scholarly journals Phosphorylation Sites in the Hypervariable Domain in Chikungunya Virus nsP3 Are Crucial for Viral Replication

2021 ◽  
Vol 95 (9) ◽  
Author(s):  
Mona Teppor ◽  
Eva Žusinaite ◽  
Andres Merits
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Sindbis Virus ◽  
Chikungunya Virus ◽  
Semliki Forest Virus ◽  
Virus Infectivity ◽  
Valuable Insight ◽  
Phosphorylation Sites ◽  
Nonstructural Proteins ◽  
Host Proteins

ABSTRACT Chikungunya virus (CHIKV, family Togaviridae) is a mosquito-transmitted alphavirus. The positive-sense RNA genome of CHIKV encodes four nonstructural proteins (nsP1 to nsP4) that are virus-specific subunits of the RNA replicase. Among nsP functions, those of nsP3 are the least understood. The C-terminal hypervariable domain (HVD) in nsP3 is disordered and serves as a platform for interactions with multiple host proteins. For Sindbis virus (SINV) and Semliki Forest virus (SFV), the nsP3 HVD has been shown to be phosphorylated. Deletion of phosphorylated regions has a mild effect on the growth of SFV and SINV in vertebrate cells. Using radiolabeling, we demonstrated that nsP3 in CHIKV and o’nyong-nyong virus is also phosphorylated. We showed that the phosphorylated residues in CHIKV nsP3 are not clustered at the beginning of the HVD. The substitution of 20 Ser/Thr residues located in the N-terminal half of the HVD or 26 Ser/Thr residues located in its C-terminal half with Ala residues reduced the activity of the CHIKV replicase and the infectivity of CHIKV in mammalian cells. Furthermore, the substitution of all 46 potentially phosphorylated residues resulted in the complete loss of viral RNA synthesis and infectivity. The mutations did not affect the interaction of the HVD in nsP3 with the host G3BP1 protein; interactions with CD2AP, BIN1, and FHL1 proteins were significantly reduced but not abolished. Thus, CHIKV differs from SFV and SINV both in the location of the phosphorylated residues in the HVD in nsP3 and, significantly, in their effect on replicase activity and virus infectivity. IMPORTANCE CHIKV outbreaks have affected millions of people, creating a need for the development of antiviral approaches. nsP3 is a component of the CHIKV RNA replicase and is involved in interactions with host proteins and signaling cascades. Phosphorylation of the HVD in nsP3 is important for the virulent alphavirus phenotype. Here, we demonstrate that nsP3 in CHIKV is phosphorylated and that the phosphorylation sites in the HVD are distributed in a unique pattern. Furthermore, the abrogation of some of the phosphorylation sites results in the attenuation of CHIKV, while abolishing all the phosphorylation sites completely blocked its replicase activity. Thus, the phosphorylation of nsP3 and/or the phosphorylation sites in nsP3 have a major impact on CHIKV infectivity. Therefore, they represent promising targets for antiviral compounds and CHIKV attenuation. In addition, this new information offers valuable insight into the vast network of virus-host interactions.

scholarly journals Proteomic Discovery of VEEV E2-Host Partner Interactions Identifies GRP78 Inhibitor HA15 as a Potential Therapeutic for Alphavirus Infections

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 283
Author(s):  
Michael D. Barrera ◽  
Victoria Callahan ◽  
Ivan Akhrymuk ◽  
Nishank Bhalla ◽  
Weidong Zhou ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Trafficking ◽  
Viral Protein ◽  
Sindbis Virus ◽  
Encephalitis Virus ◽  
Host Proteins ◽  
Atp Production ◽  
Equine Encephalitis Virus ◽  
E2 Glycoprotein ◽  
Late Stages

Alphaviruses are a genus of the Togaviridae family and are widely distributed across the globe. Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), cause encephalitis and neurological sequelae while chikungunya virus (CHIKV) and Sindbis virus (SINV) cause arthralgia. There are currently no approved therapeutics or vaccines available for alphaviruses. In order to identify novel therapeutics, a V5 epitope tag was inserted into the N-terminus of the VEEV E2 glycoprotein and used to identify host-viral protein interactions. Host proteins involved in protein folding, metabolism/ATP production, translation, cytoskeleton, complement, vesicle transport and ubiquitination were identified as VEEV E2 interactors. Multiple inhibitors targeting these host proteins were tested to determine their effect on VEEV replication. The compound HA15, a GRP78 inhibitor, was found to be an effective inhibitor of VEEV, EEEV, CHIKV, and SINV. VEEV E2 interaction with GRP78 was confirmed through coimmunoprecipitation and colocalization experiments. Mechanism of action studies found that HA15 does not affect viral RNA replication but instead affects late stages of the viral life cycle, which is consistent with GRP78 promoting viral assembly or viral protein trafficking.

scholarly journals Effects of Palmitoylation of Replicase Protein nsP1 on Alphavirus Infection

2000 ◽  
Vol 74 (15) ◽  
pp. 6725-6733 ◽  
Author(s):  
Tero Ahola ◽  
Pekka Kujala ◽  
Minna Tuittila ◽  
Titta Blom ◽  
Pirjo Laakkonen ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Semliki Forest Virus ◽  
Rna Replication ◽  
Cell Types ◽  
Alkaline Solutions ◽  
Nonstructural Proteins ◽  
Replication Complex ◽  
Replication Sites ◽  
Infected Cells ◽  
Alphavirus Infection

ABSTRACT The membrane-associated alphavirus RNA replication complex contains four virus-encoded subunits, the nonstructural proteins nsP1 to nsP4. Semliki Forest virus (SFV) nsP1 is hydrophobically modified by palmitoylation of cysteines 418 to 420. Here we show that Sindbis virus nsP1 is also palmitoylated on the same site (cysteine 420). When mutations preventing nsP1 palmitoylation were introduced into the genomes of these two alphaviruses, the mutant viruses remained viable and replicated to high titers, although their growth was slightly delayed. The subcellular distribution of palmitoylation-defective nsP1 was altered in the mutant: it no longer localized to filopodial extensions, and a fraction of it was soluble. The ultrastructure of the alphavirus replication sites appeared normal, and the localization of the other nonstructural proteins was unaltered in the mutants. In both wild-type- and mutant-virus-infected cells, SFV nsP3 and nsP4 could be extracted from membranes only by alkaline solutions whereas the nsP2-membrane association was looser. Thus, the membrane binding properties of the alphavirus RNA replication complex were not determined by the palmitoylation of nsP1. The nsP1 palmitoylation-defective alphaviruses produced normal plaques in several cell types, but failed to give rise to plaques in HeLa cells, although they induced normal apoptosis of these cells. The SFV mutant was apathogenic in mice: it caused blood viremia, but no infectious virus was detected in the brain.

scholarly journals Molecular Defects Caused by Temperature-Sensitive Mutations in Semliki Forest Virus nsP1

2008 ◽  
Vol 82 (18) ◽  
pp. 9236-9244 ◽  
Author(s):  
Valeria Lulla ◽  
Dorothea L. Sawicki ◽  
Stanley G. Sawicki ◽  
Aleksei Lulla ◽  
Andres Merits ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Minus Strand ◽  
Nonstructural Proteins ◽  
Mutant Proteins ◽  
Recombinant Viruses ◽  
Mrna Capping

ABSTRACT Alphavirus replicase protein nsP1 has multiple functions during viral RNA synthesis. It catalyzes methyltransferase and guanylyltransferase activities needed in viral mRNA capping, attaches the viral replication complex to cytoplasmic membranes, and is required for minus-strand RNA synthesis. Two temperature-sensitive (ts) mutations in Semliki Forest virus (SFV) were previously identified within nsP1: ts10 (E529D) and ts14 (D119N). Recombinant viruses containing these individual mutations reproduced the features of the original ts strains. We now find that the capping-associated enzymatic activities of recombinant nsP1, containing ts10 or ts14 lesions, were not ts. The mutant proteins and polyproteins also were membrane bound, mutant nsP1 interacted normally with the other nonstructural proteins, and there was no major defect in nonstructural polyprotein processing in the mutants, although ts14 surprisingly displayed slightly retarded processing. The two mutant viruses were specifically defective in minus-strand RNA synthesis at the restrictive temperature. Integrating data from SFV and Sindbis virus, we discuss the domain structure of nsP1 and the relative positioning of and interactions between the replicase proteins. nsP1 is suggested to contain a specific subdomain involved in minus-strand synthesis and interaction with the polymerase nsP4 and the protease nsP2.

scholarly journals Human DICER helicase domain recruits PKR and modulates its antiviral activity

2021 ◽  
Vol 17 (5) ◽  
pp. e1009549
Author(s):  
Thomas C. Montavon ◽  
Morgane Baldaccini ◽  
Mathieu Lefèvre ◽  
Erika Girardi ◽  
Béatrice Chane-Woon-Ming ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Mammalian Cells ◽  
Sindbis Virus ◽  
Rna Binding ◽  
Semliki Forest Virus ◽  
Rna Binding Proteins ◽  
Helicase Domain ◽  
Type I ◽  
Dependent Manner ◽  
Rna Helicases

The antiviral innate immune response mainly involves type I interferon (IFN) in mammalian cells. The contribution of the RNA silencing machinery remains to be established, but several recent studies indicate that the ribonuclease DICER can generate viral siRNAs in specific conditions. It has also been proposed that type I IFN and RNA silencing could be mutually exclusive antiviral responses. In order to decipher the implication of DICER during infection of human cells with alphaviruses such as the Sindbis virus and Semliki forest virus, we determined its interactome by proteomics analysis. We show that DICER specifically interacts with several double-stranded RNA binding proteins and RNA helicases during viral infection. In particular, proteins such as DHX9, ADAR-1 and the protein kinase RNA-activated (PKR) are enriched with DICER in virus-infected cells. We demonstrate that the helicase domain of DICER is essential for this interaction and that its deletion confers antiviral properties to this protein in an RNAi-independent, PKR-dependent, manner.

scholarly journals NAP1L1 and NAP1L4 binding to Hypervariable Domain of Chikungunya Virus nsP3 Protein is bivalent and requires phosphorylation

2021 ◽  
Author(s):  
Francisco Dominguez ◽  
Nikita Shiliaev ◽  
Tetyana Lukash ◽  
Peter Agback ◽  
Oksana Palchevska ◽  
...  
Keyword(s):  
Viral Replication ◽  
Chikungunya Virus ◽  
Family Members ◽  
Host Factors ◽  
Nonstructural Proteins ◽  
Host Proteins ◽  
Chikv Infection ◽  
Binding Motifs ◽  
Specific Host ◽  
Wide Range

Chikungunya virus (CHIKV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Within the last two decades, CHIKV has expanded its presence to both hemispheres and is currently circulating in both Old and New Worlds. Despite the severity and persistence of the arthritis it causes in humans, no approved vaccines or therapeutic means have been developed for CHIKV infection. Replication of alphaviruses, including CHIKV, is determined not only by their nonstructural proteins, but also by a wide range of host factors, which are indispensable components of viral replication complexes (vRCs). Alphavirus nsP3s contain hypervariable domains (HVDs), which encode multiple motifs that drive recruitment of cell- and virus-specific host proteins into vRCs. Our previous data suggested that NAP1 family members are a group of host factors that may interact with CHIKV nsP3 HVD. In this study, we performed a detailed investigation of the NAP1 function in CHIKV replication in vertebrate cells. Our data demonstrate that i) the NAP1-HVD interactions have strong stimulatory effects on CHIKV replication; ii) both NAP1L1 and NAP1L4 interact with the CHIKV HVD; iii) NAP1 family members interact with two motifs, which are located upstream and downstream of the G3BP-binding motifs of CHIKV HVD; iv) NAP1 proteins interact only with a phosphorylated form of CHIKV HVD and HVD phosphorylation is mediated by CK2 kinase; v) NAP1 and other families of host factors redundantly promote CHIKV replication and their bindings have additive stimulatory effects on viral replication.

scholarly journals Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells

2008 ◽  
Vol 82 (20) ◽  
pp. 10088-10101 ◽  
Author(s):  
Rodion Gorchakov ◽  
Natalia Garmashova ◽  
Elena Frolova ◽  
Ilya Frolov
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Sindbis Virus ◽  
Protein Complexes ◽  
Nonstructural Proteins ◽  
Cell Nuclei ◽  
High Concentration ◽  
Mosquito Cells ◽  
Infected Cells ◽  
Rna Complexes

ABSTRACT Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions.

scholarly journals Mutations in the Endodomain of Sindbis Virus Glycoprotein E2 Define Sequences Critical for Virus Assembly

2006 ◽  
Vol 80 (9) ◽  
pp. 4458-4468 ◽  
Author(s):  
John West ◽  
Raquel Hernandez ◽  
Davis Ferreira ◽  
Dennis T. Brown
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Sindbis Virus ◽  
Insect Cells ◽  
Virus Assembly ◽  
Virus Production ◽  
Initial Step ◽  
Membrane Composition ◽  
Blood Sucking

ABSTRACT Envelopment of Sindbis virus at the plasma membrane is a multistep process in which an initial step is the association of the E2 protein via a cytoplasmic endodomain with the preassembled nucleocapsid. Sindbis virus is vectored in nature by blood-sucking insects and grows efficiently in a number of avian and mammalian vertebrate hosts. The assembly of Sindbis virus, therefore, must occur in two very different host cell environments. Mammalian cells contain cholesterol which insect membranes lack. This difference in membrane composition may be critical in determining what requirements are placed on the E2 tail for virus assembly. To examine the interaction between the E2 tail and the nucleocapsid in Sindbis virus, we have produced substitutions and deletions in a region of the E2 tail (E2 amino acids 408 to 415) that is initially integrated into the endoplasmic reticulum. This sequence was identified as being critical for nucleocapsid binding in an in vitro peptide protection assay. The effects of these mutations on virus assembly and function were determined in both vertebrate and invertebrate cells. Amino acid substitutions (at positions E2: 408, 410, 411, and 413) reduced infectious virus production in a position-dependent fashion but were not efficient in disrupting assembly in mammalian cells. Deletions in the E2 endodomain (Δ406-407, Δ409-411, and Δ414-417) resulted in the failure to assemble virions in mammalian cells. Electron microscopy of BHK cells transfected with these mutants revealed assembly of nucleocapsids that failed to attach to membranes. However, introduction of these deletion mutants into insect cells resulted in the assembly of virus-like particles but no assayable infectivity. These data help define protein interactions critical for virus assembly and suggest a fundamental difference between Sindbis virus assembly in mammalian and insect cells.

scholarly journals Chikungunya Virus Nonstructural Protein 2 Inhibits Type I/II Interferon-Stimulated JAK-STAT Signaling

2010 ◽  
Vol 84 (20) ◽  
pp. 10877-10887 ◽  
Author(s):  
Jelke J. Fros ◽  
Wen Jun Liu ◽  
Natalie A. Prow ◽  
Corinne Geertsema ◽  
Maarten Ligtenberg ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Sindbis Virus ◽  
Chikungunya Virus ◽  
Nuclear Translocation ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Type I ◽  
Nonstructural Protein 2 ◽  
Stat Signaling ◽  
Host Shutoff

ABSTRACT Chikungunya virus (CHIKV) is an emerging human pathogen transmitted by mosquitoes. Like that of other alphaviruses, CHIKV replication causes general host shutoff, leading to severe cytopathicity in mammalian cells, and inhibits the ability of infected cells to respond to interferon (IFN). Recent research, however, suggests that alphaviruses may have additional mechanisms to circumvent the host's antiviral IFN response. Here we show that CHIKV replication is resistant to inhibition by interferon once RNA replication has been established and that CHIKV actively suppresses the antiviral IFN response by preventing IFN-induced gene expression. Both CHIKV infection and CHIKV replicon RNA replication efficiently blocked STAT1 phosphorylation and/or nuclear translocation in mammalian cells induced by either type I or type II IFN. Expression of individual CHIKV nonstructural proteins (nsPs) showed that nsP2 was a potent inhibitor of IFN-induced JAK-STAT signaling. In addition, mutations in CHIKV-nsP2 (P718S) and Sindbis virus (SINV)-nsP2 (P726S) that render alphavirus replicons noncytopathic significantly reduced JAK-STAT inhibition. This host shutoff-independent inhibition of IFN signaling by CHIKV is likely to have an important role in viral pathogenesis.

scholarly journals Replication of Alphaviruses: A Review on the Entry Process of Alphaviruses into Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Jason Yat-Sing Leung ◽  
Mary Mah-Lee Ng ◽  
Justin Jang Hann Chu
Keyword(s):  
Viral Entry ◽  
Sindbis Virus ◽  
Chikungunya Virus ◽  
Semliki Forest Virus ◽  
Genetic Material ◽  
Endocytic Pathway ◽  
Human Pathogens ◽  
Potential Threat ◽  
Equine Encephalitis Virus ◽  
Alphavirus Entry

Alphaviruses are small, enveloped viruses, ~70 nm in diameter, containing a single-stranded, positive-sense, RNA genome. Viruses belonging to this genus are predominantly arthropod-borne viruses, known to cause disease in humans. Their potential threat to human health was most recently exemplified by the 2005 Chikungunya virus outbreak in La Reunion, highlighting the necessity to understand events in the life-cycle of these medically important human pathogens. The replication and propagation of viruses is dependent on entry into permissive cells. Viral entry is initiated by attachment of virions to cells, leading to internalization, and uncoating to release genetic material for replication and propagation. Studies on alphaviruses have revealed entry via a receptor-mediated, endocytic pathway. In this paper, the different stages of alphavirus entry are examined, with examples from Semliki Forest virus, Sindbis virus, Chikungunya virus, and Venezuelan equine encephalitis virus described.

scholarly journals Antagonism of the Sodium-Potassium ATPase Impairs Chikungunya Virus Infection

mBio ◽  
2016 ◽  
Vol 7 (3) ◽  
Author(s):  
Alison W. Ashbrook ◽  
Anthony J. Lentscher ◽  
Paula F. Zamora ◽  
Laurie A. Silva ◽  
Nicholas A. May ◽  
...  
Keyword(s):  
Virus Infection ◽  
High Throughput ◽  
Cardiac Glycoside ◽  
High Throughput Screening ◽  
Sindbis Virus ◽  
Chikungunya Virus ◽  
Nonstructural Proteins ◽  
Chikv Infection ◽  
Sodium Potassium ◽  
Potassium Atpase

ABSTRACTChikungunya virus (CHIKV) is a reemerging alphavirus that has caused epidemics of fever, arthralgia, and rash worldwide. There are currently no licensed vaccines or antiviral therapies available for the prevention or treatment of CHIKV disease. We conducted a high-throughput, chemical compound screen that identified digoxin, a cardiac glycoside that blocks the sodium-potassium ATPase, as a potent inhibitor of CHIKV infection. Treatment of human cells with digoxin or a related cardiac glycoside, ouabain, resulted in a dose-dependent decrease in infection by CHIKV. Inhibition by digoxin was cell type-specific, as digoxin treatment of either murine or mosquito cells did not diminish CHIKV infection. Digoxin displayed antiviral activity against other alphaviruses, including Ross River virus and Sindbis virus, as well as mammalian reovirus and vesicular stomatitis virus. The digoxin-mediated block to CHIKV and reovirus infection occurred at one or more postentry steps, as digoxin inhibition was not bypassed by fusion of CHIKV at the plasma membrane or infection with cell surface-penetrating reovirus entry intermediates. Selection of digoxin-resistant CHIKV variants identified multiple mutations in the nonstructural proteins required for replication complex formation and synthesis of viral RNA. These data suggest a role for the sodium-potassium ATPase in promoting postentry steps of CHIKV replication and provide rationale for modulation of this pathway as a broad-spectrum antiviral strategy.IMPORTANCEMitigation of disease induced by globally spreading, mosquito-borne arthritogenic alphaviruses requires the development of new antiviral strategies. High-throughput screening of clinically tested compounds provides a rapid means to identify undiscovered, antiviral functions for well-characterized therapeutics and illuminate host pathways required for viral infection. Our study describes the potent inhibition of Chikungunya virus and related alphaviruses by the cardiac glycoside digoxin and demonstrates a function for the sodium-potassium ATPase in Chikungunya virus infection.

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