DOCSLIB.ORG

In Vitro Evolution of High-Titer, Virus-Like Vesicles Containing a Single Structural Protein

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
In Vitro Evolution of High-Titer, Virus-Like Vesicles Containing a Single Structural Protein In vitro evolution of high-titer, virus-like vesicles containing a single structural protein Nina F. Rosea, Linda Buonocorea, John B. Schella, Anasuya Chattopadhyaya, Kapil Bahla, Xinran Liub, and John K. Rosea,1 aDepartment of Pathology, Yale University School of Medicine, New Haven, CT 06510; and bCenter for Cellular and Molecular Imaging, Yale University School of Medicine, New Haven, CT 06510 Edited* by Robert A. Lamb, Northwestern University, Evanston, IL, and approved October 22, 2014 (received for review August 6, 2014) Self-propagating, infectious, virus-like vesicles (VLVs) are gener- RNA. These proteins form a complex that directs replication of ated when an alphavirus RNA replicon expresses the vesicular the genomic RNA to form antigenomic RNA, which is then stomatitis virus glycoprotein (VSV G) as the only structural protein. copied to form full-length positive strand RNA and a subgenomic The mechanism that generates these VLVs lacking a capsid protein mRNA that encodes the structural proteins. The capsid protein has remained a mystery for over 20 years. We present evidence encases the genomic RNA in the cytoplasm and then buds from that VLVs arise from membrane-enveloped RNA replication facto- the cell surface in a membrane containing the SFV glycoproteins. ries (spherules) containing VSV G protein that are largely trapped Alphavirus RNA replication occurs inside light-bulb shaped, on the cell surface. After extensive passaging, VLVs evolve to membrane-bound compartments called spherules that initially grow to high titers through acquisition of multiple point muta- form on the cell surface and are then endocytosed to form cyto- tions in their nonstructural replicase proteins. We reconstituted pathic vacuoles containing multiple spherules (9). The replicase these mutations into a plasmid-based system from which high- proteins appear localized near the cytoplasmic side of the spher- ules (10). Positive-strand genomic RNA produced in the spherules titer VLVs can be recovered. One of these mutations generates is packaged into nucleocapsids before SFV budding. Alphavirus a late domain motif (PTAP) that is critical for high-titer VLV RNA replicons lacking structural protein genes can replicate ef- production. We propose a model in which the VLVs have evolved ficiently inside a cell, but they are incapable of propagating beyond in vitro to exploit a cellular budding pathway that is hijacked by the cell. many enveloped viruses, allowing them to bud efficiently from the We have been interested in developing VLVs as a vaccine cell surface. Our results suggest a basic mechanism of propagation platform (5, 6). However, the relatively low titers generated were MICROBIOLOGY that may have been used by primitive RNA viruses lacking capsid a major limitation of the system. We undertook the extensive proteins. Capsids may have evolved later to allow more efficient serial passaging studies described here to determine whether packaging of RNA, greater virus stability, and evasion of innate VLVs could evolve in culture to grow to high titers. We succeeded immunity. in generating VLVs that grow to at least 1,000-fold higher titers. In the process of studying these VLVs, we generated data sug- VSV glycoprotein | evolution | SFV replicon | late domain gesting the mechanism of VLV formation. In addition, our data suggest that the high-titer VLVs have evolved through passaging nveloped RNA viruses have highly organized structures. One to use a cellular budding machinery that is exploited by many Eor more capsid proteins encase their RNA, matrix proteins enveloped viruses to drive efficient budding. often lie between the capsid and the membrane, and one or more Many enveloped RNA viruses use components of a cellular vesicular budding machinery to drive efficient budding from the transmembrane glycoproteins can interact with the matrix or cell surface (11–13). Short sequence motifs called late domains capsid proteins to direct efficient particle assembly (1). Once the in their structural proteins recruit cellular protein complexes particles are released from cells, one or more glycoproteins in called ESCRT (endosomal sorting complex required for trans- the viral envelope bind cellular receptors and catalyze membrane port). The ESCRT complexes are normally involved in budding fusion to allow the viruses to enter new cells (2). Vesicular stomatitis virus (VSV) is a negative-strand RNA virus Significance that encodes a single membrane glycoprotein (G), a matrix protein, and a nucleocapsid protein as well as two proteins that form the viral polymerase (3). Remarkably, when cells are transfected with All known membrane-enveloped RNA viruses have capsid an alphavirus RNA replicon encoding only the alphavirus non- proteins that encase their RNA genomes. This paper shows that structural replicase proteins and the VSV G protein, infectious, infectious, membrane-enveloped, virus-like vesicles with RNA self-propagating membrane-enveloped vesicles containing the VSV genomes can evolve in vitro to grow to high titers without G protein are generated (4). These infectious, virus-like vesicles a capsid protein. The infectious vesicles are apparently gener- (VLVs) grow to only low titers of 104 to 105 infectious units (i.u.) ated from RNA replication factories called spherules that bud per mL, but propagate like a virus in tissue culture cells. The from the cell surface. They evolve in vitro to bud with high vesicles contain the genomic RNA and VSV G protein, but unlike efficiency through the acquisition of multiple mutations in the known enveloped RNA viruses, they lack a capsid protein encasing non-structural replicase proteins. One mutation generates their RNA resulting in a low buoyant density (4). The mechanism a critical motif found in many viral structural proteins. This by which these VLVs are generated has never been determined. A motif is involved in recruiting cellular machinery to drive effi- nonspecific packaging was postulated because the VLVs contained cient budding. Prior to the evolution of capsid proteins, prim- both genomic RNA and subgenomic mRNA (4) and the titers itive RNA viruses may have used this budding mechanism. could be increased by sonication of the cells. Despite the low titers, VLVs expressing other proteins have proven useful as experi- Author contributions: N.F.R. and J.K.R. designed research; N.F.R., L.B., J.B.S., A.C., K.B., X.L., mental vaccines (5, 6). and J.K.R. performed research; N.F.R., L.B., J.B.S., and J.K.R. analyzed data; and N.F.R., L.B., The alphavirus replicon used in the studies described above and J.K.R. wrote the paper. was derived from Semliki Forest Virus (SFV), a positive-strand, The authors declare no conflict of interest. membrane-enveloped RNA virus that encodes four nonstructural *This Direct Submission article had a prearranged editor. proteins called nsP 1–4 and three structural proteins: capsid, and 1To whom correspondence should be addressed. Email: [email protected]. – the E1 and E2 transmembrane glycoproteins (7, 8). The nsP 1 4 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. proteins are translated from the first two-thirds of the genomic 1073/pnas.1414991111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1414991111 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 of vesicles into cellular multivesicular bodies (MVB) as well as We compared the growth rate of the VLVs to VSV using tra- other cellular processes (12). Retroviruses, paramyxoviruses, filo- ditional one-step growth curves and found that the maximal VLV viruses, and rhabdoviruses have been shown to use components of titers (∼108 pfu/mL) were typically reached by between 12 and 24 h the ESCRT pathway to drive budding (11–13). However, other after high multiplicity infection. In contrast, maximal VSV titers 9 enveloped RNA viruses including SFV and influenza virus do not (∼10 pfu/mL) were reached between 10 and 12 h after infection. use the ESCRT pathway in budding (14–16). Purified p50 VLVs Contain VSV G as Their Structural Protein. To de- Results termine what structural proteins were in the p50 VLVs, we × 6 Extensive Passaging Generates High-Titer VLVs. A plasmid with an infected 2 10 BHK cells with p50 VLVs or with VSV at a efficient promoter driving synthesis of SFV replicon RNA inside multiplicity of infection (MOI) of 10. After 24 h when the cells showed extensive cytopathic effect (c.p.e.), the VSV particles or cells was described previously (17). After transfection of this DNA the VLVs in the medium were purified through two rounds of onto cells, the replicon RNA is synthesized in the nucleus, and ultracentrifugation. The proteins in the VLVs and VSV particles then moves to the cytoplasm where it is translated and begins the were then analyzed by SDS/PAGE followed by silver staining. As replication cycle. We previously constructed a derivative of this shown in Fig. 2A, VSV virions contained the five proteins N, P, M, vector with two subgenomic SFV mRNA promoters (6). The first G, and L, whereas the purified p50 VLVs showed VSV G protein promoter drives expression of VSV G mRNA and the second as the only apparent structural protein. There was no trace of the promoter can drive expression of mRNAs encoding other antigens. SFV capsid protein (29.8 kDa) consistent with the loss of its ex- The infectious VLVs derived from these constructs are potent pression during early passages. The total yield of VLV protein experimental vaccines, but they released relatively low titers of only from 5 × 106 BHK cells infected with the p50 VLVs was ∼20 μg, about 104 to 105 infectious units (i.u.)/mL These titers could be whereas the same number of cells infected with VSV yielded increased about 100-fold by sonicating the cells (6). about 60 μg of total viral protein. We did not recover measurable To determine whether VLVs might be able to evolve to grow to protein yields from medium derived from uninfected BHK cells.
Load more

Recommended publications
  • Interactions De La Protéine Nsp1 Du Virus Chikungunya Avec Les Membranes De L’Hôte Et Conséquences Fonctionnelles William Bakhache
    Interactions de la protéine nsP1 du virus Chikungunya avec les membranes de l’hôte et conséquences fonctionnelles William Bakhache To cite this version: William Bakhache. Interactions de la protéine nsP1 du virus Chikungunya avec les membranes de l’hôte et conséquences fonctionnelles. Médecine humaine et pathologie. Université Montpellier, 2020. Français. NNT : 2020MONTT008. tel-03132645 HAL Id: tel-03132645 https://tel.archives-ouvertes.fr/tel-03132645 Submitted on 5 Feb 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE POUR OBTENIR LE GRADE DE DOCTEUR DE L’UNIVERSITÉ DE M ONTPELLIER En Biologie Santé École Doctorale Sciences Chimiques et Biologiques pour la Santé UMR 9004 - Institut de Recherche en Infectiologie de Montpellier Interactions de la protéine nsP1 du virus Chikungunya avec les membranes de l’hôte et conséquences fonctionnelles Présentée par William Bakhache Le 27 Mars 2020 Sous la direction de Laurence Briant Devant le jury composé de Jean-Luc Battini, Directeur de Recherche, IRIM, Montpellier Président de jury Ali Amara, Directeur de recherche, U944 INSERM, Paris Rapporteur Juan Reguera, Chargé de recherche, AFMB, Marseille Rapporteur Raphaël Gaudin, Chargé de recherche, IRIM, Montpellier Examinateur Yves Rouillé, Directeur de Recherche, CIIL, Lille Examinateur Bruno Coutard, Professeur des universités, UVE, Marseille Co-encadrant de thèse Laurence Briant, Directeur de recherche, IRIM, Montpellier Directeur de thèse 1 Acknowledgments I want to start by thanking the members of my thesis jury.
    [Show full text]
  • Viral Strategies to Arrest Host Mrna Nuclear Export
    Viruses 2013, 5, 1824-1849; doi:10.3390/v5071824 OPEN ACCESS viruses ISSN 1999-4915 www.mdpi.com/journal/viruses Review Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export Sharon K. Kuss *, Miguel A. Mata, Liang Zhang and Beatriz M. A. Fontoura Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; E-Mails: [email protected] (M.A.M.); [email protected] (L.Z.); [email protected] (B.M.A.F) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-214-633-2001; Fax: +1-214-648-5814. Received: 10 June 2013; in revised form: 27 June 2013 / Accepted: 11 July 2013 / Published: 18 July 2013 Abstract: Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. Keywords: virus; influenza virus; vesicular stomatitis virus; VSV; NS1; matrix protein; nuclear export; nucleo-cytoplasmic trafficking; mRNA export; NXF1; TAP; CRM1; Rae1 1. Introduction Nucleo-cytoplasmic trafficking of proteins and RNA is critical for proper cellular functions and survival.
    [Show full text]
  • Emerging Roles for Lipid Droplets in Immunity and Host-Pathogen Interactions
    CB28CH16-Valdivia ARI 5 September 2012 17:10 Emerging Roles for Lipid Droplets in Immunity and Host-Pathogen Interactions Hector Alex Saka and Raphael Valdivia Department of Molecular Genetics and Microbiology and Center for Microbial Pathogenesis, Duke University Medical Center, Durham, North Carolina 27710; email: [email protected] Annu. Rev. Cell Dev. Biol. 2012. 28:411–37 Keywords First published online as a Review in Advance on eicosanoids, interferon, autophagy May 11, 2012 The Annual Review of Cell and Developmental Abstract Biology is online at cellbio.annualreviews.org Lipid droplets (LDs) are neutral lipid storage organelles ubiquitous to Access provided by Duke University on 10/14/19. For personal use only. This article’s doi: eukaryotic cells. It is increasingly recognized that LDs interact exten- 10.1146/annurev-cellbio-092910-153958 sively with other organelles and that they perform functions beyond Annu. Rev. Cell Dev. Biol. 2012.28:411-437. Downloaded from www.annualreviews.org Copyright c 2012 by Annual Reviews. passive lipid storage and lipid homeostasis. One emerging function for All rights reserved LDs is the coordination of immune responses, as these organelles par- 1081-0706/12/1110-0411$20.00 ticipate in the generation of prostaglandins and leukotrienes, which are important inflammation mediators. Similarly, LDs are also beginning to be recognized as playing a role in interferon responses and in antigen cross presentation. Not surprisingly, there is emerging evidence that many pathogens, including hepatitis C and Dengue viruses, Chlamydia, and Mycobacterium, target LDs during infection either for nutritional purposes or as part of an anti-immunity strategy.
    [Show full text]
  • Lessons Learned from Ebola Virus
    membranes Review SARS-CoV-2 Cellular Infection and Therapeutic Opportunities: Lessons Learned from Ebola Virus Jordana Muñoz-Basagoiti 1,†, Daniel Perez-Zsolt 1,†, Jorge Carrillo 1 , Julià Blanco 1,2 , Bonaventura Clotet 1,2,3 and Nuria Izquierdo-Useros 1,* 1 IrsiCaixa AIDS Research Institute, Germans Trias I Pujol Research Institute (IGTP), Can Ruti Campus, 08916 Badalona, Spain; [email protected] (J.M.-B.); [email protected] (D.P.-Z.); [email protected] (J.C.); [email protected] (J.B.); [email protected] (B.C.) 2 Infectious Diseases and Immunity Department, Faculty of Medicine, University of Vic (UVic-UCC), 08500 Vic, Spain 3 Infectious Diseases Department, Germans Trias i Pujol Hospital, 08916 Badalona, Spain * Correspondence: [email protected] † These authors contribution is equally to this work. Abstract: Viruses rely on the cellular machinery to replicate and propagate within newly infected individuals. Thus, viral entry into the host cell sets up the stage for productive infection and disease progression. Different viruses exploit distinct cellular receptors for viral entry; however, numerous viral internalization mechanisms are shared by very diverse viral families. Such is the case of Ebola virus (EBOV), which belongs to the filoviridae family, and the recently emerged coronavirus SARS- CoV-2. These two highly pathogenic viruses can exploit very similar endocytic routes to productively infect target cells. This convergence has sped up the experimental assessment of clinical therapies against SARS-CoV-2 previously found to be effective for EBOV, and facilitated their expedited clinical testing. Here we review how the viral entry processes and subsequent replication and egress strategies of EBOV and SARS-CoV-2 can overlap, and how our previous knowledge on antivirals, Citation: Muñoz-Basagoiti, J.; antibodies, and vaccines against EBOV has boosted the search for effective countermeasures against Perez-Zsolt, D.; Carrillo, J.; Blanco, J.; the new coronavirus.
    [Show full text]
  • A SARS-Cov-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing
    A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing Supplementary Information Supplementary Discussion All SARS-CoV-2 protein and gene functions described in the subnetwork appendices, including the text below and the text found in the individual bait subnetworks, are based on the functions of homologous genes from other coronavirus species. These are mainly from SARS-CoV and MERS-CoV, but when available and applicable other related viruses were used to provide insight into function. The SARS-CoV-2 proteins and genes listed here were designed and researched based on the gene alignments provided by Chan et. al. 1 2020 .​ Though we are reasonably sure the genes here are well annotated, we want to note that not every ​ protein has been verified to be expressed or functional during SARS-CoV-2 infections, either in vitro or in vivo. ​ ​ In an effort to be as comprehensive and transparent as possible, we are reporting the sub-networks of these functionally unverified proteins along with the other SARS-CoV-2 proteins. In such cases, we have made notes within the text below, and on the corresponding subnetwork figures, and would advise that more caution be taken when examining these proteins and their molecular interactions. Due to practical limits in our sample preparation and data collection process, we were unable to generate data for proteins corresponding to Nsp3, Orf7b, and Nsp16. Therefore these three genes have been left out of the following literature review of the SARS-CoV-2 proteins and the protein-protein interactions (PPIs) identified in this study.
    [Show full text]
  • The Role of F-Box Proteins During Viral Infection
    Int. J. Mol. Sci. 2013, 14, 4030-4049; doi:10.3390/ijms14024030 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Review The Role of F-Box Proteins during Viral Infection Régis Lopes Correa 1, Fernanda Prieto Bruckner 2, Renan de Souza Cascardo 1,2 and Poliane Alfenas-Zerbini 2,* 1 Department of Genetics, Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21944-970, Brazil; E-Mails: [email protected] (R.L.C.); [email protected] (R.S.C.) 2 Department of Microbiology/BIOAGRO, Federal University of Viçosa, Viçosa, MG 36570-000, Brazil; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +55-31-3899-2955; Fax: +55-31-3899-2864. Received: 23 October 2012; in revised form: 14 December 2012 / Accepted: 17 January 2013 / Published: 18 February 2013 Abstract: The F-box domain is a protein structural motif of about 50 amino acids that mediates protein–protein interactions. The F-box protein is one of the four components of the SCF (SKp1, Cullin, F-box protein) complex, which mediates ubiquitination of proteins targeted for degradation by the proteasome, playing an essential role in many cellular processes. Several discoveries have been made on the use of the ubiquitin–proteasome system by viruses of several families to complete their infection cycle. On the other hand, F-box proteins can be used in the defense response by the host. This review describes the role of F-box proteins and the use of the ubiquitin–proteasome system in virus–host interactions.
    [Show full text]
  • Thiopurines Activate an Antiviral Unfolded Protein Response That
    bioRxiv preprint doi: https://doi.org/10.1101/2020.09.30.319863; this version posted October 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Thiopurines activate an antiviral unfolded protein response that blocks viral glycoprotein 2 accumulation in cell culture infection model 3 Patrick Slaine1, Mariel Kleer 2, Brett Duguay 1, Eric S. Pringle 1, Eileigh Kadijk 1, Shan Ying 1, 4 Aruna D. Balgi 3, Michel Roberge 3, Craig McCormick 1, #, Denys A. Khaperskyy 1, # 5 6 1Department of Microbiology & Immunology, Dalhousie University, 5850 College Street, Halifax 7 NS, Canada B3H 4R2 8 2Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, 3330 9 Hospital Drive NW, Calgary AB, Canada T2N 4N1 10 3Department of Biochemistry and Molecular Biology, 2350 Health Sciences Mall, University of 11 British Columbia, Vancouver BC, Canada V6T 1Z3 12 13 14 #Co-corresponding authors: C.M., [email protected], D.A.K., [email protected] 15 16 17 Running Title: Drug-induced antiviral unfolded protein response 18 Keywords: virus, influenza, coronavirus, SARS-CoV-2, thiopurine, 6-thioguanine, 6- 19 thioguanosine, unfolded protein response, hemagglutinin, neuraminidase, glycosylation, host- 20 targeted antiviral 21 22 23 ABSTRACT 24 Enveloped viruses, including influenza A viruses (IAVs) and coronaviruses (CoVs), utilize the 25 host cell secretory pathway to synthesize viral glycoproteins and direct them to sites of assembly. 26 Using an image-based high-content screen, we identified two thiopurines, 6-thioguanine (6-TG) 27 and 6-thioguanosine (6-TGo), that selectively disrupted the processing and accumulation of IAV 28 glycoproteins hemagglutinin (HA) and neuraminidase (NA).
    [Show full text]
  • Annual Conference 2016
    Annual Conference 2016 POSTER ABSTRACT BOOK 21-24 MARCH 2016 ACC, LIVERPOOL, UK ANNUAL CONFERENCE 2016 SESSION 1 – MEMBRANE TRANSPORTERS S1/P1 the pump in this complex and it is conserved between bacterial species, with an average of 78.5% identity between the DNA Novel tripartite tricarboxylate transporters sequences and approximately 80% similarity between the amino acid sequences amongst Enterobacteriaceae. This pump acts as from Rhodopseudomonas palustris a drug-proton antiporter, four residues have been previously Leonardo Talachia Rosa, John Rafferty, reported as essential for proton translocation in Escherichia coli AcrB: D407, D408, K940 and T978. AcrB of E. coli has an identity David Kelly of 86% and a 94% similarity to that of S. Typhimurium. Based on The University of Sheffield, Sheffield, UK these data, we constructed an AcrB D408A chromosomal mutant in S. Typhimurium SL1344. Western blotting confirmed that the Rhodopseudomonas palustris is a soil non-sulfur purple mutant had the same level of expression of AcrB as the parental bacterium, with ability to degrade lignin-derived compounds and wild type strain. The mutant had no growth deficiencies either in also to generate high yields of hydrogen gas, what raises several LB or MOPS minimal media. However, compared with wild type biotechnological interests in this bacterium. Degradation SL1344, the mutant had decreased efflux activity and was pathways, though, must begin with substrate uptake. In this multi-drug hyper-susceptible. Interestingly, the phenotype of the context, Soluble Binding Proteins (SBP`s) dependant AcrB D408A mutant was almost identical to that of an ΔacrB transporters are responsible for high-affinity and specificity mutant.
    [Show full text]
  • Functional Studies of the Coronavirus Nonstructural Proteins Yanglin QIU and Kai XU*
    REVIEW ARTICLE Functional studies of the coronavirus nonstructural proteins Yanglin QIU and Kai XU* Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023, P. R. China. *Correspondence: [email protected] https://doi.org/10.37175/stemedicine.v1i2.39 ABSTRACT Coronaviruses, including SARS-CoV, SARS-CoV-2, and MERS-CoV, have caused contagious and fatal respiratory diseases in humans worldwide. Notably, the coronavirus disease 19 (COVID-19) caused by SARS-CoV-2 spread rapidly in early 2020 and became a global pandemic. The nonstructural proteins of coronaviruses are critical components of the viral replication machinery. They function in viral RNA transcription and replication, as well as counteracting the host innate immunity. Studies of these proteins not only revealed their essential role during viral infection but also help the design of novel drugs targeting the viral replication and immune evasion machinery. In this review, we summarize the functional studies of each nonstructural proteins and compare the similarities and differences between nonstructural proteins from different coronaviruses. Keywords: Coronavirus · SARS-CoV-2 · COVID-19 · Nonstructural proteins · Drug discovery Introduction genes (18). The first two major ORFs (ORF1a, ORF1ab) The coronavirus (CoV) outbreaks among human are the replicase genes, and the other four encode viral populations have caused three major epidemics structural proteins that comprise the essential protein worldwide, since the beginning of the 21st century. These components of the coronavirus virions, including the spike are the epidemics of the severe acute respiratory syndrome surface glycoprotein (S), envelope protein (E), matrix (SARS) in 2003 (1, 2), the Middle East respiratory protein (M), and nucleocapsid protein (N) (14, 19, 20).
    [Show full text]
  • Virus-Host Interaction: the Multifaceted Roles of Ifitms And
    Virus-Host Interaction: The Multifaceted Roles of IFITMs and LY6E in HIV Infection DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jingyou Yu Graduate Program in Comparative and Veterinary Medicine The Ohio State University 2018 Dissertation Committee: Shan-Lu Liu, MD, PhD, Advisor Patrick L. Green, PhD Jianrong Li, DVM., PhD Jesse J. Kwiek, PhD Copyrighted by Jingyou Yu 2018 Abstract With over 1.8 million newly infected people each year, the worldwide HIV-1 epidemic remains an imperative challenge for public health. Recent work has demonstrated that type I interferons (IFNs) efficiently suppress HIV infection through induction of hundreds of interferon stimulated genes (ISGs). These ISGs target distinct infection stages of invading pathogens and shape innate immunity. Among these, interferon induced transmembrane proteins (IFITMs) and lymphocyte antigen 6 complex, locus E (LY6E) have been shown to differentially modulate viral infections. However, their effects on HIV are not fully understood. In my thesis work, I provided evidence in Chapter 2 showing that IFITM proteins, particularly IFITM2 and IFITM3, specifically antagonize the HIV-1 envelope glycoprotein (Env), thereby inhibiting viral infection. IFITM proteins interacted with HIV-1 Env in viral producer cells, leading to impaired Env processing and virion incorporation. Notably, the level of IFITM incorporation into HIV-1 virions did not strictly correlate with the extent of inhibition. Prolonged passage of HIV-1 in IFITM-expressing T lymphocytes led to emergence of Env mutants that overcome IFITM restriction. The ability of IFITMs to inhibit cell-to-cell infection can be extended to HIV-1 primary isolates, HIV-2 and SIVs; however, the extent of inhibition appeared to be virus- strain dependent.
    [Show full text]
  • Kunjin Virus Replicon Vectors for Human Immunodeficiency Virus Vaccine Development† Tracey J
    JOURNAL OF VIROLOGY, July 2003, p. 7796–7803 Vol. 77, No. 14 0022-538X/03/$08.00ϩ0 DOI: 10.1128/JVI.77.14.7796–7803.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved. Kunjin Virus Replicon Vectors for Human Immunodeficiency Virus Vaccine Development† Tracey J. Harvey,1,2 Itaru Anraku,1,2,3 Richard Linedale,1,2 David Harrich,1 Jason Mackenzie,1,2 Andreas Suhrbier,3 and Alexander A. Khromykh1,2* Downloaded from Sir Albert Sakzewski Virus Research Centre, Royal Children’s Hospital,1 Clinical Medical Virology Centre,2 and The Australian Centre for International and Tropical Health and Nutrition, Queensland Institute of Medical Research,3 University of Queensland, Brisbane, Queensland, Australia Received 2 December 2002/Accepted 29 April 2003 We have previously demonstrated the ability of the vaccine vectors based on replicon RNA of the Australian flavivirus Kunjin (KUN) to induce protective antiviral and anticancer CD8؉ T-cell responses using murine polyepitope as a model immunogen (I. Anraku, T. J. Harvey, R. Linedale, J. Gardner, D. Harrich, A. Suhrbier, http://jvi.asm.org/ and A. A. Khromykh, J. Virol. 76:3791-3799, 2002). Here we showed that immunization of BALB/c mice with KUN replicons encoding HIV-1 Gag antigen resulted in induction of both Gag-specific antibody and protective Gag-specific CD8؉ T-cell responses. Two immunizations with KUNgag replicons in the form of virus-like particles (VLPs) induced anti-Gag antibodies with titers of >1:10,000. Immunization with KUNgag replicons delivered as plasmid DNA, naked RNA, or VLPs induced potent Gag-specific CD8؉ T-cell responses, with one -immunization of KUNgag VLPs inducing 4.5-fold-more CD8؉ T cells than the number induced after immu nization with recombinant vaccinia virus carrying the gag gene (rVVgag).
    [Show full text]
  • Rotavirus Strategies to Take Over the Host Cell Response
    Rotavirus strategies to take over the host cell response Instituto de Biotecnología Universidad Nacional Autónoma de México Battle between viruses and host cells Viral counter-measures Virus Host Host cell defenses During rotavirus infection the cellular protein synthesis is shutoff Virus - + During infection NSP3 displaces PABP from eIF4G and prevents cellular protein synthesis mRNA AAAA NSP3PABP 4A eIF4G Cap 4E eIF4F During RV infection, PABP relocalizes to the nucleus, and this depends on NSP3 a-PABP a-eIF4GI merge - virus control a-NSP2 + virus siIrr siNSP3 Montero H et al, 2006, J Virol. 80:9031-9038 polyA+ mRNAs are also relocalized in the nucleus of the cell during infection probe :Oligo dT No probe No Virus siIrr aNSP3 Merge + Virus siIrr + Virus siNSP3 Rubio RM et al, 2013. J Virol. During RV infection cellular mRNAs accumulate in the nucleus GRP78 GRP94 Cytop 5 Nuc 4 1.0 3 2 0.5 1 Relative amount Relative of amount Relative amount Relative of amount RNA units)(arbitrary RNA RNA units)(arbitrary RNA 0 0.0 Mock 6h 12h s/v 6h 12h Hours postinfection Hours postinfection Rotavirus prevents host translation by blocking the nucleo-cytoplasmic transport of poly(A)-containing mRNAs Rubio RM et al, 2013. J Virol eIF2a is phosphorylated during rotavirus infection hr post-infection: s/v 1 2 3 4 5 6 7 8 10 12 anti-eIF2ap Rojas M et al, 2010. J Virol. Translation factor eIF2 a GTP eIF2B GTP GDP a GDP GDP Met a a eIF2B GTP GDP Global translation inhibition Selective synthesis Translation initiation Rotavirus infection induces phosphorylation of eIF2a in a PKR-dependent manner MEFs: PERK-/- WT PKR-/- MA104 - Tg Ar Tg - Tg - Irr siPKR α-eIF2α-P eIF2a-P eIF2a PKR Rojas M et al, 2010.
    [Show full text]