Title of the Thesis

In vitro Inhibition of Feline Leukaemia Virus Infection by Synthetic Peptides Derived from the Transmembrane Domain

Graduate School for Cellular and Biomedical Sciences University of Bern PhD Thesis

Submitted by Eva Johanna Bönzli from Tschugg (BE)

Thesis advisor

Prof. Dr. Hans Lutz Clinical Laboratory Vetsuisse Faculty of the University of Zürich

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Accepted by the Faculty of Medicine, the Faculty of Science and the Vetsuisse Faculty of the University of Bern at the request of the Graduate School for Cellular and Biomedical Sciences

Bern, Dean of the Faculty of Medicine

Bern, Dean of the Faculty of Science

Bern, Dean of the Vetsuisse Faculty Bern

“The cat is domestic only as far as suits its own ends”

Saki (Hector H. Munro, 1870-1916)

TABLE OF CONTENTS

SUMMARY ...... 1 INTRODUCTION & AIM ...... 4 The Feline Leukaemia Virus (FeLV) ...... 5 Retroviruses ...... 5 Origin & Structure of FeLV ...... 5 FeLV Entry into the Host Cell...... 6 “Competitor” Peptides Interfering with the FeLV Entry Step ...... 9 Aims of the Thesis ...... 11 Motivation and Significance ...... 11 Importance of FeLV in Veterinary Medicine ...... 11 Importance of FeLV in Human Medicine ...... 13 References ...... 14 RESULTS: MANUSCRIPT ...... 20 DISCUSSION ...... 39 References ...... 41 PERSPECTIVES & OUTLOOK ...... 43 Perspectives in Veterinary Medicine ...... 44 Perspectives in Human Medicine ...... 45 Future Antiviral Peptides ...... 46 References ...... 47 ACKNOWLEDGEMENTS ...... 52 CURRICULUM VITAE ...... 54 DECLARATION OF ORIGINALITY ...... 57

SUMMARY

Introduction. Exposure to the feline leukaemia virus (FeLV), a naturally occurring gammaretrovirus of felines worldwide, may cause persistent viraemia and associated fatal diseases in a portion of infected cats. Several effective vaccines exist today, but currently there is no therapy for FeLV-viraemic cats. Infected cats consequently are either euthanized or die painfully. Peptides derived from the viral transmembrane (TM) protein, which effectively inhibit viral entry in vitro, have been identified for human and animal lentiviruses (e.g. human immunodeficiency virus (HIV), and feline immunodeficiency virus (FIV)), which are closely related to FeLV. It has been shown that these peptides interfere with some early steps in the viral replication cycle, the fusion of the viral and the cell membrane, which is crucial for a virus to infect a host cell. Aim. In this thesis, the insight gained with the synthetic peptides against HIV and FIV were intended to be transferred to the design and in vitro testing of synthetic peptides to block infection of feline cells by FeLV. Materials and Methods. Nine overlapping peptides from the FeLV TM were selected and modified. Their sequences were between 15 and 37 amino acids (aa) long. Peptides were challenged with FeLV subtype A on different feline cells as a substrate. Results were evaluated by enzyme-linked immunosorbent assay (ELISA) by quantifying the viral protein p27 in the cell culture supernatants after one week. Results. A 37 aa wildtype peptide EPK364 with sequence homology to the known HIV fusion inhibitor T20, displayed inhibitory effect of up to 88% if added to a culture of cat fibroblasts several hours after infection by FeLV subtype A. None of the shorter peptides decreased the viral replication. Further studies were performed with modifications of the sequence of EPK364 to support its stability, amphiphilic characteristics, and to increase its solubility. These modified variants of EPK364 indeed showed good inhibitory activity (58% and 27%, respectively), but less than the wildtype peptide. A cell viability test in the presence or absence of the three peptides was also performed. It revealed that the presence of peptide is not toxic for the cells; the cellular growth was neither influenced by EPK364, EPK397, nor by EPK398. Discussion. The inhibitory potential of the three 37 aa peptides may be due to the fact that longer peptides are in general more stable compared to short peptides. The reduced inhibitory effect of the modified variants may be due to loss of essential amino acids in both peptide sequences, generating a trade-off between increasing peptide stability/solubility due to modifications, and peptide binding capacity. It might be also considered that either the peptide working mechanism or the entry mechanism of gammaretroviruses is different from lentiviruses.

Perspectives. Inhibitory peptides might become important not only for veterinary use as therapeutic agent for viraemic cats, but also for the human medicine. Today, xenotransplantation is a method to fill the gap of the shortage of appropriate human organs. Animal (e.g. pig, baboon) organs serve as substitutes, but the transfer to the human acceptor is threatened by undetected gammaretroviruses that may induce infections and possibly tumours in humans. An antiviral therapy that inhibits these types of infections in immunosuppressed patients may increase the safety in xenotransplantations.

INTRODUCTION & AIM

The Feline Leukaemia Virus (FeLV)

Retroviruses

Viruses are small infectious agents that replicate in living cells of all organisms, animals (including humans), plants, bacteria, and even archaea [1]. Viral particles consist of genetic material made from either DNA or RNA, a capsid (protein coat) that encloses the genetic material, and in some cases an envelope of a lipid bilayer, i.e. a membrane, which surrounds the capsid. The envelope is typically derived from portions of the host cell membranes but include some viral glycoproteins. Among the viruses, the family of the Retroviridae (retroviruses) is a large and diverse group of enveloped viruses with an RNA genome. They are found in all vertebrates including humans. A common attribute of all retroviruses is a virus-transcribed enzyme, the reverse transcriptase (RNA-dependent DNA polymerase) [2] which is needed for an important replication step within the host: after entry into the host cell cytosol, the genomic single- stranded RNA of the retrovirus is first reverse-transcribed by the reverse transcriptase into double-stranded DNA, and then inserted by another viral enzyme, the integrase, into the host chromosomal DNA located in the nucleus of the cell. Integration is crucial for the “survival” of the virus in the cell. The viral insert (called “provirus”) serves as a template and is transcribed by the host‟s enzymes into new viral RNA molecules which leave the nucleus and re-enter the cytosol. Some RNA molecules are translated by host ribosomes into proteins which are necessary for the assembly of the new virus particles. Finally, the new retroviral particles leave the cell by budding. During this process, they take some host cell membrane with them to complete the new viral envelope. The assembly process is finished when the virus has escaped from the cell. Infection and all the events associated with the replication cycle normally do not cause cell death of the host cell.

Origin & Structure of FeLV

The feline leukaemia virus (FeLV) belongs to the family of the retroviruses and the genus of the Gammaretroviridae (gammaretroviruses). It was first isolated and described in 1964 by Jarrett and co-workers [3]. FeLV is a naturally occurring gammaretrovirus affecting cats worldwide. Therefore, it is of great veterinary importance. Besides FeLV, there are some other viruses affecting cats, but only the feline immunodeficiency virus (FIV) from the genus of the Lentiviridae (lentiviruses) is considered to be of similar veterinary importance. Four subtypes (FeLV-A, -B, -C, -T) can be distinguished by specific differences in their envelope proteins and by the receptors they require for entry into the host cell defining their

host cell tropism [4-6]. FeLV-A is the transmitted form of FeLV, whereas the other subtypes arise through mutations in the gene encoding the FeLV-A envelope protein, or by recombination with endogenous FeLV (enFeLV) [7, 8] which integrated during cat evolution in the germline cells of the cat. FeLV is an enveloped, positive sense, single-stranded RNA virus. The viral genome carries three genes: gag-pol-env. Gag (group specific antigen) encodes a precursor protein which is cleaved to four inner structural core and matrix proteins termed CA (p27), NC (p10), MA (p15), and p12; pol (polymerase) encodes the enzymes protease, reverse transcriptase, and integrase which are also synthesized from a precursor protein; and env (envelope) encodes a precursor which is cleaved to form the two envelope proteins, the surface protein SU (gp70), and the transmembrane protein TM (p15E). In contrast to TM, SU is glycosylated [9]. SU and TM are linked together by disulphide bonds and form a heterodimer. SU represents the major antigen of the virus, and is responsible for binding to the correct host cell receptor, whereas TM anchors SU in the membrane, and is responsible for the processes of the viral entry (infection) step.

FeLV Entry into the Host Cell

To ensure its further existence, FeLV has to replicate within the host cell. Thus, it has to overcome the host cell membrane, a lipid bilayer representing the first defence mechanism of cells against viruses. In the entry step of FeLV, this problem is addressed by binding of the virus to a sensitive host cell that has a surface receptor enabling the virus to bind, followed by fusion of the viral membrane with the host cell membrane. Fusion of two lipid bilayers requires that they come into close contact by local membrane bending, which is the result of different conformational rearrangements in the viral envelope protein. The proteins on the viral surface solely provide the energy necessary for this membrane deformation and bending [10]. There is little knowledge about the entry mechanism of gammaretroviruses. However, it is assumed that retroviruses have evolved a common entry mechanism [11-14], which represents the basis for this study. This common entry mechanism is illustrated in Figure A and described as follows: Membrane fusion is mediated by the viral envelope proteins, SU and TM. Binding of SU to the corresponding cell surface receptor(s) converts the whole envelope protein from a “resting” or “non-fusogenic” state into a “fusion-active” state. First, virus binding induces conformational changes in SU (SU is for the further steps not important any more, because its only mission was to bind to a correct host cell which was “tagged” by a matching receptor), which leads to exposure of the TM and insertion of a small part at the N

(amino)-terminal end of the TM, i.e. the fusion peptide (Fig. 1 A in chapter “Results”), into the host cell membrane. The fusion peptide insertion anchors the virus in the host cell membrane, and prepares it for the entry. The exposed TM conformation spanning between both bilayers is called “prehairpin intermediate state” (Fig. A, b). Being hydrophobic due to exposure, the prehairpin intermediate refolds shortly afterwards into a more stabilized conformation called the “trimer-of-hairpins” which represents the fusion-active form. The trimer-of-hairpins is the result of the refolded structure of the TM. To be able to refold into the structure of the trimer-of-hairpins, both domains of the TM, namely the TM C (carboxy)- terminal domain near the viral membrane, and the N (amino)-terminal domain near the fusion peptide (Fig. 1 in chapter “Results” & Fig. A), are needed. The C-terminal helices fold back and pack in antiparallel direction into the grooves formed by the N-terminal helices. In this final trimer-of-hairpins or “six helix bundle” structure (Fig. A, d), the central N-terminal helices are termed the “core coiled-coil” (Fig. 1 B in chapter “Results”). This process is an important step, because during this rearrangement mechanism, both bilayers are forced together for the induction of a fusion pore. The mechanism which finally leads to formation of the fusion pore is still unclear. The initial contact site of both bilayers may induce monolayer rupture that allows mixing of lipids from the two outer monolayers, generating a so-called “fusion stalk” [15, 16]. Expansion of this stalk may lead to direct opening of a fusion pore, or to the formation of another intermediate, the “hemifusion diaphragm”, a local bilayer resulting from the contact between the two inner monolayers of the fusing membranes [15]. The break of the hemifusion diaphragm may result in fusion pore formation. This fusion pore may be further enlarged to complete the fusion. The pore represents the gate for the viral genome to enter into the host cell cytosol to continue its replication cycle.

Figure A

The common entry mechanism of retroviruses. The dark bilayer represents the viral membrane, the white bilayer represents the host cell membrane. Each picture shows a cross-section of the helical arrangement of the TM during viral entry. a. The viral envelope protein in its native state; SU (olive- shaped) covers some parts of the TM. b. After binding of SU (SU is not shown in the following pictures) to the cell, the TM integrates into the cell membrane (prehairpin intermediate state). The white part represents the C (carboxy)-terminal domain, the grey part represents the N (amino)-terminal domain. c. Soon after, the prehairpin collapses by folding the C-terminal helices back to the N-terminal

helices. Due to this movement, the membranes are pulled together. d. The collapsed viral TM is represented by the trimer-of-hairpins structure or “six-helix bundle” (cross-section: The N-terminal helices in grey represent the core coiled-coil). The membranes were forced close enough to undergo an intermediate fusion, the “fusion stalk”. e. The extended fusion stalk (hemifusion of the two membranes) finally leads to formation of a fusion pore (f).

“Competitor” Peptides Interfering with the FeLV Entry Step

Based on this complex cascade of conformational rearrangements, peptides against human and animal retroviruses were identified, e.g. HIV (human immunodeficiency virus; lentivirus), HTLV (human T-cell leukaemia virus; deltaretrovirus), SIV (simian immunodeficiency virus; lentivirus), and FIV (feline immunodeficiency virus; lentivirus) [17-20] that effectively inhibit viral replication, probably by blocking the fusion of the two membranes. Thus, these peptides with antiviral potential were termed “fusion inhibitors” or “entry inhibitors”. Fusion inhibitors mimic regions of the viral N- and C-terminal domains of the TM. A 36 amino acid long peptide termed T-20 corresponding to a region in the C-terminal part of the HIV TM was detected to block de novo infection of HIV [21]. This peptide has been approved for use in the treatment of HIV as additional component of the highly active antiretroviral therapy (HAART) [22]. Clinical trials have shown that peptides are safe and have a low toxicity profile [23, 24]. In the lentivirus FIV, a fusion inhibitor termed C8 was detected in the C-terminal region of the FIV TM inhibiting viral replication in vitro [20]. The envelope glycoprotein of FeLV subtype A has previously been cloned and sequenced [25], which gave the opportunity to compare the TM sequences of FeLV with the TM sequences of HIV and FIV. In the FeLV C-terminal region which is homologous to HIV and FIV, structural similarities were found, e.g. a motif comprising three of the relatively rare aromatic amino acid Tryptophan (W) in close proximity. In FIV, this motif with the Tryptophans was shown to be essential for the virus to infect the cell [26]. Thus it was concluded that this motif has a special function in the viral entry process, and possibly in inhibiting the entry process. The working mechanism of antiviral peptides is not completely understood. This study was based on the hypothesis that antiviral peptides interfere in a competitive manner with the C- terminal helices during the refolding process of the prehairpin intermediate. Derived from the C-terminal region of the TM, these antiviral peptides may bind to the N-terminal regions of the TM during the prehairpin intermediate state (Fig. B). Inhibitory peptides that bind into the grooves of the core coiled-coil, may block the backfold of the C-terminal helices to the N- helices. Having a competitor peptide present, the whole prehairpin may be stiffened and

therefore, rearrangement into the fusion-active trimer-of-hairpins structure might not be possible any more (Fig. B). The hypothesis for this study was constructed as follows: synthetic peptides derived from the C-terminal domain of the FeLV TM are able to prevent host cell infection by FeLV.

Figure B

Inhibition of the entry step of FeLV. The dark bilayer represents the viral membrane, the white bilayer represents the host cell membrane. The cross-sections in each picture show the helical arrangement of the TM during the first steps in viral entry, and in the presence of entry inhibitors. a. If synthetic entry inhibitors (represented as white peptides with a putative helical secondary structure) mimicking parts of the C (carboxy)-terminal domains are present, the virus particle with its envelope protein in the native state, might not be affected at all. b. However, if the virus is in the prehairpin intermediate state after binding to a host cell, synthetic peptides may act and bind to their target at the exposed N (amino)-terminal domain. The cross-section shows that entry inhibitors bind into the grooves formed by the N-helices, as usually the C-terminal helices during refolding would do to form the trimer-of-hairpins. c. Refolding is not possible any more and viral entry is blocked.

Aims of the Thesis

The aims were defined as follows: Design and synthesis of peptides derived from the FeLV envelope protein predicted to interfere with the viral TM and therefore prevent virus entry into the cell Assessment of the direct antiviral activity of the peptides in vitro Improvement of the most potent antiviral peptides by modification of their sequences

In detail, the first aim of this thesis was to find peptides derived from the viral transmembrane protein of FeLV that have the potential to reduce viral replication by inhibiting the fusion of the viral and the cell membrane. With the knowledge that there are already synthetic peptides that effectively inhibit entry of several viruses, especially lentiviruses (HIV, FIV), the idea was to find the first antiviral peptides for inhibition of FeLV, a gammaretrovirus. Thus, one part of the study aimed to analyse the amino acid sequence of the FeLV TM, and to compare it with the homologous sequences in the TM of HIV and FIV. During this analysis, similar motives as in HIV and FIV peptides attracted special attention. Thus, 11 different peptides from the C-terminal domain of the FeLV TM were chosen and produced synthetically. The aim of the second part was to establish an in vitro assay to test the synthetic peptides for their activity, and to find the most potent antiviral compound. To address this part of the project, several approaches using feline cells as base to test the peptide activity were developed and evaluated. The method to measure the results was quantification of the viral antigen p27. The last aim after the detection of a potent peptide was to improve its effectiveness by increasing its alphahelicity, solubility, as well as its stability. This aim was achieved by replacing specific amino acids in the peptide sequence to have an optimised structure with amphiphilic properties. An amphiphilic peptide structure was considered to be optimal for target binding.

Motivation and Significance

Importance of FeLV in Veterinary Medicine

Knowledge about the entry mechanism of gammaretroviruses is poor, and knowledge about the working mechanism of synthetic peptides against gammaretroviruses is even worse. Therefore, this study on the inhibition of FeLV entry may contribute to a better understanding of the underlying molecular processes of gammaretrovirus entry and inhibition mechanism. Since antiviral peptides may have therapeutic potential, the importance in veterinary medicine is obvious. FeLV still represents a significant source of mortality and is the leading

cause of infectious deaths in cats. FeLV is mainly transmitted directly from cat to cat via the oro-nasal route, where transmission occurs through saliva, which represents the main infection source. Infected saliva is transmitted via licking, mutual grooming, by bites during fights, or it is transmitted indirectly by sharing of food- and water dishes [27, 28]. FeLV can also be transmitted by faeces and milk [29, 30]. FeLV RNA was also detected in the urine of sick cats, but its infection potential has not been demonstrated so far [31]. In the body of the cat, the virus replicates first in lymphoid tissues such as local lymph nodes. Through infected lymphocytes, it reaches the bone marrow. From there, it can perfectly spread throughout the body, because it is integrated in blood cells. After arriving at epithelial cells, e.g. the salivary glands, it can be transmitted to a new host. The clinical signs induced by FeLV are highly diverse and paradoxical. The pathological picture comprises cytosuppressive as well as cytoproliferative disorders, depending on the main FeLV subtype circulating in the cat. To summarize, FeLV has been associated with anaemia, lymphoma, and other manifestations like immune-mediated diseases as haemolytic anaemia, glomerulonephritis and polyarthritis, peripheral lymphadenopathy, chronic enteritis with degeneration of intestinal epithelial cells and crypt necrosis, and reproductive disorders like foetal resorption, abortion, neonatal death and the “fading kitten syndrome”, but these reproductive disorders are observed rarely today; moreover, neurological diseases that occur mainly as peripheral neuropathies, and direct neuropathogenic manifestations, e.g. abnormal vocalization and paralysis [32-40]. Furthermore, FeLV induces immunosuppression in persistently infected cats, including an AIDS-like immunodeficiency syndrome [41-43]. Once a cat is infected, it is at risk to develop these malignant FeLV-related diseases. There is no therapy so far for use in cats. Antiretroviral drugs available for humans are not suitable for cats due to high toxicity and low efficacy. FeLV not only infects all breeds of the domesticated cat (felis catus), but also other felids as the European wildcat (felis silvestris), the Bobcat (lynx rufus), the European lynx (lynx lynx), the Iberian lynx (lynx pardinus), and the Florida puma (puma concolor coryi) [23, 24, 28-30]. Especially the Iberian lynx and the Florida puma are threatened with extinction [48, 49]. The FeLV prevalence may be influenced by the density of cats and there may be noticeable geographical and local variation. In some European countries, the USA and Canada, the prevalence of FeLV infection in single-cat households seems to be very low, usually less than 1 % [50-52]. FeLV prevalence in Switzerland was found to be 3% in healthy cats and up to 13% in ill cats [52]. However, in large multi-cat households without specific prevention, the prevalence may exceed 20%. In the last years, the prevalence and importance of FeLV in Europe has been decreasing. Decreasing prevalence is the consequence of identification and segregation of infected cats, and of extensive vaccination programs. Today, several vaccines against FeLV are commercially available [44-47].

However, no vaccine is 100% efficacious. Moreover, many owners choose not to vaccinate their cats for different reasons. The unvaccinated cat population (including the stray cat population) remains at risk for infection by FeLV, and is a source for FeLV transmission. Thus, synthetic antiviral peptides may become important as a therapeutic approach for FeLV-infected domestic cats, and also for related felids.

Importance of FeLV in Human Medicine

In addition to the importance of antiretroviral peptides in veterinary medicine, the potential of peptides that act effectively against gammaretroviruses may become significant also in human medicine. Because FeLV is a pathogen with a multifaceted range of clinical signs, it serves as a model for pathogenic activities of HIV [7, 53]. Thus, further knowledge on the FeLV entry mechanisms and related processes could improve the understanding and characterization of human retroviruses including HIV. Moreover, FeLV serves as a model for understanding the mechanisms of cancer initiation and development, and for a better knowledge of the role of viruses in the aetiology of human cancers [54, 55]. Today, there is a need of appropriate donor organs for patients that suffer from organ failure and/or other diseases that result in an organ transplantation. However, many patients never receive a new organ due to a huge shortage of donors. To address this problem, animal organs from specific pathogen-free breedings (e.g. the pig) are considered to be used to temporally replace human donor organs [56, 57]. Such xenotransplantations (the surgical transfer of cells, tissues, or whole organs from one species to another) may find their way into regular human medicine. Especially pigs have several advantages compared to other animals: they are available in large quantities, and they have numerous anatomical and physiological similarities to humans [58]. However, xenotransplantation of pig organs might bring risks, because pigs harbour replication-competent endogenous gammaretroviruses termed PERV (pig endogenous retrovirus) [59]. There are several strategies such as the selection of low-virus producing pigs [60] to prevent transmission of PERV; however, it has to be considered that in pigs there are approximately 50 proviral integration sites in the pig genome [61, 62], which makes it difficult to produce pigs that are completely devoid of all PERV-related elements. Although PERV-related infections could not be detected so far in small laboratory animals and non-human primates after they were exposed to PERV [56], there might be a possibility that infections arise as a consequence of xenotransplanation [63] in immunosuppressed patients. A peptide-based therapy to prevent infections and diseases caused by gammaretroviruses may contribute to the safety in xenotransplantations. Therefore, it is important to know whether there are inhibitory peptides that are successfully

acting in blocking the FeLV entry. In the chapter “Perspectives & Outlook”, the possible use of inhibitory peptides in xenotransplantations is also presented as a future perspective.

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RESULTS: MANUSCRIPT

In vitro inhibition of feline leukaemia virus infection by synthetic peptides derived from the transmembrane domain

Eva Boenzli1*, Céline Robert-Tissot1, Giuseppina Sabatino2,3, Valentino Cattori1, Marina Luisa Meli1, Bernd Gutte4, Paolo Rovero2, Norman Flynn5, Regina Hofmann-Lehmann1, and Hans Lutz1

1Clinical Laboratory, Faculty of Veterinary Medicine, University of Zurich, Zurich, Switzerland

2Laboratory of Peptide and Protein Chemistry and Biology, University of Florence, Florence, Italy

3EspiKem Srl, Prato, Italy

4Institute of Biochemistry, University of Zurich, Zurich, Switzerland

5Retrovirus Research Laboratory, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Glasgow, United Kingdom

Running head

Inhibition of FeLV infection by synthetic peptides

*Corresponding author.

Eva Boenzli, Clinical Laboratory, Faculty of Veterinary Medicine, University of Zurich, Winterthurerstrasse 260, CH-8057 Zurich, Switzerland. Phone: +41 44 635 83 67. Fax: +41 44 635 89 06. E-mail: [email protected]

Abstract Background: The feline leukaemia virus (FeLV) is a gammaretrovirus commonly affecting cats. Infection with this virus often leads to fatal outcomes and, so far, no cure is available for this disease. Synthetic peptides with structures mimicking the transmembrane protein of the viral surface proteins hold the potential to effectively interfere with viral entry by hampering the fusion of viral and host cell membranes and constitute a novel approach for the treatment of infections with retroviruses. We identified and synthetically produced 11 FeLV peptides and evaluated their potential to block FeLV infection in vitro. Methods: Cell cultures were exposed to FeLV subgroup A prior to the addition of the peptides. The inhibitory effect of the peptides was assessed by measuring FeLV gag protein in the supernatant of peptide versus mock-treated cell cultures using an enzyme-linked immunosorbent assay (ELISA). Results: A peptide (EPK364) of 37 amino acids in length, with sequence homology to the HIV fusion inhibitor T-20, significantly suppressed viral replication by 88%, while no effects were found for shorter peptides. Two structurally modified variants of EPK364 also inhibited viral replication by up to 58% (EPK397) and 27% (EPK398). Conclusions: Our data support the identification of synthetic FeLV peptides that have the potential for a curative short-term therapy of viraemic cats. In addition, these peptides may become an important tool in xenotransplantation, where endogenous gammaretroviruses of the donor species may be able to infect the host.

Introduction Retroviruses usually establish life-long infections that frequently result in fatal diseases (e.g. acquired immunodeficiency syndrome, AIDS). Feline leukaemia virus (FeLV) [1] is a naturally occurring gammaretrovirus of domestic cats and wild felids [2-4] including endangered species such as the Iberian Lynx [3] and the Florida puma [5]. While some cats develop persistent viraemia (so-called progressor cats), characterized by high viral loads in blood and tissues, others are able to control viremia (regressor cats) by an efficient immune system. Viraemia may cause fatal diseases including tumours, immunodeficiency, and degenerative diseases of the haematopoietic system [6]. Due to its multifaceted pathogenesis, FeLV infection serves as a model for the study of tumour development and immune suppression [2], as well as for risk assessment in xenotransplantation [7, 8]. As xenotransplantations may gain in importance in the next years, risks linked to the transfer of animal retroviruses to humans have to be assessed beforehand.

For most retrovirus-induced diseases, therapies are either unavailable or prone to the development of resistance [9]. Most current antiretroviral drugs target viral enzymes that are essential for replication [10-12]. In contrast, “entry/fusion inhibitors” constitute a different antiretroviral drug class characterized by its potential to act during very early phases of the viral life cycle [13-15]. These compounds consist of small synthetic peptides that interfere with three major initial steps of viral infection: binding of the virus to the surface of the host cell, fusion of virus and host cell membranes, and viral entry into the host cell. The molecular mechanism linked to entry of FeLV remains poorly understood. However, it is assumed to be similar to the well-described adsorption process of the human immunodeficiency virus (HIV), a closely related retrovirus that belongs to the genus of the lentiviruses. Thus, the entry step is likely to be mediated by the host cell receptor and viral glycoproteins on the surface of the virus. Both the amino-terminal surface subunit (SU), and the carboxy-terminal transmembrane subunit (TM) are most probably involved in virus adsorption and entry [16]. The more detailed model that has been described with respect to the entry mechanisms of HIV [16, 17] suggests that binding of the HIV SU to both the host cell receptor and at least one co-receptor leads to conformational changes in SU. A cascade of rearrangements in TM is then triggered, leading to the insertion of its N-terminal fusion peptide into the target cell membrane. The corresponding TM conformation is called „prehairpin‟, which is, due to its hydrophobic nature, a transient structure that collapses soon after its generation and refolds into a stabilized „trimer-of-hairpins‟. During the latter process, the N-terminal region (NHR) of the TM (downstream of the fusion peptide) first forms a central hydrophobic triple-stranded coiled-coil [18], and the C-terminal region (CHR) then folds in antiparallel orientation in the grooves between two N-helices to complete the FeLV trimer-of-hairpins (Fig. 1 B). This structure is believed to draw the viral and cell membranes together and induce the final membrane fusion [19]. In various virus models, synthetic peptides that mimic parts of the viral TM have been designed with the aim to interfere with viral entry. These peptides may directly interact with the central coiled-coil of the TM, thereby preventing the formation of the trimer-of-hairpins and thus, the induction of the membrane fusion [17]. Effective inhibitory peptides, mapped to an amphipatic alpha-helical region in the ectodomain of the viral TM, have been described e.g. for HIV [13, 14] and for the feline immunodeficiency virus (FIV) [15, 20]. Moreover, a HIV inhibitor (T-20) has been produced commercially (Hoffmann-LaRoche and Trimeris, March 2003) [13, 14, 21] and is currently included in antiretroviral therapy protocols. Very little has been reported to date regarding inhibitory peptides in the context of gammaretrovirus infections. Ou and Silver [22] reported that externally added TM peptides of Moloney murine leukaemia virus (Mo-MLV) did not inhibit viral replication. In contrast, Wallin and co-workers

[23] observed inhibitory activity with peptides constructed to mimic either the NHR or the membrane-spanning region of Mo-MLV TM, but not with various CHR peptides. The peptides used in this study were similarly structured to known entry inhibitors (HIV, FIV). Thus, when the sequences of successful HIV and FIV peptides were compared, a common feature was discerned: a region containing 3 tryptophan residues in close proximity. Giannecchini and co-workers mapped the antiviral activity of their FIV peptide to this region [15]. A similar, though not identical motif is present in the FeLV TM. We therefore designed and tested overlapping peptides from this region. However, some of these peptides were found to become more potent if their sequence was remodelled by replacing specific amino acids at non-conserved sites [24]. The aim of the present study was to evaluate the potential of peptides, specifically derived from the FeLV TM, to inhibit entry of FeLV into host cells in vitro in order to assess their possible use in antiretroviral therapy in vivo, similar to that already included in treatment protocols against HIV [25, 26].

Materials and Methods Cell lines Three immortalized feline cell lines were used. FEA and CrFK were maintained in RPMI- 1640 medium (Sigma-Aldrich, Buchs, Switzerland) supplemented with 10% foetal calf serum (FCS), 1% L-Glutamine (200mM), and 1% penicillin/streptomycin (all from Invitrogen, Luzern, Switzerland). QN10s cells were maintained in D-MEM (Dulbecco‟s modified eagle medium, Sigma-Aldrich) supplemented with 10% FCS, 1% L-Glutamine (200mM), 1% MEM sodium pyruvate, 100IU/ml penicillin, and 100µg/ml streptomycin (all Invitrogen).

Virus strain The molecularly cloned FeLV subgroup A/Glasgow-1 [1], kindly provided by Prof. Os Jarrett was used. Virus was titrated beforehand to determine 50% tissue culture infectious doses 0 (TCID50). To this aim, stock virus was diluted in RPMI-1640 medium to concentrations of 10 , 10-1, 10-2, 10-3, 10-4, and 10-5 and added to 5 x 103 FEA cells/well in a 96-well microtiter plate (six wells per dilution), seeded the day before. Supernatants were tested every second day for the presence of virus with p27 ELISA. TCID50 was calculated according to the Spearman- Karber Formula (M = xk +d[0.5-(1/n)(r)]; xk = dose of highest dilution; r = sum of negative responses; d = spacing between dilutions; n = wells per dilution).

Chemicals 24

To improve the solubility of the peptide EPK211, the organic solvents Dimethylsulfoxide (DMSO), Acetonitrile (MeCN), and Dimethylformamide (DMF) (all from Sigma-Aldrich GmbH, Buchs, Switzerland), were used in final concentrations of 0-10% in RPMI-1640 or water.

Selection and modification of peptides Eleven peptides derived from the FeLV TM were designed (Fig. 1 A). All compounds were obtained from EspiKem (Florence, Italy) and were N-terminally acetylated and C-terminally amidated except for EPK364, EPK397 and EPK398 which had a free C terminus. Modifications of the peptide EPK364 are described in Figures 1A und B. The peptides were synthesized by solid-phase peptide synthesis and purified to levels greater than 85% by analytical high-performance liquid chromatography.

Viral replication inhibition test Viral inhibition experiments were conducted on all three cell lines (FEA, CrFK, QN10s). To evaluate the inhibitory potential of the peptides, experiments were carried out over the period of 9 days. On day 0, 5 x 103 cells/well were seeded in 96-well flat-bottom microtiter plates.

On day 1, 10 TCID50 of FeLV stock virus was diluted in culture medium and added to the cells (100µl/well). The cells were incubated with the virus at 37°C in a 5% CO2 environment (Forma Incubator, Brouwer, Luzern, Switzerland) for periods of 0, 1, 3 and 8 hrs, after which the virus was removed by washing once with HBSS (Hank‟s Balanced Salt Solution 1x, Invitrogen). The peptides were diluted in medium to final concentrations of 50, 5, 0.5 and 0.05µg/ml (which correspond to 12, 1.2, 0.12 and 0.012µM) and added to the infected cells (100µl/well). Control cells were inoculated with FeLV-A and treated with the diluent and/or a control peptide. On day 3, supernatants were discarded and substituted with 100µl of fresh peptide dilutions (final concentrations: 50, 5, 0.5 and 0.05 µg/ml). On day 6, only half of the content of each well was removed and replaced by the same volume of fresh medium without peptide. On day 8, the supernatants were harvested. Each initial incubation period (0, 1, 3, 8 hrs) was tested in triplicates. All experiments were repeated at least twice.

Test for a possible toxic effect of the peptides In order to test for a possible toxic effect of EPK364, EPK397 and EPK398, viable cell counts after treatment with the peptides were compared to those after treatment with a control. For these experiments, 4 x 104 cells/well were first seeded on day 0 in 12-well flat-bottom plates (Milian, Genf, Switzerland). One day later, the cells were treated either with 500µl of peptide in culture medium (final concentration: 50µg/ml), or with medium alone as control. On day 3, an additional 500µl medium that contained peptide was added to the wells (final 25

concentration: 50µg/ml). On day 6, half of the supernatant was removed from each well and replaced by fresh medium without peptide. On day 8, cells were washed once with HBSS to remove all dead cells. Attached, viable cells were detached with 50µl of 0.05% Trypsin (Invitrogen, Luzern, Switzerland). The cell count was determined using a Sysmex XT 2000 (Digitana AG, Hamburg, Germany). Each peptide was tested in triplicate wells. All experiments were repeated at least twice.

Detection of FeLV p27 by an enzyme-linked immunosorbent assay (ELISA) The presence of p27 in the culture supernatant was determined using a sandwich ELISA as previously described [27]. Results are represented as percentages of a defined positive control (culture supernatant of FL-74 feline lymphoblast cell line permanently expressing FeLV), which was designated as 100% positive. Samples had to surpass a threshold of 5% of the positive control to be considered positive [28]. Collected data were processed in MS Excel (Office 2007).

Statistical analysis Data were compiled with Microsoft Excel (Office 2007) and statistical analyses were carried out using PASW® statistics software version 18.0.2 (Polar Engineering and Consulting, Nikiski, USA). The peptide-treated group was compared with the mock-treated group by a Mann-Whitney-U test. P-values < 0.05 were considered to be statistically significant.

Results Effect of different solvents on peptide solubility and cell viability Peptide EPK211, a very promising but highly hydrophobic compound, formed precipitates in water as well as in solutions containing less than 2% DMSO (Dimethylsulfoxide), MeCN (Acetonitrile) and DMF (Dimethylformamide). As cell culture growth was heavily disturbed by concentrations of 2% or more of any organic solvent, further tests with peptide EPK211 unfortunately had to be abandoned. However, the tryptophan-rich region of EPK211 was still present in the sequences of EPK237 and EPK364. Tests with all other peptides were performed using water or cell culture medium as a solvent.

Evaluation of various assay procedures First, the optimal conditions for our virus inhibition assay had to be established using FEA, CrFK and QN10s cells. We chose to use FEA cells for further tests because these cells showed an overall steady growth, and thus were considered most appropriate to reproduce 26

inhibition of virus replication. We could also show inhibition with the other two cell lines, but not to the same extent as with FEA cells (data not shown). Three procedures to determine the effect of the peptides were evaluated in vitro: Simultaneous administration of peptide and FeLV-A, peptide administration prior to viral inoculation, and virus administration prior to treatment with the peptide. Results indicated that the last approach produced most effective inhibition.

Dose dependency of peptide inhibition The test peptides were diluted in water or medium to final concentrations of 50, 5, 0.5 and 0.05µg/ml. As best inhibition of FeLV replication was always induced at a final concentration of 50µg/ml, all further tests were carried out with this concentration.

Effect of peptides on viral replication EPK205, EPK206, EPK207, EPK208, EPK209 und EPK210 did not show any inhibitory effect, or even enhanced viral replication, especially after 1 and 3 hrs of virus incubation (Figure 2A-F). EPK237, designed in an attempt to increase the solubility of EPK211 while conserving its structural qualities, displayed antiviral activity although only at a moderate level (Fig 2G). EPK364, an analogue of the commercially available HIV fusion inhibitor T-20, indicated the highest inhibitory potential, inducing a decrease in FeLV replication of up to 88% (Fig. 2H). Interestingly, the two modified variants of EPK364, namely EPK397 and EPK398, were less effective than expected. EPK397 and EPK398, both modified to enhance and stabilize secondary structure, inhibited viral replication at levels of up to 58% (Fig. 2I) and 27% (Fig. 2K) respectively. Protocol variations with different pH, temperature, and virus incubation times were performed with all peptides (data not shown). Nevertheless, none of these factors improved their inhibitory capacity; on the contrary, most of these changes were incompatible with our cellular assay, most likely due to excessive disruption of cellular homeostasis.

Effect of peptides on cellular growth Incubation of FEA cells with a concentration of 50ug/ml peptide for one week did not significantly affect cell growth when compared to mock-treated cells (Fig. 3). It was concluded that the peptides were not toxic to the cells and that reduced viral propagation was the consequence of specific inhibition by the peptides and not linked to decreased cell viability.

Discussion Using an in vitro assay, we found that peptides of 15-25 aa in length derived from the FeLV TM failed to show antiviral activity, whereas 37 aa peptides significantly inhibited FeLV replication in feline cells. The short peptides (≤ 25 aa) had been included in the evaluation because an eight aa peptide exhibited antiviral activity in a FIV system [15]. However, insufficient stability of the shorter peptides might have caused reduced antiviral activity in our experiments, as longer peptides may generally possess a better conformational stability. This is usually linked to the lower tendency of short peptides to fold in a preferred conformation, such as an alpha-helix or a hairpin. In proteins or long peptides, these conformations are stabilized by a large network of weak interactions, such as hydrogen bonds, ionic or dipolar bonds, and hydrophobic interactions between side chains. Further, longer peptides provide more possible interactions with the target amino acids. Indeed, the HIV gp41 inhibitory peptides T-20 and C34 are 36, and 34 aa in length respectively [29]. Considering this, the short peptide effective against FIV [15] may be an exception, most probably due to its stabilization in a specific conformation by a strong hydrophobic interaction between two tryptophan (W) side chains. With our changes to the sequence of EPK364, we aimed at stabilizing the secondary structure, increasing the amphiphilic content, and thus optimizing the interaction mechanism between the inner coiled-coil trimer of the virus TM and the peptide inhibitor (Fig. 1 B). The helical wheel plot of EPK364 reveals an amphiphilic helix with most of the hydrophobic residues clustered on the same face of the helix (Fig. 1 C.1). The conservatively modified EPK397 was designed to generate a cluster of hydrophilic residues (K, E) on one side of the helix (Fig. 1 C.2). Replacements in EPK398 further optimized the amphiphilic arrangement of the helix (Fig. 1 C.3). However, EPK397 and EPK398 gained no obvious benefit from these modifications. Our observations lead to speculate that conservation of the unusual clustering of a rare aromatic aa W in the CHR is a more important property of peptides for viral inhibition than optimization of their secondary structure at the expense of this W residue. The peptide concentration of 50µg/ml (12µM) required for viral inhibition remained relatively high when compared to the reported HIV peptide concentration of 80ng/ml (18nM) [13]. This may be due to self-aggregation of peptides, or to the sterically and kinetically restricted exposure of the prehairpin intermediate state during FeLV entry. The need to arrest the gammaretrovirus Mo-MLV (Moloney murine leukaemia virus) at an intermediate stage of its entry to reach a decrease in viral replication by synthetic peptides [23] suggests that the prehairpin collapses soon after its generation, leaving only little time for antiviral peptides to interact. Concerning the kinetics of the entry mechanism, lentiviruses may behave differently, i.e. it was shown that the prehairipin intermediate 28

state in HIV entry lasts for 15 to 20 minutes [29]. In addition to possible differences in the stability of the prehairpin intermediate state, a completely different mechanism of peptides, i.e. without involvement of the prehairpin state, should be considered. Indeed, it was shown that peptides derived from different domains of the same virus can result in diverging inhibition mechanisms [24]. Thus, the virus may not only be inhibited during its entry step, but as shown also during budding. The increase in viral replication observed one hour post inoculation of peptide-treated cells is consistent with findings previously reported [30]. It is unclear by what mechanisms the divergent effects are caused. Whereas HIV-positive patients require lifelong application of inhibitory peptides, it is reasonable to assume that FeLV-infected cats can overcome viraemia after a short-term peptide treatment. This hypothesis is supported by the spontaneous overcome of viraemia in many cats. Therefore, we speculate that in cats that have not been able to overcome viraemia, the lowering of viral burden with short-term peptide treatment may give the immune system the opportunity to mount a successful immune response and suppress productive viral replication and viraemia. In addition to this potentially new therapeutic approach in veterinary medicine, peptides against gammaretroviruses may gain much importance in human medicine as well. It is well possible that after xeontransplantation which may grow in importance, an endogenous animal gammaretrovirus may cross the species barrier and severely infect humans. This could develop because once in humans the glycosylation of the emerging virus is made by human cells and thus, the host‟s immune system may not recognize the emerging virus as foreign [31]. The presence of undetected replication- competent gammaretroviruses of animal origin in human recipients may lead to systemic infections and tumours. Hence, a therapy based on synthetic inhibitory peptides may become of primordial importance in the process of organ transfer, as such a treatment holds the potential to inhibit gammaretroviruses. Altogether, we have developed a synthetic peptide that indicated promising potential in cell culture. Future studies will determine the feasibility to use this peptide in vivo. Also, our study supports the need to further understand the differences in entry mechanisms of members of the retrovirus family in order to construct effective peptide inhibitors for viruses with pathogenic potential in animals and humans.

Acknowledgments We thank Beatrice Weibel, Enikö Goenczi and Theres Meili Prodan for their contributions in laboratory work. We are grateful to Dr. Maik Hadorn for useful discussions and completion of 29

the figures. We thank Prof. Os Jarrett and Prof. Margaret Hosie, Glasgow, who kindly provided the FeLV-A virus stock. The laboratory work was carried out using the logistics of the Centre of Clinical Studies, Vetsuisse Faculty, University of Zurich. This work was supported by a grant obtained from the Promedica Foundation, Chur, Switzerland. Regina Hofmann-Lehmann was the recipient of a professorship from the Swiss National Foundation (PP00P3-119136). This study was conducted by E. Boenzli in partial fulfilment of the requirements for a PhD thesis at the Vetsuisse Faculty, University of Bern.

Disclosure statement The authors declare no competing interests.

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24. He Y, Xiao Y, Song H, et al. Design and evaluation of sifuvirtide, a novel HIV-1 fusion inhibitor. J Biol Chem 2008; 283:11126-11134.

25. Kilby JM, Hopkins S, Venetta TM, et al. Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry. Nat Med 1998; 4:1302-1307.

26. Molto J, Ruiz L, Valle M, et al. Increased antiretroviral potency by the addition of enfuvirtide to a four-drug regimen in antiretroviral-naive, HIV-infected patients. Antivir Ther 2006; 11:47-51.

27. Lutz H, Pedersen NC, Durbin R, Theilen GH. Monoclonal antibodies to three epitopic regions of feline leukemia virus p27 and their use in enzyme-linked immunosorbent assay of p27. J Immunol Methods 1983; 56:209-220.

28. Hofmann-Lehmann R, Huder JB, Gruber S, et al. Feline leukaemia provirus load during the course of experimental infection and in naturally infected cats. J Gen Virol 2001; 82:1589-1596.

29. Hrin R, Montgomery DL, Wang F, et al. Short communication: In vitro synergy between peptides or neutralizing antibodies targeting the N- and C-terminal heptad repeats of HIV Type 1 gp41. AIDS Res Hum Retroviruses 2008; 24:1537-1544.

30. Mizukoshi F, Baba K, Goto Y, et al. Antiviral activity of membrane fusion inhibitors that target gp40 of the feline immunodeficiency virus envelope protein. Vet Microbiol 2009; 136:155-159.

31. Weiss RA. Transgenic pigs and virus adaptation. Nature 1998; 391:327-328.

32. Otaka A, Nakamura M, Nameki D, et al. Remodeling of gp41-C34 peptide leads to highly effective inhibitors of the fusion of HIV-1 with target cells. Angew Chem Int Ed Engl 2002; 41:2937-2940.

Figure 1

Design and modifications of peptides. A: Schematic representation of the FeLV TM, the peptides covering the amino acids (aa) 541-585 (GenBank: AAA93093.1), and the HIV peptide T-20. C-C: Cysteine-loop. B. Schematic representation of the FeLV trimer-of-hairpins according to the helical wheel concept as described previously [32]. The projection represents the central triple stranded coiled-coil (grey) with each helix turning clockwise, and the outer triplet (black) with each helix turning anti-clockwise. The aa are plotted in a rotating manner from a-g, where the angle of rotation between consecutive aa is 100°. Bonds between the inner and outer helices are formed between positions d and g as well as between e and a. An inhibitory peptide holds the position of one of the helices of the outer triplet by binding into the groove formed by two central helices. C. Based on the helical wheel projection, EPK364, EPK397 and EPK398 are presented in 1 C.1-3 to visualize possible hydrophobic and hydrophilic interactions between the inner coiled-coil trimer [18] and an inhibitory peptide (1 B). Aa at positions b, c, f and g are located at potential solvent- accessible sites and were replaced with glutamic acid (E) or lysine (K). The negatively (E) and positively (K) charged aa were arranged between positions i and i+4 along the peptide sequence in order to create an optimal environment for aa interactions. The high propensity of E & K to form alphahelical structures allow the generation of intrahelical salt bridges to stabilize the helical conformation and increase the solubility of the peptide. Aa at positions a, d and e are facing the hydrophobic groove of the coiled-coil trimer (1 B) and usually are not replaced. In EPK364 (C.1), no aa was replaced. Aa facing the hydrophobic groove of the coiled-coil were maintained in EPK397 but aa at solvent-accessible sites were substituted with E and K (C.2). In EPK398, the aa facing the hydrophobic groove were partially replaced by leucine (L) (C.3).

Figure 2

Inhibitory effect of synthetic peptides on the replication of FeLV. p27 antigen was measured 7 days after inoculation with FeLV-A. Negative inhibition (increase of virus replication, white bars) and positive inhibition (decrease of virus replication, grey bars) is given as percentage after 1, 3 and 8 hrs of virus incubation compared to a positive control (100%). Peptide concentration: 50µg/ml. Cell line: FEA. Significance levels: *p ≤ 0.05; **p ≤ 0.01. Data are shown as means of triplicates.

Figure 3

Cell viability. Influence of peptides EPK364, EPK397, and EPK398 on FEA growth. Results represent the cell numbers/μl * 1000. White bars: untreated cells; grey bars: peptide treated cells. Viable cells were counted 7 days after treatment with peptides. Peptide concentration: 50µg/ml. All data are shown as means of triplicates.

DISCUSSION

In this study, the efficacy of inhibitory peptides to block replication of FeLV in vitro was shown for the first time. This study further raises some issues discussed in the following paragraphs. The lack of efficacy of the short 15 amino acid (aa) peptides to inhibit/reduce the virus replication was unexpected. The short peptides even seem to increase the replication (the reason for the increase might be explained by different unspecific interactions between peptide, virus, and cell [S. Giannecchini, personal communication]), whereas replication is decreased by up to 88% by the long, i.e. 37 aa long, peptides. In general, short peptides are probably less stable than long peptides because of fewer interactions between the amino acids. This might support our observation that only the longer peptides may be stable enough to bind and interfere with the virus. Because some of the short peptides have the same sequence as part of the long and efficient EPK364 (Fig. 1 in chapter “Results”), it is likely that the differences in the inhibitory potential of long and short peptides are caused by their length and not their sequence alone. Indeed, also the HIV antiviral peptides T-20 and C34 are 36 and 34 aa in length, respectively [1]. A very short eight amino acid peptide is known to inhibit FIV in vitro [2], although this might be an exception. EPK364 added to feline cells after challenge with FeLV-A showed an efficacy of up to 88% in blocking viral replication. Challenge of cells with FeLV before addition of peptides mimics what might happen in a viraemic cat. To further increase this efficacy, the wildtype peptide was thought to become more soluble, stable, and amphiphilic if specific modulations in the sequence were performed (modified variants: EPK397, EPK398). The idea of having an amphiphilic molecule with a hydrophobic face on the one side of the helix, and a hydrophilic face on the other side, is based on its putative binding site at the exposed viral TM during the prehairpin intermediate state, according to theoretical processes of FeLV entry [3]. Because knowledge of FeLV entry is poor, the idea of the FeLV entry mechanism is mainly based on the common putative entry mechanism of retroviruses, e.g. HIV [4, 5] (Fig. A). Figure 1 in the chapter “Results” represents the design and structure of the modified peptides, as well as their putative binding site. However, results clearly indicated that optimisation of the wildtype structure was – against our expectations – not beneficial for the antiviral capacity of the peptide. The most obvious explanation is the replacement of some important amino acids in the peptide sequence that were done to optimise the structure. The work of Giannecchini and co-workers further supports this interpretation. They showed that the triplet of rare aromatic amino acid Tryptophan (W) in the sequence is important for the efficacy of a FIV peptide from the homologous region, and that deletion of these amino acids lead to decreased inhibitory effect, or no inhibitory effect at all [2, 6]. This may explain the observation that EPK397 and EPK398 are less effective, because they lack one W residue, and additional modifications in EPK398 may lead to even more decrease in efficacy. Another explanation

for the reduction in the efficacy of EPK397 and EPK398 may be that the binding target is not necessarily located on the exposed TM prehairpin, but on another structure/transitional conformation. In other words, although lentiviruses (HIV/FIV) are closely related to gammaretroviruses (e. g. FeLV), and their entry mechanisms may be common, FeLV peptides may act differently than homologous peptides of HIV/FIV. Peptides may directly interact with the core-coiled-coil of the TM, thereby disrupting the conformational rearrangements required for membrane fusion. However, there may be at least two other ways of working mechanisms: peptides may act by blocking the interaction of the envelope glycoprotein with the cell surface receptor, or they may disrupt the interaction of the TM with another membrane component that supports membrane fusion [4]. Further investigations are needed to conclude about the precise mechanism for gammaretroviruses. In this context, it is known that the HIV prehairpin intermediate state last for 15-20 minutes, before it slowly resolved into a more stable conformation [1]. FeLV may differ in the entry kinetics from HIV, i.e. the intermediate may be shorter. If this is the case, synthetic peptides have to find their way to their binding site in a relatively short period of time. This in turn may explain the requirement of a high amount of peptide in our assay, which is about 600-fold higher compared to the required peptide concentration in the HIV assay [7]. Further investigations might focus on the wildtype peptide EPK364. This peptide has excellent potential in the inhibition of virus entry in vitro. Evaluation of its exact working mechanism during viral entry may lead to improvements of its efficacy, or it might help to improve the efficacy of future antiviral peptides. Moreover, EPK364 might be tested in vivo as possible therapeutic agent in viraemic cats (see chapter “Perspectives & Outlook”). Like peptides mimicking the C (carboxy)-terminal domain of the FeLV TM, peptides derived from parts of the N (amino)-terminal domain of the FeLV TM might be also tested for their capacity to act as antiviral agents; compared to C-peptides, N-peptides may be competitors for the N- terminal helices in order to inhibit conformational rearrangements.

References

1. Hrin R., Montgomery D.L., Wang F., Condra J.H., An Z., Strohl W.R., Bianchi E., Pessi A., Joyce J.G., and Wang Y.J. Short communication: In vitro synergy between peptides or neutralizing antibodies targeting the N- and C-terminal heptad repeats of HIV Type 1 gp41. 2008. AIDS Res Hum Retroviruses. 24(12): p. 1537-44.

2. Giannecchini S., Di Fenza A., D'Ursi A.M., Matteucci D., Rovero P., and Bendinelli M. Antiviral activity and conformational features of an octapeptide derived from the

membrane-proximal ectodomain of the feline immunodeficiency virus transmembrane glycoprotein. 2003. J Virol. 77(6): p. 3724-33.

3. Otaka A., Nakamura M., Nameki D., Kodama E., Uchiyama S., Nakamura S., Nakano H., Tamamura H., Kobayashi Y., Matsuoka M., and Fujii N. Remodeling of gp41-C34 peptide leads to highly effective inhibitors of the fusion of HIV-1 with target cells. 2002. Angew Chem Int Ed Engl. 41(16): p. 2937-40.

4. Eckert D.M. and Kim P.S. Mechanisms of viral membrane fusion and its inhibition. 2001. Annu Rev Biochem. 70: p. 777-810.

5. Chan D.C. and Kim P.S. HIV entry and its inhibition. 1998. Cell. 93(5): p. 681-4.

6. Giannecchini S., Bonci F., Pistello M., Matteucci D., Sichi O., Rovero P., and Bendinelli M. The membrane-proximal tryptophan-rich region in the transmembrane glycoprotein ectodomain of feline immunodeficiency virus is important for cell entry. 2004. Virology. 320(1): p. 156-66.

7. Wild C.T., Shugars D.C., Greenwell T.K., McDanal C.B., and Matthews T.J. Peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection. 1994. Proc Natl Acad Sci U S A. 91(21): p. 9770-4.

PERSPECTIVES & OUTLOOK

The following chapter describes not only perspectives for veterinary and human medicine, but also how the complexity of the virus-host cell interaction could be reduced by introducing a novel model system to test the inhibitory effect of peptides in simulacra.

Perspectives in Veterinary Medicine

Future clinical (in vivo) studies in veterinary medicine may profit from the results of this in vitro study. The most promising perspective may be a novel therapeutic approach using synthetic peptides for the control of FeLV infection in the domesticated cat. Like for many other diseases caused by retroviruses, there is until now only palliative treatment for cats suffering from FeLV-related diseases. The replication cycle, i.e. the integration of the viral genome into the host cell, causes the major problems in the context of retrovirus infection. Once integrated it is impossible for an organism to get rid of the virus. Thus, virions can be synthesised de novo for the whole lifetime of an organism, causing fatal viraemia especially when the immune system of the host is reduced. However, FeLV is unique among retroviruses, because it is known that some cats successfully overcome viraemia spontaneously by a powerful answer of their immune system [H. Lutz, personal communication]. With this in mind, we hypothesized that synthetic peptides may become a therapeutic agent for infected cats that are at risk of succumbing from malignant virus-related diseases: administration of peptides in high dose for several days to viraemic cats may decrease the viral load by preventing new infections, and help the cat to overcome viraemia spontaneously. Thus, application of synthetic peptides may make the difference whether a cat suffers from lifelong de novo FeLV infections and may die from malignant diseases, or whether they may overcome viraemia and are able to live a normal life, and are not a risk of infection for other cats. However, it is known from HIV studies that not all compounds with antiviral activity in vitro are effective in vivo [1-3]. Thus it will be necessary to precisely evaluate the efficacy of antiviral peptides for their use in vivo. The behaviour of the peptides in the body including the kinetics of its bioavailability, its degradation, its side effects, and its immunogenicity has to be monitored. Tests may include studies (i) whether peptides have the potential to be a therapeutic agent as described above, or (ii) whether peptides are rather able to induce an effective immune answer if presented to the host immune system, and thus protect the cat from virulent FeLV, like a vaccination does. To test the therapeutic potential of peptides, two different groups of naïve kittens known to be highly susceptible to FeLV [4, 5] may be inoculated with FeLV subtype A. Two weeks after they had become viraemic, which can be monitored by measuring viral protein p27 and viral load, the first group will be treated with a specific concentration of peptide that has been

calculated beforehand according to the body weight of each kitten, over a period of several days. Kittens of the second group will be treated with saline solution as a control. Modulations of the viral load has to be monitored using enzyme-linked immunosorbent assay (ELISA) for p27 virus protein detection, reverse transcriptase (RT)-PCR to detect cell-free virus, real-time PCR to detect cell-associated provirus, and virus isolation to gain knowledge of the infectivity of the viral particles. Eventually, the immune system of the kittens might be additionally supported by immune factors such as IL-12 [6, 7] to stimulate components of the immune system (Th1 cells), and to support the peptides in clearing the body from the viral burden. To evaluate the immunogenicity and antigenicity of the peptide, two groups of naïve kittens may be treated – one group with the antiviral peptide under optimal conditions and possibly with a suitable adjuvant [8], the other group with saline solution as a control. Before challenging the kittens with FeLV subtype A, they may be monitored for side effects caused by the peptide, and for the kinetics of peptide degradation. It has to be considered that peptides may be degraded rapidly in the body. Nevertheless, they may have an impact on the immune system and may cause a humoral immune answer and elicit peptide-specific antibodies. After the monitoring of the peptide behaviour in the body, both groups of kittens are inoculated with FeLV-A, and monitored for any signs of infection. This idea is based on several observations. Sera from FIV-infected cats usually contain high levels of antibodies that recognize regions of the TM, and such regions have been shown to be immunogenic when presented to cats as a fusion protein [9-11]. Moreover, immunization of the cats with an immunodominant TM peptide has delayed the infection after challenge with FIV [12]. Additionally, cats naturally infected with FeLV developed strong antibodies to the TM [5]. Such in vivo studies also provide the opportunity to look at all different kinds of host virus- specific immune responses [13-16] before, during, and after the treatment with the peptides. With such knowledge, one could get valuable insights into the role of various immune mechanisms in viral clearance.

Perspectives in Human Medicine

Synthetic peptides may also gain in importance in xenotransplanations. Until now, human gammaretroviruses have not been identified. However, there are animal gammaretroviruses that are considered to have the potential to cross the species barrier, and to become an issue in transplanting animal organs by inducing severe diseases and possibly tumours in humans [17, 18]. Due to the anatomical and physiological conformity with humans, pigs and baboons are under discussion as organ donors on a large scale. However in both species the safety issue of transferring animal gammaretroviruses to the human has to be

considered. According to recent work in the field [19] the porcine endogenous retrovirus (PERV) represents a major safety issue in transferring organs from porcine origin. On the other hand, baboons harbour the gammaretrovirus-like BaEV (baboon endogenous virus) which is known to be able to replicate in human cells in vitro and thus, baboon tissues or organs might become a zoonotic risk as well [20]. The fact that (xeno-) transplanted patients are immunosuppressed to prevent the organ from rejection by the own immune system may facilitate the replication of these gammaretroviruses and therefore to cause infections and diseases. The potential to treat immunosuppressed xenotransplant recipients with antiviral peptides, similarly to the treatment of cats with anti-FeLV peptides to prevent infection caused by undetected gammaretroviruses, may offer a different approach in dealing with safety issues associated with xenotransplantation.

Future Antiviral Peptides

Today, the design of antiviral drugs, especially synthetic peptides that are directed against retroviruses, is currently done by in vitro studies in biological model systems made of natural cells as done in this study [21-23]. In this context, the interaction of the virus envelope protein and specific host cell receptors has been studied extensively [24, 25]. However, research using materials of biological origin, such as living cells, has to deal with a huge number of various processes that occur simultaneously. In addition to numerous factors related to the cells itself and the virus that may change the outcome of the assay, there are even more factors in the environment influencing the system. Concerning the number of all these processes and components that are involved in the virus-cell interaction, detailed insights into the underlying molecular mechanism are impeded. Thus, the design and test of antiretroviral peptides is mainly based on hypotheses today. However, a detailed insight in the underlying mechanisms is needed to optimally design well-working peptides that are specifically directed against a distinct stage of the entry process, e.g. the prehairpin intermediate state, or another specific conformation. In the last years, efforts were made to replace the natural virus-cell system by an artificial virus-liposome model to reduce the complexity. Liposomes have been employed as models for biological systems and thus, to tackle biological issues in simulacra [26-29]. Liposomes provide an aqueous compartment separated from an aqueous environment surrounded by a phospholipid membrane [30]. However, the complete absence of cellular components in the liposome systems has unfavourable effects on both the validity and the comparability with the virus-cell in vitro system used so far. Thus, the idea is to have a combined virus-liposome model system which is simple to understand on a physical level but physiologically complex

enough to draw conclusions for the natural system: Liposomes can be filled with commercially available cell-free expression systems that provide the minimal cellular protein machinery to express proteins in vitro [31-37]. The idea is to artificially express the cellular receptor(s) for FeLV, which have been identified for each FeLV subtype [38-48], in the liposome membrane, and to study the binding and fusion of natural FeLV virions with the liposome. Because the cellular complexity will be reduced to minimum and provides nothing but the basic components for the virus-cell interaction on a molecular level, the evaluation of FeLV entry and its blocking by synthetic peptides will technically become easier. The future use of a simple well-defined system may improve the understanding of the components and their interaction.

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ACKNOWLEDGEMENTS

I am very grateful to my thesis advisor Prof. Dr. Hans Lutz for being an excellent supervisor. I admired especially his pleasure in his work and his endless ability to create solutions and ideas whenever I got stuck.

My mentor Prof. Dr. Hanspeter Nägeli and my co-referee Prof. Dr. Ulrich Hübscher gave me scientific and administrative support during my PhD study.

With Prof. Dr. Paolo Rovero and Prof. Dr. Bernd Gutte, I had helpful, exciting and interesting discussions on protein biochemistry.

I thank Dr. Marlene Wolf and Gabrielle Favre from the Graduate School for Biomedical Sciences, Bern, for a friendly and helpful collaboration.

Isabel Ziegler and Christina Mazzola supported me in administrative concerns.

Thanks to Annette Ryser, Bettina Roth, Marco Bürgin, Nadine Russell, and Theres Meili Prodan (in alphabetical order) for their irreplaceable friendship.

I am deeply grateful to my siblings Maja Borner & Tobias Bönzli, my parents Jeannette Bönzli and Peter Bönzli, and my boyfriend Maik Hadorn for supporting me in every possible way. Thank you!

Last but not least, thanks to the cats Onassis, Olympia, Pablo Panther and Portus for cheering me up after a hard working day.

CURRICULUM VITAE

Personal data

First name: Eva Last name: Bönzli Citizenship: Swiss Birth date: March 26, 1979

Scientific employment

2006 – 2010

PhD student, Clinical Laboratory, Faculty of Veterinary Medicine, University of Zurich, Switzerland.

Education

2006 – 2010

PhD, Faculty of Veterinary Medicine, University of Zurich, Switzerland. Thesis: “In vitro inhibition of feline leukaemia virus infection by peptides derived from the transmembrane domain”

Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland

2004 – 2005

Diploma Thesis (equivalent to MSc), Institute of Molecular Zoology, University of Basel, Switzerland. Thesis: “A possible role for Notch in glial tumour formation in Drosophila melanogaster?”

2000 – 2004

Study (MSc) in Biology, "Integrative biology", University of Basel, Switzerland.

1991 – 2000

Gymnasium Solothurn (grammar school graduation), Typus C, Switzerland.

Publication List

Peer-reviewed publications

Boenzli E, Robert-Tissot C, Sabatino G, Cattori V, Meli ML, Gutte B, Rovero P, Flynn N, Hofmann-Lehmann T, & Lutz H. In vitro inhibition of feline leukaemia virus infection by peptides derived from the transmembrane domain. Accepted in Antiviral Therapy.

Major A, Cattori V, Boenzli E, Riond B, Ossent P, Meli ML, Hofmann-Lehmann T, & Lutz H. (2010). Exposure of cats to low doses of FeLV: seroconversion as the sole parameter of infection. Veterinary Research 41 (2): 10.

Conference proceedings

Boenzli E, Cattori V, Meli ML, Gutte B, Rovero P, Hofmann-Lehmann T, & Lutz H. 10th International Feline Retrovirus Research Symposium (IFRRS). Charleston, SC, USA, May 23 – 26. Oral presentation.

Boenzli E, Cattori V, Meli ML, Gutte B, Rovero P, Hofmann-Lehmann T, & Lutz. 4th Graduate School student‟s symposium. Bern, Switzerland, January 27. Poster presentation.

Boenzli E, Cattori V, Meli ML, Gutte B, Rovero P, Hofmann-Lehmann T, & Lutz H. InnLab. Berlin, Germany, January 21 - February 1. Poster presentation.

Boenzli E, Cattori V, Meli ML, Gutte B, Rovero P, Hofmann-Lehmann T, & Lutz. 3rd Graduate School student‟s symposium. Bern, Switzerland, January 28. Oral presentation.

Boenzli E, Cattori V, Meli ML, Gutte B, Rovero P, Hofmann-Lehmann T, & Lutz H. 9th International Feline Retrovirus Research Symposium (IFRRS). Vienna, Austria, August 24 - 27. Poster presentation.

Boenzli E, Cattori V, Meli ML, Gutte B, Rovero P, Hofmann-Lehmann T, & Lutz. 2nd Graduate School student‟s symposium. Bern, Switzerland, January 31. Poster presentation.

Boenzli E, Cattori V, Meli ML, Gutte B, Rovero P, Hofmann-Lehmann T, & Lutz. 1st Graduate School student‟s symposium. Bern, Switzerland, March 16. Poster presentation.

Declaration of Originality

Last name, first name: Bönzli, Eva

Matriculation number: 00-052-761

I hereby declare that this thesis represents my original work and that I have used no other sources except as noted by citations. All data, tables, figures and text citations which have been reproduced from any other source, including the internet, have been explicitly acknowledged as such. I am aware that in case of non-compliance, the Senate is entitled to divest me of the doctorate degree awarded to me on the basis of the present thesis, in accordance with the “Statut der Universität Bern (Universitätsstatut; UniSt)”, Art. 20, of 17 December 1997.

Place, date Signature

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