Multiple Host Factors Interact with the Hypervariable Domain of Chikungunya Virus nsP3 and Determine Viral Replication in Cell-Specific Mode

ABSTRACTAlphaviruses are widely distributed in both hemispheres and circulate between mosquitoes and amplifying vertebrate hosts. Geographically separated alphaviruses have adapted to replication in particular organisms. The accumulating data suggest that this adaptation is determined not only by changes in their glycoproteins but also by the amino acid sequence of the hypervariable domain (HVD) of the alphavirus nsP3 protein. We performed a detailed investigation of chikungunya virus (CHIKV) nsP3 HVD interactions with host factors and their roles in viral replication in vertebrate and mosquito cells. The results demonstrate that CHIKV HVD is intrinsically disordered and binds several distinctive cellular proteins. These host factors include two members of the G3BP family and their mosquito homolog Rin, two members of the NAP1 family, and several SH3 domain-containing proteins. Interaction with G3BP proteins or Rin is an absolute requirement for CHIKV replication, although it is insufficient to solely drive it in either vertebrate or mosquito cells. To achieve a detectable level of virus replication, HVD needs to bind members of at least one more protein family in addition to G3BPs. Interaction with NAP1L1 and NAP1L4 plays a more proviral role in vertebrate cells, while binding of SH3 domain-containing proteins to a proline-rich fragment of HVD is more critical for virus replication in the cells of mosquito origin. Modifications of binding sites in CHIKV HVD allow manipulation of the cell specificity of CHIKV replication. Similar changes may be introduced into HVDs of other alphaviruses to alter their replication in particular cells or tissues.IMPORTANCEAlphaviruses utilize a broad spectrum of cellular factors for efficient formation and function of replication complexes (RCs). Our data demonstrate for the first time that the hypervariable domain (HVD) of chikungunya virus nonstructural protein 3 (nsP3) is intrinsically disordered. It binds at least 3 families of cellular proteins, which play an indispensable role in viral RNA replication. The proteins of each family demonstrate functional redundancy. We provide a detailed map of the binding sites on CHIKV nsP3 HVD and show that mutations in these sites or the replacement of CHIKV HVD by heterologous HVD change cell specificity of viral replication. Such manipulations with alphavirus HVDs open an opportunity for development of new irreversibly attenuated vaccine candidates. To date, the disordered protein fragments have been identified in the nonstructural proteins of many other viruses. They may also interact with a variety of cellular factors that determine critical aspects of virus-host interactions.

Download Full-text

Structural and Functional Characterization of Host FHL1 Protein Interaction with Hypervariable Domain of Chikungunya Virus nsP3 Protein

Journal of Virology ◽

10.1128/jvi.01672-20 ◽

2020 ◽

Vol 95 (1) ◽

Author(s):

Tetyana Lukash ◽

Tatiana Agback ◽

Francisco Dominguez ◽

Nikita Shiliaev ◽

Chetan Meshram ◽

...

Keyword(s):

Viral Replication ◽

Binding Site ◽

Cell Lines ◽

Binding Sites ◽

Chikungunya Virus ◽

Biological Significance ◽

Host Factors ◽

Published Data ◽

Wide Range ◽

Infection Spread

ABSTRACT Decades of insufficient control have resulted in unprecedented spread of chikungunya virus (CHIKV) around the globe, and millions have suffered from the highly debilitating disease. Nevertheless, the current understanding of CHIKV-host interactions and adaptability of the virus to replication in mosquitoes and mammalian hosts is still elusive. Our new study shows that four-and-a-half LIM domain protein (FHL1) is one of the host factors that interact with the hypervariable domain (HVD) of CHIKV nsP3. Unlike G3BPs, FHL1 is not a prerequisite of CHIKV replication, and many commonly used cell lines do not express FHL1. However, its expression has a detectable stimulatory effect(s) on CHIKV replication, and Fhl1 knockout (KO) cell lines demonstrate slower infection spread. Nuclear magnetic resonance (NMR)-based studies revealed that the binding site of FHL1 in CHIKV nsP3 HVD overlaps that of another proviral host factor, CD2AP. The structural data also demonstrated that FHL1-HVD interaction is mostly determined by the LIM1 domain of FHL1. However, it does not mirror binding of the entire protein, suggesting that other LIM domains are involved. In agreement with previously published data, our biological experiments showed that interactions of CHIKV HVD with CD2AP and FHL1 have additive effects on the efficiency of CHIKV replication. This study shows that CHIKV mutants with extensive modifications of FHL1- or both FHL1- and CD2AP-binding sites remain viable and develop spreading infection in multiple cell types. Our study also demonstrated that other members of the FHL family can bind to CHIKV HVD and thus may be involved in viral replication. IMPORTANCE Replication of chikungunya virus (CHIKV) is determined by a wide range of host factors. Previously, we have demonstrated that the hypervariable domain (HVD) of CHIKV nsP3 contains linear motifs that recruit defined families of host proteins into formation of functional viral replication complexes. Now, using NMR-based structural and biological approaches, we have characterized the binding site of the cellular FHL1 protein in CHIKV HVD and defined the biological significance of this interaction. In contrast to previously described binding of G3BP to CHIKV HVD, the FHL1-HVD interaction was found to not be a prerequisite of viral replication. However, the presence of FHL1 has a stimulatory effect on CHIKV infectivity and, subsequently, the infection spread. FHL1 and CD2AP proteins were found to have overlapping binding sites in CHIKV HVD and additive proviral functions. Elimination of the FHL1-binding site in the nsP3 HVD can be used for the development of stable, attenuated vaccine candidates.

Download Full-text

Structural and Functional Characterization of Host FHL1 Protein Interaction with Hypervariable Domain of Chikungunya Virus nsP3 Protein

10.1101/2020.09.29.319343 ◽

2020 ◽

Author(s):

Tetyana Lukash ◽

Tatiana Agback ◽

Francisco Dominguez ◽

Nikita Shiliaev ◽

Chetan Meshram ◽

...

Keyword(s):

Viral Replication ◽

Binding Site ◽

Cell Lines ◽

Binding Sites ◽

Chikungunya Virus ◽

Host Factors ◽

Vaccine Candidates ◽

Positive Effects ◽

Wide Range ◽

Infection Spread

ABSTRACTDecades of insufficient control resulted in unprecedented spread of chikungunya virus (CHIKV) around the globe and millions already suffered from the highly debilitating disease. Nevertheless, the current understanding of CHIKV-host interactions and adaptability of the virus to replication in mosquitoes and mammalian hosts is still elusive. Our new study shows that four-and-a-half LIM domain protein (FHL1) is one of the host factors that interact with hypervariable domain (HVD) of CHIKV nsP3. Unlike G3BPs, FHL1 is not a pre-requisite of CHIKV replication, and many commonly used cell lines do not express FHL1. However, its expression has detectable stimulatory effect(s) on CHIKV replication, and the Fhl1 KO cell lines demonstrate slower infection spread. The NMR-based studies revealed that the binding site of FHL1 in CHIKV nsP3 HVD overlaps with that of another pro-viral host factor, CD2AP. The structural data also demonstrated that FHL1-HVD interaction is mostly determined by LIM1 domain of FHL1. However, it does not mirror binding of the entire protein, suggesting that other LIM domains are involved. In agreement with previously published data, our biological experiments showed that interactions of CHIKV HVD with CD2AP and FHL1 have additive positive effects on the efficiency of CHIKV replication. This study shows that CHIKV mutants with extensive modifications of FHL1- or both FHL1- and CD2AP- binding sites remain viable and develop spreading infection in multiple cell types. Thus, such modifications of HVD may improve live CHIKV vaccine candidates in terms of their safety and stability of the attenuated phenotype.IMPORTANCEReplication of chikungunya virus (CHIKV) is determined by a wide range of host factors. Previously, we have demonstrated that the hypervariable domain (HVD) of CHIKV nsP3 protein contains linear motifs that recruit defined families of host proteins into formation of functional viral replication complexes. Now, using NMR-based structural and biological approaches, we have characterized the binding site of cellular FHL1 protein in CHIKV HVD and defined the biological significance of this interaction. In contrast to previously described binding of G3BP to CHIKV HVD, the FHL1-HVD interaction was found to not be a prerequisite of viral replication. However, the presence of FHL1 has a stimulatory effect on CHIKV infectivity and subsequently, the infection spread. FHL1 and CD2AP proteins were found to have overlapping binding sites in CHIKV HVD and additive pro-viral functions. Elimination of FHL1-binding site in nsP3 HVD can be used for the development of stable, live attenuated vaccine candidates.

Download Full-text

Kaposi's Sarcoma-Associated Herpesvirus ori-Lyt-Dependent DNA Replication: Involvement of Host Cellular Factors

Journal of Virology ◽

10.1128/jvi.01319-07 ◽

2008 ◽

Vol 82 (6) ◽

pp. 2867-2882 ◽

Cited By ~ 57

Author(s):

Yan Wang ◽

Hong Li ◽

Qiyi Tang ◽

Gerd G. Maul ◽

Yan Yuan

Keyword(s):

Dna Replication ◽

Viral Replication ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Cellular Proteins ◽

Cellular Factors ◽

Virally Encoded ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Lytic Dna Replication ◽

Parp 1

ABSTRACT Herpesvirus lytic DNA replication requires both the cis-acting element, the origin, and trans-acting factors, including virally encoded origin-binding protein, DNA replication enzymes, and auxiliary factors. Two lytic DNA replication origins (ori-Lyt) of Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified, and two virally encoded proteins, namely, RTA and K8, have been shown to bind to the origins. In this study, we sought to identify cellular factors that associate with ori-Lyt by using DNA affinity purification and mass spectrometry. This approach led to identification of several cellular proteins that bind to KSHV ori-Lyt. They include topoisomerases (Topo) I and II, MSH2/6, RecQL, poly(ADP-ribose) polymerase I (PARP-1), DNA-PK, Ku86/70 autoantigens, and scaffold attachment factor A (SAF-A). RecQL appears to associate with prereplication complexes and be recruited to ori-Lyt through RTA and K8. Topoisomerases, MSH2, PARP-1, DNA-PK, and Ku86 were not detected in prereplication complexes but were present in replication initiation complexes on ori-Lyt. All these cellular proteins accumulate in viral replication compartments in the nucleus, indicating that these proteins may have a role in viral replication. Topo I and II appear to be essential for viral DNA replication as inhibition of their activities with specific inhibitors (camptothecin and ellipticine) blocked ori-Lyt-dependent DNA replication. Furthermore, inhibition of PARP-1 with chemical inhibitors (3-aminobenzamide and niacinamide) resulted in decreased ori-Lyt-dependent DNA replication, whereas hydroxyurea, which raises PARP-1 activity, caused an increase in the DNA replication, suggesting a positive role for PARP-1 in KSHV lytic DNA replication.

Download Full-text

Network-Guided Discovery of Influenza Virus Replication Host Factors

mBio ◽

10.1128/mbio.02002-18 ◽

2018 ◽

Vol 9 (6) ◽

Cited By ~ 14

Author(s):

Emily E. Ackerman ◽

Eiryo Kawakami ◽

Manami Katoh ◽

Tokiko Watanabe ◽

Shinji Watanabe ◽

...

Keyword(s):

Influenza Virus ◽

Viral Replication ◽

Virus Replication ◽

Drug Targets ◽

Antiviral Drug ◽

Viral Proteins ◽

Host Factors ◽

Ppi Network ◽

Host Proteins ◽

Influenza Virus Replication

ABSTRACTThe positions of host factors required for viral replication within a human protein-protein interaction (PPI) network can be exploited to identify drug targets that are robust to drug-mediated selective pressure. Host factors can physically interact with viral proteins, be a component of virus-regulated pathways (where proteins do not interact with viral proteins), or be required for viral replication but unregulated by viruses. Here, we demonstrate a method of combining human PPI networks with virus-host PPI data to improve antiviral drug discovery for influenza viruses by identifying target host proteins. Analysis shows that influenza virus proteins physically interact with host proteins in network positions significant for information flow, even after the removal of known abundance-degree bias within PPI data. We have isolated a subnetwork of the human PPI network that connects virus-interacting host proteins to host factors that are important for influenza virus replication without physically interacting with viral proteins. The subnetwork is enriched for signaling and immune processes distinct from those associated with virus-interacting proteins. Selecting proteins based on subnetwork topology, we performed an siRNA screen to determine whether the subnetwork was enriched for virus replication host factors and whether network position within the subnetwork offers an advantage in prioritization of drug targets to control influenza virus replication. We found that the subnetwork is highly enriched for target host proteins—more so than the set of host factors that physically interact with viral proteins. Our findings demonstrate that network positions are a powerful predictor to guide antiviral drug candidate prioritization.IMPORTANCEIntegrating virus-host interactions with host protein-protein interactions, we have created a method using these established network practices to identify host factors (i.e., proteins) that are likely candidates for antiviral drug targeting. We demonstrate that interaction cascades between host proteins that directly interact with viral proteins and host factors that are important to influenza virus replication are enriched for signaling and immune processes. Additionally, we show that host proteins that interact with viral proteins are in network locations of power. Finally, we demonstrate a new network methodology to predict novel host factors and validate predictions with an siRNA screen. Our results show that integrating virus-host proteins interactions is useful in the identification of antiviral drug target candidates.

Download Full-text

Susceptibility Genes to Plant Viruses

Viruses ◽

10.3390/v10090484 ◽

2018 ◽

Vol 10 (9) ◽

pp. 484 ◽

Cited By ~ 20

Author(s):

Hernan Garcia-Ruiz

Keyword(s):

Virus Replication ◽

Defense Responses ◽

Plant Viruses ◽

Host Factors ◽

Replication Proteins ◽

Replication Complex ◽

Virion Assembly ◽

Rna Translation ◽

Cellular Factors ◽

Genetically Determined

Plant viruses use cellular factors and resources to replicate and move. Plants respond to viral infection by several mechanisms, including innate immunity, autophagy, and gene silencing, that viruses must evade or suppress. Thus, the establishment of infection is genetically determined by the availability of host factors necessary for virus replication and movement and by the balance between plant defense and viral suppression of defense responses. Host factors may have antiviral or proviral activities. Proviral factors condition susceptibility to viruses by participating in processes essential to the virus. Here, we review current advances in the identification and characterization of host factors that condition susceptibility to plant viruses. Host factors with proviral activity have been identified for all parts of the virus infection cycle: viral RNA translation, viral replication complex formation, accumulation or activity of virus replication proteins, virus movement, and virion assembly. These factors could be targets of gene editing to engineer resistance to plant viruses.

Download Full-text

Temporal SILAC-based quantitative proteomics identifies host factors involved in chikungunya virus replication

PROTEOMICS ◽

10.1002/pmic.201400581 ◽

2015 ◽

Vol 15 (13) ◽

pp. 2267-2280 ◽

Cited By ~ 10

Author(s):

Emmely E. Treffers ◽

Ali Tas ◽

Florine E.M. Scholte ◽

Myrthe N. Van ◽

Matthias T. Heemskerk ◽

...

Keyword(s):

Virus Replication ◽

Quantitative Proteomics ◽

Chikungunya Virus ◽

Host Factors

Download Full-text

Wolbachia wStri Blocks Zika Virus Growth at Two Independent Stages of Viral Replication

mBio ◽

10.1128/mbio.00738-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 17

Author(s):

M. J. Schultz ◽

A. L. Tan ◽

C. N. Gray ◽

S. Isern ◽

S. F. Michael ◽

...

Keyword(s):

Dengue Virus ◽

Viral Replication ◽

Yellow Fever ◽

Virus Replication ◽

Zika Virus ◽

Chikungunya Virus ◽

Yellow Fever Virus ◽

Rna Viruses ◽

Content Type ◽

Infected Cells

ABSTRACTMosquito-transmitted viruses are spread globally and present a great risk to human health. Among the many approaches investigated to limit the diseases caused by these viruses are attempts to make mosquitos resistant to virus infection. Coinfection of mosquitos with the bacteriumWolbachia pipientisfrom supergroup A is a recent strategy employed to reduce the capacity for major vectors in theAedesmosquito genus to transmit viruses, including dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV). Recently, a supergroup BWolbachia wStri, isolated fromLaodelphax striatellus, was shown to inhibit multiple lineages of ZIKV inAedes albopictuscells. Here, we show thatwStri blocks the growth of positive-sense RNA viruses DENV, CHIKV, ZIKV, and yellow fever virus by greater than 99.9%.wStri presence did not affect the growth of the negative-sense RNA viruses LaCrosse virus or vesicular stomatitis virus. Investigation of the stages of the ZIKV life cycle inhibited bywStri identified two distinct blocks in viral replication. We found a reduction of ZIKV entry intowStri-infected cells. This was partially rescued by the addition of a cholesterol-lipid supplement. Independent of entry, transfected viral genome was unable to replicate inWolbachia-infected cells. RNA transfection and metabolic labeling studies suggested that this replication defect is at the level of RNA translation, where we saw a 66% reduction in mosquito protein synthesis inwStri-infected cells. This study’s findings increase the potential for application ofwStri to block additional arboviruses and also identify specific blocks in viral infection caused byWolbachiacoinfection.IMPORTANCEDengue, Zika, and yellow fever viruses are mosquito-transmitted diseases that have spread throughout the world, causing millions of infections and thousands of deaths each year. Existing programs that seek to contain these diseases through elimination of the mosquito population have so far failed, making it crucial to explore new ways of limiting the spread of these viruses. Here, we show that introduction of an insect symbiont,Wolbachia wStri, into mosquito cells is highly effective at reducing yellow fever virus, dengue virus, Zika virus, and Chikungunya virus production. Reduction of virus replication was attributable to decreases in entry and a strong block of virus gene expression at the translational level. These findings expand the potential use ofWolbachia wStri to block viruses and identify two separate steps for limiting virus replication in mosquitos that could be targeted via microbes or other means as an antiviral strategy.

Download Full-text

The time course analysis of morphological changes induced by Chikungunya virus replication in mammalian and mosquito cells

Acta Virologica ◽

10.4149/av_2018_403 ◽

2018 ◽

Vol 62 (04) ◽

pp. 360-373 ◽

Cited By ~ 1

Author(s):

A. Ghosh ◽

P. A. Alladi ◽

G. Narayanappa ◽

R. Vasanthapuram ◽

A. Desai

Keyword(s):

Virus Replication ◽

Time Course ◽

Chikungunya Virus ◽

Morphological Changes ◽

Mosquito Cells

Download Full-text

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

10.1101/2021.05.19.444900 ◽

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.

Download Full-text

Reconstitution of an RNA Virus Replicase in Artificial Giant Unilamellar Vesicles Supports Full Replication and Provides Protection for the Double-Stranded RNA Replication Intermediate

Journal of Virology ◽

10.1128/jvi.00267-20 ◽

2020 ◽

Vol 94 (18) ◽

Author(s):

Nikolay Kovalev ◽

Judit Pogany ◽

Peter D. Nagy

Keyword(s):

Virus Replication ◽

Rna Synthesis ◽

Rna Viruses ◽

Host Factors ◽

Replication Process ◽

Double Stranded Rna ◽

Replication Intermediate ◽

Cellular Factors ◽

Positive Strand Rna

ABSTRACT Positive-strand RNA [(+)RNA] viruses are important pathogens of humans, animals, and plants and replicate inside host cells by coopting numerous host factors and subcellular membranes. To gain insights into the assembly of viral replicase complexes (VRCs) and dissect the roles of various lipids and coopted host factors, we have reconstituted Tomato bushy stunt virus (TBSV) replicase using artificial giant unilamellar vesicles (GUVs). We demonstrate that reconstitution of VRCs on GUVs with endoplasmic reticulum (ER)-like phospholipid composition results in a complete cycle of replication and asymmetrical RNA synthesis, which is a hallmark of (+)RNA viruses. TBSV VRCs assembled on GUVs provide significant protection of the double-stranded RNA (dsRNA) replication intermediate against the dsRNA-specific RNase III. The lipid compositions of GUVs have pronounced effects on in vitro TBSV replication, including (−) and (+)RNA synthesis. The GUV-based assay has led to the discovery of the critical role of phosphatidylserine in TBSV replication and a novel role for phosphatidylethanolamine in asymmetrical (+)RNA synthesis. The GUV-based assay also showed stimulatory effects by phosphatidylinositol-3-phosphate [PI(3)P] and ergosterol on TBSV replication. We demonstrate that eEF1A and Hsp70 coopted replicase assembly factors, Vps34 phosphatidylinositol 3-kinase (PI3K) and the membrane-bending ESCRT factors, are required for reconstitution of the active TBSV VRCs in GUVs, further supporting that the novel GUV-based in vitro approach recapitulates critical steps and involves essential coopted cellular factors of the TBSV replication process. Taken together, this novel GUV assay will be highly suitable to dissect the functions of viral and cellular factors in TBSV replication. IMPORTANCE Understanding the mechanism of replication of positive-strand RNA viruses, which are major pathogens of plants, animals, and humans, can lead to new targets for antiviral interventions. These viruses subvert intracellular membranes for virus replication and coopt numerous host proteins, whose functions during virus replication are not yet completely defined. To dissect the roles of various host factors in Tomato bushy stunt virus (TBSV) replication, we have developed an artificial giant unilamellar vesicle (GUV)-based replication assay. The GUV-based in vitro approach recapitulates critical steps of the TBSV replication process. GUV-based reconstitution of the TBSV replicase revealed the need for a complex mixture of phospholipids, especially phosphatidylserine and phosphatidylethanolamine, in TBSV replication. The GUV-based approach will be useful to dissect the functions of essential coopted cellular factors.

Download Full-text