2018
UNIVERSIDADE DE LISBOA
FACULDADE DE CIÊNCIAS
DEPARTAMENTO DE BIOLOGIA VEGETAL
Mechanism of a viral inhibitor of Toll-like Receptors
activation
Júlio Joel Laia Henriques
Mestrado em Biologia Molecular e Genética
Dissertação orientada por:
Michael Parkhouse
Margarida Telhada
i
I.
Acknowledgements
This year was full of experiments and experiences, lows and highs. All what I’ve been through this year shaped me to become a better scientist and a better citizen. Besides being a professional career, investigation is getting in contact with other humans and all of them, for the bad or for the good reasons, leave scars on my education. In this section I want to thank to all the people who left good scars on me and helped me to work on this master’s thesis.
I want to thank Mike, Michael Parkhouse, for taking me in his lab and support me in my decisions, and Sílvia Correia for giving me confidence and critical thinking required to be in a lab.
I thank Margarida Telhada, my Professor and faculty supervisor, and Rita Zilhão, my Professor and master coordinator, for support and advise.
I thank Professor Filomena Caeiro for giving me support for writing this thesis.
I thank my Rui Nascimento, Marisa Maia, Rita Simões, Lucas Padilha, Bárbara Patrício, Margarida Abreu, Catarina Gomes (Cacá), Jessica King, Cátia Alves, Ana Margarida Valente, João Santos, Jéssica Paulo for being the true friends they have always been.
I thank Yolanda Afonso and Catarina Azevedo, who have proved to be the true companions for life and that not even distances will tear us apart.
I thank Diogo Tomaz, Pedro Homem, Yara Rodrigues, Diogo Freire, Cristiana Santos, Clara Barreto, Lídia Jesus, Joana Branco dos Santos, Ana Alves, Hélio Rocha, Pâmela Borges, Ana Carvalho, Marta Silva, Gustavo Santos, Joana Loureiro, Raquel Mendes, Ojas Deshpande, Joana Carvalho and Camille van Lunen for being the new friendships I got from this project time.
I thank Patrícia Beldade for taking time listening to me, taking care of me and giving me advise. I thank my parents Júlio Henriques and Margarete Laia who have always supported me and helped me to move on. I thank my brother David for being my everlasting companion.
I thank/agradeço my grandparents Alfredo Canário and Maria de Lurdes Dias, my uncle Garreth Spragg, my aunt Paula Laia Spragg, Mia Laia Spragg, Mauro Laia, o meu tio José Laia, Sofia, Helena Almeida, Carlos Almeida, Mariana Almeida, Ana Almeida, Ana Gonçalves, Carlos Gonçalves for being family and support me/por serem família e me apoiarem, e à minha médica Isabel Jorge.
Por fim, agradeço a uma pessoa que está sempre a acompanhar a minha vida e a ajudar-me a seguir em frente nos piores e nos melhores momentos, o meu grande amigo Gonçalo Amaral.
ii
II.
Abstract
African Suine Fever Virus (AFSV) infects macrophages from Sus scrofa individuals (comprising pigs and wild boars), leading to severe haemorrhages and a high mortality of the infected pigs. In Africa, this virus also infects other species from the Suidae family, such as Potamochaerus porcus and
Phacochaerus aethiopicus. These animals are the natural reservoir in a sylvatic cycle and show no
symptoms of the disease when infected with the virus.
This disease is highly transmitted by direct pig to pig contact and by infected Ornithodoros soft ticks. Currently, an outbreak has been spreading from Eastern Europe, constituting a serious threat to the rest of the Europe. Sadly, there is not a vaccine yet, which is an urgent necessity, not only because pork is an important source of meat for humans, but also to protect wild boar subspecies and pig breeds. AFSV is a member of Asfarviridae family with a large icosahedral enveloped dsDNA virion, encoding for almost 200 proteins, depending on the isolate. Many of these proteins have evolved for manipulation of host cell biology and immunology, which several dedicated to inhibition of the interferon response14. These proteins confer an advantage to the virus through inhibition of the major antivirus immune response.
Developing a live attenuated viral vaccine through gene deletion of one or more virus genes inhibiting the interferon response is a practical solution
The ASFV transmembrane protein I329L, the focus of my thesis, was shown by our lab to have a slight homology to the Toll-like receptor 3 (TLR3) and, more importantly, to inhibit TLR3-mediated interferon production, and is therefore a candidate for construction of a gene deletion attenuated viral vaccine. Personal communication from the current research group shows an attempt to address a differential gene expression in cells expressing the viral protein I329L using DNA Microarray Analysis. However, no differential gene expression was observed. Thus, the objective of my project is to establish appropriate experimental approaches to test the mechanism of the I329L-mediated inhibition of the TLr3 response.
iii
III.
Sumário alargado
O vírus da Peste suína africana (VPSA) apresenta um elevado tropismo para macrófagos e infecta indivíduos da espécie Sus scrofa, que inclui javalis e porcos, levando a hemorragias severas e a uma elevada taxa de mortalidade dos indivíduos infectados. Esta doença é altamente transmitida entre indivíduos e através de carraças do género Ornithodoros.
Vários surtos ocorreram já, mas felizmente foram erradicados. Contudo, em 2007 na Geórgia, apareceu um surto preocupante que continua a avançar desde a Europa de Leste para o resto da Europa. A situação é alarmante porque não existe ainda vacina para esta doença. É necessário criar uma vacina tanto para proteger os porcos enquanto uma das principais fontes de alimentação para os humanos, como também para proteger e manter o património ecológico e genético das subespécies de javali e das raças de porco de forma sustentável.
Em África, este vírus infecta outras espécies da família Suidae, como o Potamocherus porcus e o
Phacochoerus aehtiopicus, que servem de reservatório natural num ciclo silvático sem mostrarem
qualquer tipo de sintoma.
O VPSA é um membro da família Asfarviridae e apresenta um virião icosaédrico, com invólucro e DNA em cadeia dupla que codifica para quase 200 proteínas, dependendo do isolado. Este vírus é responsável pela inibição da produção de interferão do tipo I e um número muito grande destas proteínas está envolvida nesta inibição da resposta imunitária. Contudo, não se conhecem ainda em detalhe os mecanismos de inibição viral, envolvendo tanto respostas serológicas como celulares.
Assim, o objectivo dos vários grupos de investigação na área do VPSA é estudar as proteínas deste vírus quanto ao mecanismo de inibição da resposta imunitária para se poder criar uma vacina viva de uma versão atenuada do vírus. Esta versão atenuada do vírus deverá conter o mínimo de delecções no seu genoma de genes que codificam para essas proteínas. Assim, é possível induzir uma resposta imunitária que reconheça o máximo de antigénios e que não seja inibida pelo vírus.
A proteína do VPSA em foco na minha tese é a proteína transmembranar I329L, que apresenta um domínio intracelular (DIC) e um domínio extracelular (DEC). O DIC apresenta um domínio do tipo receptor Toll/interleucina-1 (TIR) e o DEC apresenta domínios de repetições ricas em leucinas (LRR). Estado da arte mostra que a I329L é capaz de subverter a resposta imune, inibindo a produção de interferão do tipo I. Assim, o laboratório pretendeu saber qual o mecanismo pelo qual a I329L é capaz de subverter a resposta imune.
Sabendo que os porcos são vertebrados e que as carraças são invertebrados, e que o VPSA infecta tanto porcos como carraças, então o VPSA infecta tanto vertebrados como invertebrados. A resposta imunitária que é comum tanto vertebrados como invertebrados é a imunidade inata. Deste modo, é necessário procurar homologias da proteína viral I329L com proteínas envolvidas na resposta imune inata.
O grupo de investigação no qual decorre este projecto descobriu que a proteína I329L apresenta uma homologia vestigial com o receptor do tipo Toll 3 (TLR3).
Os receptores do tipo Toll (TLR) são receptores da imunidade inata. Os TLR são também proteínas transmembranares que apresentam um domínio rico em leucinas e um domínio TIR. Estes receptores activam-se por homodimerização após ligação de um ligando específico de cada TLR.
iv A homologia, apesar de vestigial, entre a proteína I329L e o receptor TLR3 encontra-se entre o domínio TIR do TLR3 e o domínio do tipo TIR da I329L, no domínio extracelular de cada um. O TLR3 é activado por RNA em cadeia dupla e utiliza a proteína TRIF (proteína adaptadora indutora de interferão β e que contém o domínio TIR) como proteína principal a jusante na via.
A sinalização do TLR3 divide-se numa via que utiliza a TRIF como proteína sinalizadora e noutra que é independente desta e que se serve da Src para regular a célula ao nível do ciclo celular e da locomoção. A Src, ao activar-se, autofosforila-se na tirosina 416. A via de sinalização do TLR3 pela TRIF culminará, por um lado, na degradação do IκB e na libertação do NF-κB, que migra para o núcleo e actua como factor de transcrição, permitindo a expressão de citocinas pró-inflamatórias. Por outro lado, a via de sinalização do TLR3 pela TRIF leva a que o IRF3 seja enviado para o núcleo para actuar como factor de transcrição, permitindo a expressão de interferão do tipo I, e consequentemente, de genes estimulados por interferão.
Através de ensaios que utilizam luciferase como repórter em fusão com o promotor de genes estimulados por interferão (ISGs), é possível quantificar de forma relativa a activação e a inibição da via de sinalização do TLR3. Utilizando como indutores da via o poli(I:C) (análogo artificial do RNA em cadeia dupla) e TRIF expressa ectopicamente e células transfectadas com apenas um dos domínios, o grupo concluiu que a I329L inibe a via de sinalização do TLR3 com o domínio intracelular, ligando-se ao domínio TIR do TLR3 e talvez à própria TRIF. Ainda, é possível que o domínio extracelular do I329L dimerize com o TLR3, impedindo a sua activação.
Com isto, o laboratório em que a investigação deste projecto decorre pretendeu perceber mais a fundo o impacto da I329L, não só na via de sinalização do TLR3, como também noutras vias celulares. Assim, o laboratório pretendeu analisar o impacto da I329L nos perfis de expressão génica e de fosforilação de péptidos na célula. Recorrendo a amostras de células induzidas com poli(I:C), ou não, e expressando a I329L, ou não, o grupo quis analisar o impacto da I329L na expressão génica na célula enviando estas amostras para serem analisadas por ensaio de microarranjo de DNA. A activação da via de sinalização do TLR3 e da sua inibição pela I329L nestas amostras foram confirmadas por ensaio de repórter de luciferase antes de serem enviadas. O resultado do ensaio de microarranjo de DNA mostrou que não houve nenhuma diferença na expressão de genes das amostras em relação ao controlo (células não induzidas por poli(I:C) e não transfectadas com I329L).
Assim, o objectivo da minha tese é testar a activação da sinalização do TLR3 e a sua inibição pela proteína viral I329L usando técnicas baratas.
Para tal, é necessário primeiro que o sistema de activação e inibição pela I329L da via de sinalização do TLR3 seja observável. Assim, para testar as células antes de as analisar por análise de microarranjo de DNA e por espectrometria de massa, que são técnicas caras, preciso de confirmar a activação e a inibição por I329L nas células com técnicas económicas disponíveis no laboratório. Essas técnicas são o ensaio de repórter de luciferase, immunoblot para o IκB, immunoblot para Src fosforilada na tirosina 416 e PCR de transcrição reversa para avaliar a expressão de genes estimulados por interferão.
As células utilizadas até então, as HEK-293T, não expressam TLR3, tendo que ser sempre transfectadas com plasmídeos que expressam o TLR3. As HEK-293T não mostravam mais a via de sinalização do TLR3 activada. Deste modo, testei outras linhas celulares. Em adição, procurei saber se o problema de activação das células residia na expressão do TLR3 e na degradação do poli(I:C).
Os resultados mostraram que algumas linhas celulares apresentam activação da via de sinalização, como as linhas Cos1 e RPE1. A inibição da via de sinalização do receptor TLR3 pela proteína viral I329L foi também testada em células RPE1, mas sem mostrar a via inibida.
v Este sumário não se rege pelas normas do Novo Acordo Ortográfico.
Palavras-chave: Vírus da Peste Suína Africana, via de sinalização do TLR3, I329L, inibição viral,
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List of Contents
I. Acknowledgements ... i
II. Abstract ... ii
III. Sumário alargado ... iii
1. Introduction ... 1
1.1. African swine fever ... 1
1.2. Immunity ... 2
1.2.1. Innate immunity ... 3
1.2.1.1. Toll-like receptor family ... 3
1.2.1.1.1. Toll-like receptor 3 ... 4
1.3. I329l – a viral inhibitor of the innate immune response ... 5
2. Objectives ... 6
3. Materials and Methods ... 7
4. Results and Discussion ... 9
4.1. Is TLR3 signalling pathway being activated in HEK-293T? ... 9
4.2. Why is TLR3 signalling pathway not shown to be activated in HEK-293T? ... 10
4.2.1. Poly(I:C) degradation hypothesis ... 10
4.2.2. Is TLR3 expressed in cells? ...11
4.3.4. Cell “drifting” hypothesis ... 12
4.3 Why do RPE1 cells do not show inhibition of the TLR3 signalling pathway?...14
vii
List of Figures
Figure 1- Structural diagram of the African Swine Fever Virus ... 2 Figure 2 - Blast of I329L protein sequence to other protein sequences shows two interesting homologies
with the receptor TLR3 and the cytosolic protein TRIF in the intracellular domain of I329L ... 5
Figure 3 - Ribbon vector model of the ASFV I329L protein. ... 5 Figure 4 - Activation and I329L-mediated inhibition of the TLR3 signalling pathway in HEK-293T .. 9 Figure 5 - Poly(I:C) agarose run ... 10 Figure 6 - Activation of the TLR3 signalling pathway in HEK-293T ... 11 Figure 7 - TLR3 presence in HEK-293T. HEK-293T cells were transfected with TLR3 expressing
plasmid ... 11
Figure 8 - Activation of the TLR3 signalling pathway in several cell lines ... 13 Figure 9 - Activation and I329L-mediated inhibition of the TLR3 signalling pathway inRPE1 ... 14
viii
List of Tables
ix
List of Abbrreviations and Acronyms
ASF African Swine Fever
ASFV African Swine Fever Virus
cDNA CLR
Complementary DNA
Calcitonin-like Receptor Family
dsRNA Double stranded RNA
ECD Extracellular domain
EGFR Epidermal growth factor receptor
GAS Gamma interferon activation site
element
ICD Intracellular domain
IFN Interferon
IL Interleukin
IRF Interferon regulatory factor
ISRE Interferon sensitive response element
IκB Nuclear factor of kappa light
polypeptide gene enhancer in B-cells inhibitor
LR Linker region
MyD88 Myeloid differentiation primary response 88
NF-κB NLR
Factor nuclear kappa B NOD-like Receptor Family
PAMP Pathogen-associated molecular pattern
Poly(I:C) Polyinosilic:polycytydylic acid
PRR RLR
Pattern recognition receptor RIG-1-like Receptor Family
RT-PCR Reverse transcription polymerase chain reaction
Src Cell Sarcoma virus-like protein
TIR Toll/interleukin-1 receptor
TLR Toll-like receptor
TM Transmembrane region
TNFα Tumor necrosis factor alpha
TRIF TIR-domain-containing adapter-inducing interferon-β
1
1. Introduction
1.1. African Swine Fever
African Swine Fever (ASF) is a viral transmitted disease first described in Kenya in 1910 as a highly haemorrhagic and lethal disease of Sus scrofa (domestic pig and wild boar)1. ASFV is transmitted
directly by contact between infected and non-infected pigs and also by the Orinithodoros genus soft tick, acting as vector in a sylvatic cycle2.
The mortality rate varies between isolates and can be 100% in the most virulent isolates3. Sudden deaths with few lesions (peracute cases) may be the first sign of an infection in a herd. Acute cases are characterized by a high fever, anorexia, lethargy, weakness and recumbency. Erythema can be seen and is most apparent in white pigs. Some pigs develop cyanotic skin blotching, especially on the ears, tail, lower legs or hams. Pigs may also have diarrhoea, constipation and/or signs of abdominal pain; the diarrhoea is initially mucoid and may later become bloody. There may also be visible signs of haemorrhagic tendencies, including epistaxis and haemorrhages in the skin. Respiratory signs (including dyspnoea), nasal and conjunctival discharges, and neurological signs have also been reported. Pregnant animals frequently abort; in some cases, abortions may be the first signs of an outbreak. Leukopenia and thrombocytopenia of varying severity may be detected in laboratory tests. Death often occurs within 7 to 10 days. The incubation period is 5 to 21 days after direct contact with infected pigs, but it can be less than 5 days after exposure to ticks3. Acute disease typically appears in 3 to 7 days3. Haemorrhagic lesions are associated with release of cytokines by infected monocyte/macrophages rather than direct endothelial cell damage4.
The ASFV was first reported in Europe in Portugal in 19575. Other European outbreaks have been
reported6, including in Malta, Italy, France, Belgium and the Netherlands7. Due to successful biosecurity regulations, African Swine Fever Virus (ASFV) was then eradicated from most European regions at the beginning of 1990; however, it remained endemic in Sardinia8.
In 2007, an alarming outbreak took place in European Georgia9,10. This outbreak had devastating consequences for the pig industry and is spreading throughout the Baltic Countries, the Caucasus and Eastern Europe (regions according to the United Nations Statistics Division)11. In 2014, the virus had
already been spread in Poland12. Moreover, the ASFV entered Europe again in Lithuania in February of the same year10.
Studies made in Portugal using Ornithodoros erraticus showed that the ticks may carry the ASFV five
years and three months after the removal of infectious hosts and transmit it to other Suidae individuals13.
Unfortunately, there is not a vaccine14. The available methods of disease control are slaughter of infected
and in-contact animals, safe carcass disposal, sanitation, disinfection, movement controls and quarantines, and the prevention of contact with wild suids and infected ticks3. A high priority is therefore to find a vaccine to protect the non-infected areas11, not only because pork has an economic and nutritional importance for humans, but also to maintain the heritage of the wild boar subspecies and the domestic pig breeds.
The infection of the natural host Suidae species is characterized by the absence of clinical symptoms, reflecting the long-term host-pathogen co-evolution. Suidae species that can also be infected and show no symptoms of the disease include Phacochoerus africanus, Phacochoerus aethiopicus,
2 Immunity to ASF virus (ASFV) is complex, involving both serological and cellular responses15 and the recognition of multiple antigen determinants14. Thus, construction of a vaccine comprising one or few
viral proteins may not be a feasible strategy. Development of an attenuated virus or complex DNA vaccine, on the other hand, is a practical and potentially effective approach, offering the advantage of stimulating all arms of the immune response via multiple antigenic determinants14. At the present, the most feasible strategy for development of a vaccine is the construction of an attenuated virus by deletion of one or more genes that confer advantage to the virus through inhibition of their antiviral immune response. Such vaccine demands a prior knowledge of the virus structure, the function of its components and the nature of the immune response.
Figure 1- Structural diagram of the African Swine Fever Virus (adapted from Viralzone49)
The ASFV is a double stranded DNA (dsDNA) virus from the Asfarviridae family48. The virion has a
complex multi-layered structure (Figure 1). The nucleoprotein core contains the viral genome, enzymes and other proteins necessary for the early stages of infection. This nucleoprotein core is surrounded by a matrix shell and an internal envelope into which the icosahedral capsid is then assembled16,17. ASFV infects monocytes and macrophages first by adhesion to the plasma membrane, and then, after endocytosis, the release of its capsid and genome by fusion of the envelope with the endocytic vesicle membrane. Immediate early and early genes are transcribed before DNA replication and intermediate and late genes transcribed after the initiation of DNA replication in the cytoplasm. Viral particles are then assembled in the Endoplasmic Reticulum18, migrate to the plasma membrane through the intervention of microtubules and bud through the plasma membrane19.
1.2. Immunity
For millions of years viruses have evolved under the opposing pressure of their host immune systems. These enforced changes are mirrored in the immune system itself, as vertebrates have developed highly complex strategies to eliminate invading pathogens20,21. The immune response can be divided in two principal functional arms: innate and adaptive immune responses, although they are not completely independent22. Innate immunity acts immediately and has a limited antigen receptor repertoire and no
memory. On the other hand, adaptive immunity has an enormous and specific antigen receptors repertoire and memory23, the latter being the sine qua non of a vaccine.
3
1.2.1. Innate Immunity
ASFV infects both vertebrate (Suidae species) and invertebrate species (Ornithodoros soft tick) and therefore it is reasonable that it has evolved strategies to subvert innate immunity, the only common immune response shared between its vertebrate24 and invertebrate hosts25,26,27.
The innate immune response involves the rapid recognition of pathogen associated molecular patterns (PAMPs) present in the pathogens themselves or in the infected cells28. The PAMPs are recognized by an array of different pattern recognition receptors (PRRs), namely the Toll-like Receptor Family, the NOD-like Receptor Family (NLR), the RIG-1-like Receptor Family (RLR) and the Calcitonin-like Receptor Family (CLR)28. One of the most important consequences of PAMP-PRR interactions is the activation of interferon and proinflammatory cytokine expression.
Interferons (IFN) are crucial for viral defence29. Type I IFN is secreted by essentially all cells in response to virus infection. PRRs signalling pathways activate the nuclear factor-kappa B (NF-κB), the interferon regulatory factor 3 (IRF3) and the interferon regulatory factor 7 (IRF7) transcription factors. These transcription factors promote the expression and secretion of proinflammatory cytokines and chemokines. Also, the interferon-β (IFN-β) promoter14, the interferon-sensitive response element (ISRE)30 and the γ-activation site (GAS) are ultimately activated to express interferons, and consequent induction of both innate and adaptive immune responses14.
1.2.1.1. Toll-like Receptor Family
Mammalian Toll-like receptors (TLRs) are homologues of Toll receptors found in the Drosophila invertebrate model31. TLRs are type I transmembrane receptors which contain an extracellular leucine-rich repeat (LRR) domain responsible for TLR dimerization and an intracellular Toll-IL-1 receptor (TIR) domain that transmits downstream signals via the recruitment of TIR-containing adaptor proteins, namely the myeloid differentiation primary response gene 88 (MyD88) or the TIR domain-containing adaptor-inducing interferon (INF) (TRIF)32. Then, interferon-stimulated genes (ISGs) are expressed in cells upon interferon production29. Several mammalian Toll-like receptors (TLRs) recognize several types of nucleic acids and microbial associated lipids, which permit the detection of most types of viruses and bacteria (Table 1)34–36.
4
1.2.1.1.1. Toll-like Receptor 3
TLR3 is the TLR known to play a vital key role in the response to viral infection, recognizing double stranded RNA (dsRNA), a frequent product during viral replication636,37. The extracellular domain
(ECD) contains a 23 leucine-rich repeats (LRRs) domain41. Further downstream events in TLR3 activation include phosphorylation of the exposed tyrosines in the intracellular domain (ICD) linker region (LR) by EGFR (Y858) and c-Src (Y759)39. Src autophosphorylates in its tyrosine 416 after phosphorylating tyrosine 759 of the TLR332. There are two major TLR3 pathways: TRIF-dependent and TRIF-independent Src-mediated pathways40,41. In TRIF-dependent pathways, TRIF binds the TIR
domain, signals to different pathways and culminates in the production of type I INFs, TNFα, IL-1 and other cytokines42,43. On the other hand, in the TRIF-independent pathways, Src will promote pathways for cells motility and adhesion with no gene induction required39.
TLR
Table 1- TLRs and their ligands
Ligand
Species
TLR1 Triacyl lipopeptides; Homo sapiens/ Mus
musculus
TLR2
Diacyl and tryacillipopeptides; peptidoglycans;
Homo sapiens/ Mus musculus lipoteichoic acid; phenol-soluble modulin;
4Manalpha-Po(4)-containing phosphoglycans, atypical LPS; zymosan; phospholipomannan; phospholipomannan; GPI anchors; galbeta-1;
Glycolipids
TLR3 dsRNA Homo sapiens/ Mus
musculus
TLR4
LPS; Glucoronoxylomannan; GPI anchors; F protein of respiratorial syncytial virus (RSV);
Homo sapiens/ Mus musculus F protein of respiratorial syncytial virus (RSV),
gp52 nad gp36 envelope proteins of mouse
mammary tumor virus (MMTV); HMGB1; Hsp70;
TLR5 Flagellin Homo sapiens/ Mus
musculus
TLR6 Diacyllipopeptides; lipoteichoic acid; Homo sapiens/ Mus
musculus
TLR7 PolyU and GU-rich RNA Homo sapiens/ Mus
musculus
TLR8 PolyU and GU-rich ssRNA; imidazoquinolines Homo sapiens/ Mus
musculus TLR9 CpG-rich DNA; short DNA-RNA hybrid (<400?); Hemozoin Homo sapiens/ Mus
musculus
TLR10 Unknown Homo sapiens
TLR11 profilin-like protein Mus musculus
TLR12 profilin-like protein Mus musculus
5
1.3. I329L – a viral inhibitor of the innate immune response
ASFV impairs IFNβ intermediate activation by a variety of mechanisms14,44. For example, A276R viral
protein targets IRF3 signalling, but not IRF7, in an NF-κB independent manner. Also, A528R inhibits both IRF3 and IRF7 signalling pathways and also the JAK/STAT signalling pathway14.
Previous studies have shown a marginal homology of the ASFV viral I329L intracellular domain with the TIR domain in TLR345 (Figure 2). I329L is a highly glycosylated type I transmembrane protein with a 17 aminoacids signal peptide sequence (aminoacids. 1-17), followed by an N-terminal extracellular domain (aminoacids 18-239), a transmembrane domain (aminoacids 240-260), and a 69-aminoacid C-terminal intracellular domain (aminoacids. 261-329)45 (Figure 3).
Figure 2 – Blast of I329L protein sequence to other protein sequences shows two interesting homologies with the receptor TLR3 and the cytosolic protein TRIF in the intracellular domain of I329L (Oliveira et al., 2011)45
Figure 3 - Ribbon vector model of the ASFV I329L protein. The I329L shows an extracellular domain (ECD), a transmembrane region and an intracellular domain (ICD)39
6 More importantly, the viral protein I329L has shown to impair IFN production14,38,45. Both the I329L
intracellular (ICD) and the extracellular (ECD) domains have been shown to inhibit the TLR3-mediated IRF3 signaling38. The I329L ICD inhibits through binding to TRIF due to its TIR-like domain. The ECD also inhibits but using a yet unknown mechanism. Both the ECD and the ICD have inhibited the poly(I:C)-mediated activation of TLR3. Whereas the ICD, but not the ECD, inhibited TLR3 signalling stimulated by ectopic expression of TRIF.
Master’s Thesis investigation performed by Pedro Moura38 in the present research group concluded that
the ICD inhibits the TLR3 signalling pathway by binding to TRIF and that the ECDTM inhibits the TLR3 signalling upstream of the ICD inhibition, possibly by dimerizing with TLR3 ECD. His work used HEK-293T cells as a model because they are mammalian cells and the TLR3 signalling pathway is conserved in vertebrate and invertebrate cells, mammals being vertebrates. Stimulation of TLR3 transfected cells can be achieved by inducing them with poly(I:C), a dsRNA analogue. As these cells do not express TLRs, this was achieved by TLR3-expressing plasmid transfection.
Surprisingly, when such control and poly(I:C)-stimulated TLR3-transfected cells were submitted to gene expression microarray analysis, no differential gene expression was observed in the poly(I:C)-treated sample, thus invalidating the main objective of the experiment (Sílvia Correia, Michael Parkhouse group, personal communication); namely to define the impact of I329L on the TLR3 intracellular signalling pathway.
2. Objectives
My priority in the lab was therefore to develop simple and economical approaches to confirm TLR3 signalling pathway activation and its I329L-mediated inhibition in cells. The approaches were Luciferase Reporter Assay of IRF3 signalling and IκB degradation, Src tyrosine 41646 and ubiquitous
phosphorylation profiles by immunoblot and differential expression of interferon stimulated genes (ISGs) by RT-PCR.
7
3. Materials and Methods
Cells: HEK-293T, Cos 1, Cos 7 and NIH3T3 cell lines were cultured in 5% CO2 at 37 ◦C in Dulbecco’s
modified Eagle medium (DMEM) Low Glucose with L-Glutamine and with Sodium Pyruvate and 1000 mg/l glucose, supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS) and 100 U/ml and 100 µg/ml penicillin and streptomycin, respectively (Thermofisher).
RPE1 cells were cultured in 5% CO2 at 37 ◦C in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) (Thermofisher).
Plasmids: pCDNA3-HA (empty vector (EV)), pCDNA3-I329L-HA (I329L-expressing plasmid),
pCDNA3-I329LECDTM-HA (I329L extracellular domain + transmembrane region (ECDTM) expressing plasmid) pCDNA3-I329LICDTM-HA (I329L intracellular domain + transmembrane region (ICDTM) expressing plasmid), pCDNA3-TLR3-HA and pUNO-TLR3-HA (TLR3-expressing plasmids), IFN-β promoter (pIFΔ(-125/+72)lucter (luciferase-expressing plasmid), pCMVβ (β-galactosidase-expressing plasmid (β-Gal-expressing plasmid)
Stimuli: polyinosinic:polycytidylic acid (poly (I:C)) (PeproTech)
Immunoblotting: Cells were cultured in 6-well plates at 2.5 x 105 cells/mL. Cells were transfected with
plasmid DNA according to FuGENE® 6 Transfection Reagent E2691 protocol 24 hr later with 1 µg of EV plasmid, I329L-expressing plasmid, ECDTM plasmid or ICDTM plasmid and 100 ng of TLR-3 expressing plasmid). After 48 hr incubation, cells were induced with 500 ng/mL poly (I:C) during 30 min pre-lysis, and lysed in cold lysis buffer (6 M urea, 75 mM NaCl, 1 mM EDTA, 1% (v/v) NP40, 2% (v/v) glycerol, 1 mM PMSF, 1 µM DTT, 25 mM HEPES, pH 7.4) supplemented with 1x PHOSS-RO PHOSS-ROCHE PhosSTOP™ phosphatase inhibitor cocktail. Extracts were boiled (5 min, 100 ◦C) with sample buffer (1.7% [w/v] SDS, 5% [v/v] glycerol, 0.1 M DTT, bromophenol blue [0.02 mg/ml], 58 mM Tris-HCl, pH 6.8)). Proteins were then separated by 12% SDS-PAGE, electroblotted onto polyvinylidene fluoride (PVDF, BioRad) membranes and blocked with 5% (w/v) non-fat dried milk in PBS for 1 hr at room temperature (RT). The membranes were probed with the following antibodies: a mouse monoclonal antibody against β-actin (Sigma) conjugated with HRP to provide an internal control for protein loading, a rabbit monoclonal antibody against IκB (Cell Signaling 9242), a rabbit polyclonal antibody against TLR3 (abcam E2691), a rabbit monoclonal antiblody against phosphorylated tyrosine 416 of Src (Cell Signaling 2101) and a mouse pan antibody against phosphotyrosines (Milipore 05-321). Membranes were incubated with the indicated antibodies overnight, at 4 ◦C, and developed using ECL chemiluminescent reagents (Thermofisher 32106) or Luminata Forte (Merck WBLUF0500), according to the manufacturer’s instructions.
Cells were cultured in 6-well plates at 2.5 x 105 cells/mL. Cells were transfected with plasmid DNA according to X-treme 9® Transfection Reagent (Merck XTG9-RO) protocol 24 hr later with 1 µg of EV plasmid, I329L-expressing plasmid, ECDTM-expressing plasmid) or ICDTM-expressing plasmid and 100 ng of TLR3 expressing vector. Several timepoints, ranging from 15 to 200 min, each 15 min, were performed to induce cells with 500 ng/mL poly (I:C). After induction, protocol for RPE1 cells is the same as HEK-293T cells.
Luciferase reporter assays: HEK-293T and RPE1 cells were cultured in 24-well plates at 6 x 104
cells/well, and 24 hr later were transfected according to Lipofectamine® 2000 Transfection Reagent (Thermofisher 11668019) protocol with reporter plasmids for luciferase-expressing plasmid, 100 ng) and the internal control β-galactosidase (25 ng), TLR3 expressing plasmid (25 ng) and EV plasmids (300 ng), keeping the total amounts of DNA and equimolar ratios constant in all assays by adding the appropriate amount of empty vector. After 48 h, cells were either stimulated with 25 µg/ml of poly(I:C) for 5 hr or left untreated. The cells were lysed in 100 µl lysis solution (ABX210LM, Promega Systems) according to the manufacturer’s instructions, and samples were assayed for both
8 luciferase and β-galactosidase activities. Luciferase activity was normalized to the β-galactosidase activity from the co-transfected plasmid internal control and expressed as luciferase relative to galactosidase activity. Values are expressed as mean relative stimulation ± SD (calculated from triplicate determinations). A minimum of three independent assays were done for each experiment reported. Transfections were performed with equal amounts of DNA mixtures comprising 25 ng of TRIF plasmid vector (TRIF) and the above-mentioned plasmids. After 48 h, the cells were harvested and lysed, following the above protocol. Assays for other TLRs followed the first protocol but using expression plasmids for TLRs other than TLR3.
Sample preparation for Mass Spectometry Phosphopeptide Sequencing: HEK-293T cells were
cultured in 6-well plates at 2.5 x 105 cells/mL. Cells were transfected with plasmid DNA according to
FuGENE® 6 Transfection Reagent protocol 24 hr later with 1 µg of EV, I329L-expressing vector,
ECDTM plasmid or ICDTM-expressing vector and 500 ng of TLR3-expressing plasmid. After 48 hr incubation, cells were induced with 500 ng/mL poly (I:C) during 30 min pre-lysis and lysed in cold lysis buffer (6 M urea, 75 mM NaCl, 1 mM EDTA, 1% (v/v) NP40, 2% (v/v) glycerol, 1 mM PMSF, 1 µM DTT, 25 mM HEPES, pH 7.4) supplemented with 1x PHOSS-RO ROCHE PhosSTOP™ phosphatase inhibitor cocktail. Samples were stored at -80ºC.
Sample preparation for Microarrays: HEK-293T cells were cultured in 60 mm plates at 5 x 105
cells/mL and later transfected according to the FuGENE 6® protocol with 2 µg of EV, I329L exp. vector, ECDTM exp. vector or ICDTM exp.vector and 1 µg of TLR3 exp. vector. Cells were stimulated or not with poly(I:C) during 30 min at 37˚C and were then pelleted by centrifugation for 5 min at 190G 4˚C, washed in 1x PBS and centrifuged for 5 min at 190 G 4˚C and resuspended in Trizol. Samples were stored at -80ºC.
Reverse Transcription – PCR: HEK-293T cells were cultured in 6-well plates at 2,5 x 105 cells/mL. Cells were transfected with plasmid DNA according to FuGENE® 6 Transfection Reagent protocol 24
hr later with 1 µg of EV, I329L-expressing plasmid), ECDTM-expressing plasmid or
ICDTM-expressing plasmid and 100 ng of TLR3-ICDTM-expressing plasmid. After 48 hr incubation, cells were induced with 500 ng/mL poly (I:C) during 30 min pre-lysis. Cells were lysed using TRIzol Reagent (Thermofisher 15596026). purified according to NZYTech Total RNA isolation kit and cDNA was synthesised according to NZYTech First-Stranded cDNA Synthesis kit. cDNA concentration was quantified in Nanodrop. Interferon-stimulated genes ISG15, ISG54 and ISG56 were amplified using specific primers from Invitrogen and PCCR kit from. Several conditions were tested, varying the annealing and extension temperatures and magnesium concentration. Samples were run on a 1.2% agarose gel.
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4. Results and Discussion
4.1 Is TLR3 signalling pathway being activated in HEK-293T?
To address whether the TLR3 signalling pathway is activated in TLR3 transfected HEK-293T cells, cells were induced, or not, by poly(I:C) and cotransfected with a I329L-expressing plasmid, or a control plasmid. Activation and inhibition by I329L of the TLR3 signalling pathway were analysed by Luciferase reporter assay and IκB degradation, ubiquitous tyrosine phosphorylation and phosphorylation of Src profiles (Figure 4).
A six-fold activation by poly(I:C) can be seen in the Luciferase reporter assay (Figure 4 - A). Also, the I329L-mediated inhibition of the TLR3 signalling pathway is not observed since the activation of the TLR3 signalling pathway inhibition by the viral protein I329L may in fact also correspond to a six-fold activation comparing to I329L-expressing cells but not induced with poly(I:C). In parallel culture, using the exact same cells and plasmids, IκB degradation, ubiquitous tyrosine phosphorylation and phosphorylation of Src on tyrosine 416 profiles (Figure 4 - B) showed no activation of the TLR3 signalling pathway. These results show that the activation of the TLR3 signalling pathway is insignificant, having no impact on degradation of IκB nor phosphorylation in proteins involved in the TLR3 signalling pathway as Src. A B 0 2 4 6 8 10 12 EV I219L Arbitrar y ligh t un its no induction poly(I:C) 60 KDa 43 KDa 39 KDa 60 KDa
Figure 4 - Activation and I329L-mediated inhibition of the TLR3 signalling pathway in HEK-293T. A) Luciferase reporter assay of I329L-mediated inhibition of IRF3 signalling in HEK-293T. HEK-293T cells were co-transfected with TLR3 expressing plasmid, empty vector or I329L expressing plasmids, β-galactosidase and IRF3 promoter luciferase reporter plasmids. At 48-h post-transfection, cells were stimulated with 25 µg/ml Poly (I:C) for 5 h or left untreated. Luciferase activity was normalized to β-galactosidase activity as a control for transfection efficiency. Data expressed as mean relative luciferase units ± SD (triplicate determinations). B) Impact of I329L on TLR3 signalling pathway-mediated IκB degradation, c-Src phosphorylation and ubiquitous tyrosine phosphorylation in HEK-293T. HEK-293T cells were transfected with TLR3 exp. plasmid and empty vector or I329L expressing vector. 48 h post-transfection, cells were stimulated with 100 µg/ml Poly (I:C) during 3 h or left untreated, and then lysed. Samples were separated on 12% SDS-PAGE and detected with anti-IκB, pan anti-phosphotyrosines. Actin was detected as a control for protein loading, with anti-β-Actin.
10 To address whether there is a difference of expression in interferon-stimulated genes (ISGs) upon TLR3 signalling pathway induction with poly(I:C) in HEK-293T, I decided to perform a Reverse Transcription PCR. However, this approach was abandoned due to the high amount of time spent on PCR protocol optimization.
4.2 Why is TLR3 signalling pathway not shown to be activated in HEK-293T?
Possible explanations for the absence of activation of the TLR3 signalling pathway include degradation of poly(I:C), low expression of protein, mutation of TLR3-expressing plasmid or cell “drift” (mutations on cell clones present after several subcultures) of the HEK-293T cells.
4.2.1 Poly(I:C) degradation hypothesis
To address whether poly(I:C) from the lab is degraded, I tested poly(I:C) by two different approaches: poly(I:C) run on an agarose gel electrophoresis (Figure 5) and comparing TLR3 signalling pathway activation with stimulation poly(I:C) stock from the lab with TLR3 signalling pathway activation with stimulation with a control poly(I:C) stock (Figure 6).
Figure 5 - Poly(I:C) agarose run. 30 mg of poly(I:C) from the lab was run in a 1% agarose gel and gel bands were UV revealed.
Results from poly(I:C) agarose gel run (Figure 5) show poly(I:C) is not degraded because only one band is visible, and not a smear.
11 The poly(I:C) control stock aliquot comes from a stock demonstrated to induce genome deletions using a Cre-lox system. The Mx1 protein is encoded by the Mx1 gene, controlled by the Mx1 promoter. This promoter is induced by IFN. In this case, the cre-recombinase sequence was cloned downstream of the Mx1 promoter. Because poly(I:C) induces INF production, INF promotes the expression of the genes controlled by the Mx1 and the cre sequence is controlled by the Mx1 promoter, the genes that are flanked by the lox sites will be deleted by poly(I:C) induction 46. Sequencing of the target sequences confirms gene deletion, and therefore confirms poly(I:C) quality (data not shown).
Src tyrosine 416 phosphorylation profile of HEK-293T cells induced with a lab poly(I:C) or a control poly(I:C) aliquots (Figure 6) did not show activation of the TLR3 signalling pathway.
4.2.2 Is TLR3 expressed in cells?
To address whether TLR3 is expressed in cells, HEK-293T cells transfected with TLR3 were immunoblotted for TLR3 (Figure 7).
Results from immunoblot for TLR3 (Figure 7) show that TLR3 is present in HEK-293T cells transfected with TLR3-expressing plasmid.
To know whether the TLR3-expressing plasmid has no mutations, I shall sequence the plasmid. 43 KDa
60 KDa
Figure 6 - Activation of the TLR3 signalling pathway in HEK-293T. HEK-293T cells were transfected with TLR3 expressing plasmid. 48 h post-transfection, cells were stimulated with 100 µg/ml Poly (I:C) during 3 h or left untreated, and then lysed. Samples were separated on 12% SDS-PAGE and detected with, anti-P-Src Y416. Actin was detected as a control for protein loading, with anti-β-Actin.
43 KDa ~100 KDa
Figure 5 - TLR3 presence in HEK-293T. HEK-293T cells were transfected with TLR3 expressing plasmid. Sample was separated on 12% SDS-PAGE and detected with anti-TLR3. Actin was detected as a control for protein loading, with anti-β-Actin
12 To address whether I could activate the TLR3 signalling pathway in HEK-293T using other TLR3-expressing plasmids, I immunoblotted HEK-293T samples, induced or not with poly(I:C), for IκB and Src phosphorylated in tyrosine 416. The results from these immunoblots (data not shown) show no activation of the TLR3 signalling pathway.
To address whether there was no activation of the TLR3 signalling pathway due to low efficiency of TLR3-expressing plasmid, constitutively TLR3-expressing cells will be tested in the lab for activation of the TLR3 signalling pathway.
4.2.3 Cell “drifting” Hypothesis
During prolonged maintenance in culture cell lines do change, due to selection of mutated cells with a growth advantage, raising the possibility that the HEK-293T cells may have lost some part of the TLR3 signalling pathway. To address whether the TLR3 signalling pathway could be activated in other cell lines, Cos1 cells, Cos7 cells, NIH3T3 cells, Vero cells (Figure 8) and RPE1 cells (Figure 9) were tested for TLR3 signalling pathway activation by evaluating the predicted degradation of IκB and observation of an increase in Src phosphorylation.
13 Results from immunoblot on Cos1 cell line upon poly(I:C) induction show TLR3 signalling pathway activation by IκB degradation and an increase in Src phosphorylation in tyrosine 416 (Figure 8). RPE1 cells also showed activation of the TLR3 signalling pathway by IκB degradation (Figure 9). RPE1 cells were stimulated during 75 min according to García-Cattaneo, Gobert and Müller et al.47. No other cell line was showed TLR3 signalling pathway activation (Figure 8) through an increase in Src phosphorylation in tyrosine 416 and degradation of IκB upon treatment with poly(I:C).
43 KDa 60 KDa
60KDa
Figure 8 - Activation of the TLR3 signalling pathway in several cell lines. Cos1, Cos7, NIH3T3 and Vero cells stimulated with 100 µg/ml Poly (I:C) during 3 h or left untreated, and then lysed. Samples were separated on 12% SDS-PAGE and detected with anti-IκB, , anti c-Src anti-PY416 and anti-IκB. Actin was detected as a control for protein loading, with anti-β-Actin.
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The RPE1 cells were also tested for TLR3 signalling pathway I329L-mediated inhibition using the IRF3 promoter Luciferase Reporter Assay for IRF3 and IκB degradation profile (Figure 9).
The Luciferase Reporter Assay of the IRF3 signalling shows a 5-fold activation of the TLR3 signalling pathway and an inhibition by I329L resulting in a 2-fold activation of the TLR3 signalling pathway in RPE1 cells (Figure 9-A).
IκB is degraded upon poly(I:C) induction (Figure 9-B). This shows that the TLR3 signalling pathway is activated in RPE1 cells. However, IκB is still degraded when cells express I329L (Figure 9-B), which means that a 2-fold activation of the TLR3 signalling pathway (Figure 9-A) is not enough for observing such downstream events. Other reasons for IκB degradation in cells expressing I329L may rely on I329L-expressing plasmid mutations and time of induction of the TLR3 signalling pathway is not enough for observing downstream events like IκB degradation.
4.3 Why do RPE1 cells do not show inhibition of the TLR3 signalling pathway?
Sanger sequencing shows the I329L-expressing plasmid is not mutated.
Personal communication from Sílvia Correia, Potdoc from from the research group in which this project takes place, shows an attempt to test TLR3 signalling pathway activation in RPE1 by IκB degradation using other timepoints of induction of the TLR3 signalling pathway. Induction of the TLR3 signalling pathway from 15 to 200 min, taking timepoints every 15 min, shows activation of the TLR3 signalling pathway in RPE1 started at 1 hr post induction and that the TLR3 signalling pathway is no longer activated after 2 hr post induction. Also, no inhibition of the TLR3 signalling pathway by the I329L in RPE1 was observed by IκB degradation before the TLR3 signalling pathway is no longer activated.
A B
43 KDa 39 KDa
Figure 9 - Activation and I329L-mediated inhibition of the TLR3 signalling pathway in RPE1 cells. A) Luciferase reporter assay of I329L-mediated inhibition of IRF3 signalling. RPE1 cells were co-transfected empty vector or I329L expressing plasmids, β-galactosidase and and IRF3 promoter luciferase reporter plasmids. At 48-h post-transfection, cells were stimulated with 25 µg/ml poly (I:C) for 5 h or left untreated. Luciferase activity was normalized to β-galactosidase activity as a control for transfection efficiency. Data expressed as mean relative luciferase units ± SD (triplicate determinations). B) Impact of I329L on TLR3 signalling pathway-mediated IκB degradation. RPE1 cells were transfected with empty vector or I329L expressing vector. 48 h post-transfection, cells were stimulated with 100 µg/ml Poly (I:C) during 3 hr for 75 min or left untreated, and then lysed. Samples were separated on 12% SDS-PAGE and detected with anti-IκB. Actin was detected as a control for protein loading, with anti-β-Actin.
0 2 4 6 8 10 12 14 EV I329L arbri trar y l ight units no induction poly(I:C)
15
Conclusion
My goals in this project on developing simple and economical approaches to confirm TLR3 signalling pathway activation and its I329L-mediated inhibition in cells were not fulfilled. However, my work has showed to be a valuable tool in future projects concerning I329L-mediated inhibition of the TLR3 signalling pathway.
Previous results from the research group in which this project took place (Master’s thesis of Pedro Moura, 2015, Faculdade de Ciências da Universidade de Lisboa) demonstrated that HEK-293T cell line shows activation of the TLR3 signalling pathway and its inhibition by I329L. Using the same system, however, I was unable to demonstrate TLR3 activation in the HEK-293T cells.
The fact that HEK-293T cells were once used as a model in the current research group to study the TLR3 signalling pathway indicates that these cells may have suffered a genetic “drift”. An Efficiency Cloning Analysis must therefore be performed in HEK-293T to test the genetic “drift” hypothesis. Also, because these cells do not express TLRs, and they need to be transfected with TLR3-expressing plasmids. I believe TLR3-expressing plasmid degradation may also be a crucial factor to be tested.
Being the TLR3 signalling pathway highly conserved among vertebrates and invertebrates, mammalian cell lines like HEK-293T, Cos7, NIH3T3 and Vero were supposed to show TLR3 signalling pathway activation. However, my results show these cell lines are not good models for studying the TLR3 signalling pathway since TLR3 signalling pathway was not activated in these cell lines. Probably, these cells may lack proteins from the TLR3 signalling pathway or may express proteins which impair the TLR3 signalling pathway. Some hope came from the work with the RPE1 cell line, but here the difference in IκB was higher than desirable. Thus, activation of the TLR3 signalling pathway is observed in the RPE1 cell line, but not its inhibition by I329L. A inhibition of the I329L by cell proteins expressed in RPE1 may be the cause for TLR3 not being inhibited by I329L.
My thesis is therefore of a great importance for leading the research on the impact of I329L on the TLR3 signalling pathway by addressing cells lines to use as a model and cell lines not to use.
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