Outi Heikkilä

Director, Head of Manufacturing at DelSiTech, Turku, Finland

Overview

Personal data

Current position: Director, Head of Manufacturing at DelSiTech, Turku, Finland

Previous positions:
Production Manager and Senior Scientist at Biovian, Turku, Finland
Postdoctoral Fellow at Virology Department, University of Turku, Finland

Education: PhD Virology, MSc Biochemistry

Research

Virus Gene Therapy, Herpes simplex virus

Research

Herpes simplex virus

Receptor interactions and endocytosis of Coxsackievirus A9

Coxsackievirus A9(CV-A9) is a significant human pathogen that causes infections of the central nervous system, myocarditis and respiratory illnesses.

Publications

Main

  • "Integrins are not essential for entry of coxsackievirus A9 into SW480 human colon adenocarcinoma cells", Virology Journal 2016; 13: 171, authors: Outi Heikkilä, Pirjo Merilahti, Marika Hakanen, Eveliina Karelehto, Jonna Alanko, Maria Sukki, Saija Kiljunen, and Petri Susi

    Background Coxsackievirus A9 (CV-A9) is a pathogenic enterovirus type within the family Picornaviridae. CV-A9 infects A549 human epithelial lung carcinoma cells by attaching to the αVβ6 integrin receptor through a highly conserved Arg-Gly-Asp (RGD) motif, which is located at the exposed carboxy-terminus of the capsid protein VP1 detected in all studied clinical isolates. However, genetically-modified CV-A9 that lacks the RGD motif (CV-A9-RGDdel) has been shown to be infectious in some cell lines but not in A549, suggesting that RGD-mediated integrin binding is not always essential for efficient entry of CV-A9. Methods Two cell lines, A549 and SW480, were used in the study. SW480 was the study object for the integrin-independent entry and A549 was used as the control for integrin-dependent entry. Receptor levels were quantitated by cell sorting and quantitative PCR. Antibody blocking assay and siRNA silencing of receptor-encoding genes were used to block virus infection. Peptide phage display library was used to identify peptide binders to CV-A9. Immunofluorescence and confocal microscopy were used to visualize the virus infection in the cells. ResultsWe investigated the receptor use and early stages of CV-A9 internalization to SW480 human epithelial colon adenocarcinoma cells. Contrary to A549 infection, we showed that both CV-A9 and CV-A9-RGDdel internalized into SW480 cells and that function-blocking anti-αV integrin antibodies had no effect on the binding and entry of CV-A9. Whereas siRNA silencing of β6 integrin subunit had no influence on virus infection in SW480, silencing of β2-microglobulin (β2M) inhibited the virus infection in both cell lines. By using a peptide phage display screening, the virus-binding peptide identical to the N-terminal sequence of HSPA5 protein was identified and shown to block the virus infection in both A549 and SW480 cell lines. HSPA5 was also found to co-localize with CV-A9 at the SW480 cell periphery during the early stages of infection by confocal microscopy. Conclusions The data suggest that while αVβ6 integrin is essential for CV-A9 in A549 cell line, it is not required in SW480 cell line in which β2M and HSPA5 alone are sufficient for CV-A9 infection. This suggests that the choice of CV-A9 receptor(s) is dependent on the tissue/cellular environment.

  • "γ 1 34.5 neurovirulence gene of herpes simplex virus type 1 modifies the exosome secretion profile in epithelial cells", Journal of Virology 2016 Dec, authors: Heikkilä O, Ryödi E, Hukkanen V

    Recently, it has been demonstrated that herpes simplex virus 1 (HSV-1)-infected cells secrete exosomes that deliver to uninfected cells the innate immune sensor STING and viral RNAs (1, 2). Here, we report for the first time that the deletion of the viral γ134.5 neurovirulence gene affects HSV-induced exosome secretion.

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  • "Interleukin-27 Inhibits Herpes Simplex Virus Type 1 Infection by Activating STAT1 and 3, Interleukin-6, and Chemokines IP-10 and MIG", J Interferon Cytokine Res. 2016 Nov, authors: Heikkilä O, Nygårdas M, Paavilainen H, Ryödi E, Hukkanen V

    Interleukin-27 (IL-27) inhibits the replication of many viruses, but the mechanism differs according to virus and cell type. In this study, we observed that IL-27 expression was upregulated in herpes simplex virus type 1 (HSV-1)-infected SJL/J mice, which led us to further investigate the role of IL-27 in HSV-1 infection using epithelial, glioma, and immunological cells as cell models. We showed that in all studied cell lines, the IL-27 messenger RNA (mRNA) level was upregulated due to the HSV-1 infection. When the cells were primed with IL-27 before the virus infection, the virus release was prevented, indicating an antiviral role of IL-27 in HSV-1 infection. Furthermore, we observed that IL-27 secretion to the culture medium was reduced in infected epithelial and immunological cells, but not in glioma cells. Not surprisingly, HSV-1 induced type I, II, and III interferons regardless of cell line, but IL-27 itself caused varying interferon responses dependent on cell type. However, common to all cell types was the IL-27-stimulated secretion of IL-6 and chemokines IP-10 and MIG. In addition, IL-27 stimulation activated STAT1 and STAT3 in HeLa and T98G cells, suggesting that IL-27 engages the STAT1/3 pathway, which then leads to the upregulation of IL-6, IP-10, and MIG.

  • "Receptors and endocytosis of coxsackievirus A9", Annales Universitatis Turkuensis D 1067 (2013-05-25), Doctoral thesis (article-based), Institute of Biomedicine, Department of Virology, University of Turku, author: Outi Heikkilä

    Coxsackievirus A9 (CV-A9) belongs to human enteroviruses within family Picornaviridae, which are the main cause of aseptic meningitis. In addition, CV-A9 causes a wide range of other clinical manifestations of acute disease including respiratory infections, myocarditis, encephalitis and severe generalized infections in newborns.

    In this study, the functions of integrins αVβ6 and αVβ3 in the attachment and cellular entry of CV-A9 were analyzed. Further, virus and cell surface interactions and endocytosis of CV-A9 were studied in specific cell lines. Also, a method for production of GFP-expressing CV-A9 particles by long PCR-mediated mutagenesis and in vivo transcription was developed.

    The results indicated that RGD-motif (arginine-glycine-asparagine) that resides in the viral capsid is important for CV-A9 infection particularly in cell lines expressing integrin αVβ6 and that this integrin serves as a high affinity attachment receptor for the virus. CV-A9 is also capable of infecting certain cell lines independently of αV-integrins by binding to the cell surface HSPA5 protein. Regardless of the attachment stage, the internalization of the virus occurs via the same entry pathway and is dependent on β2M, dynamin, and Arf6 but independent of clathrin and caveolin-1. Furthermore, the virus internalization occurs within Arf6-containing vesicles suggesting that Arf6 is central mediator of CV-A9 endocytosis. While in this study the results of CV-A9 endocytosis were based on microscopical visualization within individual fixed cells, a rapid method for generation of a virus for real-time imaging was also described.

    Link to the book

  • "A combined method for rescue of modified enteroviruses by mutagenic primers; long PCR and T7 RNA polymerase-driven in vivo transcription", Journal of virological methods 10/2010, authors: Outi Heikkilä, Markus Kainulainen, Petri Susi
    The current methods for manipulation of enteroviral RNA genomes and production of modified virus particles include stepwise subcloning procedures and in vitro transcription and RNA transfection steps that are both time-consuming and inefficient. Several enteroviral cDNA clones with 5'-terminal T7 promoter and coxsackievirus A9 (CAV9) PCR product with the T7 promoter were transfected successfully into target cells expressing T7 RNA polymerase for the rescue of virus particles. This demonstrated the overall feasibility of the in vivo transcription method. Furthermore, a rapid method using high-fidelity DNA polymerase, Phusion™, for amplification and mutagenesis of CAV9 cDNA was generated. A long PCR method was employed together with mutagenic primers for direct introduction of a unique restriction enzyme site into the VP1-2A junction of the CAV9 cDNA clone during the PCR amplification process. Enhanced green fluorescent protein was subcloned to that site, and CAV9-eGFP cDNA was transfected to the target cells for in vivo transcription and successful rescue of CAV9-eGFP particles. The method allowed a straightforward mutagenesis and in vivo production of infectious enteroviral particles, and may be applicable routinely for rapid production of the modified picornaviruses over the use of the traditional subcloning protocols.
  • "Internalization of Coxsackievirus A9 is Mediated by {beta}2-microglobulin, Dynamin and Arf6 but not Caveolin-1 or Clathrin", Journal of virology 01/2010, authors: Outi Heikkilä, Petri Susi, Tuire Tevaluoto, Heidi Härmä, Varpu Marjomäki, Timo Hyypiä, Saija Kiljunen
    Coxsackievirus A9 (CAV9) is a member of the Human enterovirus B species within the Enterovirus genus of the family Picornaviridae. It has been shown to utilize alphaV integrins, particularly alphaVbeta6, as its receptors. The endocytic pathway by which CAV9 enters human cells after the initial attachment to the cell surface has so far been unknown. Here, we present a systematic study concerning the internalization mechanism of CAV9 to A549 human lung carcinoma cells. The siRNA silencing of integrin beta6 subunit inhibited the virus proliferation, confirming that alphaVbeta6 mediates the CAV9 infection. However, siRNAs against integrin-linked signaling molecules, such as Src, Fyn, RhoA, PI(3)K and Akt1, did not reduce CAV9 proliferation, suggesting that the internalization of the virus does not involve integrin-linked signaling events. CAV9 endocytosis was independent of clathrin or caveolin-1, but was restrained by dynasore, an inhibitor of dynamin. The RNAi silencing of beta2-microglobulin efficiently inhibited virus infection and caused CAV9 to accumulate on the cell surface. Furthermore, CAV9 infection was found to depend on Arf6, as both silencing of this molecule by siRNA and the expression of a dominant-negative construct resulted in decreased virus infection. In conclusion, the internalization of CAV9 to A549 cells follows an endocytic pathway that is dependent on integrin alphaVbeta6, beta2-microglobulin, dynamin and Arf6 but independent of clathrin and caveolin-1.
  • "Integrin {alpha}V{beta}6 is a high-affinity receptor for coxsackievirus A9", The Journal of general virology 02/2009, authors: Outi Heikkilä, Petri Susi, Glyn Stanway, Timo Hyypiä
    Coxsackievirus A9 (CAV9), a member of the genus Enterovirus in the family Picornaviridae, possesses an integrin-binding arginine-glycine-aspartic acid (RGD) motif in the C terminus of VP1 capsid protein. CAV9 has been shown to utilize integrins alphaVbeta3 and alphaVbeta6 as primary receptors for cell attachment. While CAV9 RGD-mutants (RGE and RGDdel) are capable of infecting rhabdomyosarcoma (RD) cell line, they grow very poorly in an epithelial lung carcinoma cell line (A549). In this study, the relationships between CAV9 infectivity in A549 and RD cells, receptor expression and integrin binding were analysed. A549 cells were shown to express both integrins alphaVbeta3 and alphaVbeta6, whereas alphaVbeta6 expression was not detected on the RD cells. Native CAV9 but not RGE and RGDdel mutants bound efficiently to immobilized alphaVbeta3 and alphaVbeta6. Adhesion of CAV9 but not RGE/RGDdel to A549 cells was also significantly higher than to RD cells. In contrast, no affinity or adhesion of bacterially produced VP1 proteins to the integrins or to the cells was detected. Function-blocking antibodies against alphaV-integrins blocked CAV9 but not CAV9-RGDdel infectivity, indicating that the viruses use different internalization routes; this may explain the differential infection kinetics of CAV9 and RGDdel. In an affinity assay, soluble alphaVbeta6, but not alphaVbeta3, bound to immobilized CAV9. Similarly, only soluble alphaVbeta6 blocked virus infectivity. These data suggest that CAV9 binding to alphaVbeta6 is a high-affinity interaction, which may indicate its importance in clinical infections; this remains to be determined.

Other

  • "The ERK-1 function is required for HSV-1-mediated G1/S progression in HEP-2 cells and contributes to virus growth", Sci Rep. 2017 Aug 23, authors: Colao I, Pennisi R, Venuti A, Nygårdas M, Heikkilä O, Hukkanen V, Sciortino MT.

    The herpes simplex virus 1 is able to readdress different cellular pathways including cell cycle to facilitate its replication and spread. During infection, the progression of the cell cycle from G1 to S phase makes the cellular replication machinery accessible to viral DNA replication. In this work we established that HSV-1, in asynchronized HEp-2 cells, strictly controls cell cycle progression increasing S-phase population from 9 hours post infection until the end of HSV-1 replication. The G1/S phases progression depends on two important proteins, cyclin E and CDK2. We demonstrate that their phosphorylated status and then their activity during the infection is strongly correlated to viral replication events. In addition, HSV-1 is able to recruit and distribute ERK1/2 proteins in a spatio-temporal fashion, highlighting its downstream regulatory effects on cellular processes. According with this data, using chemical inhibitor U0126 and ERK dominant negative cells we found that the lack of ERK1 activity affects cyclin E protein accumulation, viral gene transcription and percentage of the cells in S phase, during the viral replication. These data suggested a complex interaction between ERK, cell cycle progression and HSV-1 replication.

  • "Endocytosis of Integrin-Binding Human Picornaviruses", Review Article, Advances in Virology Volume 2012 (2012), authors: Pirjo Merilahti, Satu Koskinen, Outi Heikkilä, Eveliina Karelehto and Petri Susi
    Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9), echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1) has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.
  • "Interaction of {alpha}V{beta}3 and {alpha}V{beta}6 integrins with Human parechovirus 1", Journal of virology 06/2010, authors: Jani Seitsonen, Petri Susi, Outi Heikkilä, Robert S Sinkovits, Pasi Laurinmäki, Timo Hyypiä, Sarah J Butcher
    Human parechovirus (HPEV) infections are very common in early childhood and can be severe in neonates. It has been shown that integrins are important for cellular infectivity of HPEV1 through experiments using peptide blocking assays and function-blocking antibodies to alphaV-integrins. The interaction of HPEV1 with alphaV-integrins is presumably mediated by a C-terminal RGD-motif in the capsid protein VP1. We characterized the binding of integrins alphaVbeta3 andalphaVbeta6 to HPEV1 by biochemical and structural studies. We showed that although HPEV1 bound efficiently to immobilized integrins, alphaVbeta6 bound more efficiently than alphaVbeta3 to immobilized HPEV1. Moreover, soluble alphaVbeta6, but not alphaVbeta3, blocked HPEV1 cellular infectivity, indicating that it is a high-affinity receptor for HPEV1. We also showed that HPEV1 binding to integrins in vitro could be partially blocked by RGD-peptides. Using electron cryo-microscopy, and image reconstruction, we showed that HPEV1 has the typical T=1 (pseudo T=3) organization of a picornavirus. Complexes of HPEV1 and integrins indicated that both integrin footprints reside between the five-fold and three-fold symmetry axes. This result does not match the RGD position predicted from the CAV9 X-ray structure but is consistent with the predicted location of this motif in the shorter C-terminus found in HPEV1. This first structural characterization of a parechovirus indicates that the differences in receptor binding are due to the amino acid differences in the integrins rather than significantly different viral footprints.
  • "Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridiumperfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate.", Journal of molecular biology 2010 May 7, authors: Tuominen H., Salminen A., Oksanen E., Jämsen J., Heikkilä O., Lehtiö L., Magretova N. N., Goldman A., Baykov A. A. and Lahti R.
    Nucleotide-binding cystathionine beta-synthase (CBS) domains serve as regulatory units in numerous proteins distributed in all kingdoms of life. However, the underlying regulatory mechanisms remain to be established. Recently, we described a subfamily of CBS domain-containing pyrophosphatases (PPases) within family II PPases. Here, we express a novel CBS-PPase from Clostridium perfringens (CPE2055) and show that the enzyme is inhibited by AMP and activated by a novel effector, diadenosine 5',5-P1,P4-tetraphosphate (AP(4)A). The structures of the AMP and AP(4)A complexes of the regulatory region of C. perfringens PPase (cpCBS), comprising a pair of CBS domains interlinked by a DRTGG domain, were determined at 2.3 A resolution using X-ray crystallography. The structures obtained are the first structures of a DRTGG domain as part of a larger protein structure. The AMP complex contains two AMP molecules per cpCBS dimer, each bound to a single monomer, whereas in the activator-bound complex, one AP(4)A molecule bridges two monomers. In the nucleotide-bound structures, activator binding induces significant opening of the CBS domain interface, compared with the inhibitor complex. These results provide structural insight into the mechanism of CBS-PPase regulation by nucleotides.

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Useful software

BioImageXD is a free open source software for analysis, processing and 3D rendering of multi dimensional microscopy images.

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