticular feature. CHIKV infection can cause enthesitis at peripheral and axial sites. We found that enthesitis was associated with a high number of inflamed joints and reduced physical function. These results indicate that the assessment of enthesitis should be considered when monitoring disease activity and as a treatment response parameter in CHIKV-infected patients.

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RNA-Protein Interaction Analysis of SARS-CoV-2 5′ and 3′ Untranslated Regions Reveals a Role of Lysosome-Associated Membrane Protein-2a during Viral Infection

mSystems ◽

10.1128/msystems.00643-21 ◽

2021 ◽

Author(s):

Rohit Verma ◽

Sandhini Saha ◽

Shiv Kumar ◽

Shailendra Mani ◽

Tushar Kanti Maiti ◽

...

Keyword(s):

Interaction Analysis ◽

Rna Virus ◽

Untranslated Regions ◽

Host Proteins ◽

Positive Strand ◽

Replication Complex ◽

Positive Strand Rna Virus ◽

Positive Strand Rna ◽

Viral Genomic Rna

Replication of a positive-strand RNA virus involves an RNA-protein complex consisting of viral genomic RNA, host RNA(s), virus-encoded proteins, and host proteins. Dissecting out individual components of the replication complex will help decode the mechanism of viral replication. 5′ and 3′ UTRs in positive-strand RNA viruses play essential regulatory roles in virus replication.

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Distinct Poly(rC) Binding Protein KH Domain Determinants for Poliovirus Translation Initiation and Viral RNA Replication

Journal of Virology ◽

10.1128/jvi.76.23.12008-12022.2002 ◽

2002 ◽

Vol 76 (23) ◽

pp. 12008-12022 ◽

Cited By ~ 100

Author(s):

Brandon L. Walter ◽

Todd B. Parsley ◽

Ellie Ehrenfeld ◽

Bert L. Semler

Keyword(s):

Translation Initiation ◽

Rna Synthesis ◽

Rna Binding ◽

Binding Protein ◽

Rna Replication ◽

Viral Rna ◽

Host Protein ◽

Loop Structure ◽

Stem Loop ◽

Stem Loop Structure

ABSTRACT The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5′-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.

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