Virus:host interactions during chikungunya virus infection Bouma, Ellen Marleen

University of Groningen

Virus:host interactions during chikungunya virus infection Bouma, Ellen Marleen

DOI:

10.33612/diss.171018969

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2021

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Bouma, E. M. (2021). Virus:host interactions during chikungunya virus infection: Analyzing host cell factors and antiviral strategies. University of Groningen. https://doi.org/10.33612/diss.171018969

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scope of the thesis

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1 General Introduction

The (re)-emergence of mosquito-borne viruses

In the past decades, we have witnessed a drastic re-emergence of mosquito-borne viruses. From the 1950s, the incidence of mosquito-borne dengue virus (DENV) infections increased and nowadays there are an estimated 100-400 million DENV infections each year

. In the late 1990s, the mosquito-borne West Nile virus (WNV) was reported in North America and currently WNV infection is the leading cause of viral encephalitis in the USA

. Around 2005, chikungunya virus (CHIKV) started to re-emerge in the African continent and in recent years debilitating disease outbreaks have been reported in more than 100 countries around the world

. In 2015, we experienced the emergence of Zika virus (ZIKV) in the Americas and infection of pregnant woman was found to increase the risk of microcephaly and other congenital malformations in newborns

. The rise in mosquito-borne viral infections is caused by the wide-spread distribution of the mosquito vectors, virus adaptations, climate change, international trades and travel, and unplanned urbanization

.

Currently there are no antiviral therapies or broadly applicable vaccines available to treat or prevent infections caused by these or related mosquito-borne viruses.

With the risk for future outbreaks, new research initiatives are required to elucidate the underlying molecular factors and disease mechanisms of these pathogens. In this thesis I focused on the cellular and molecular events during CHIKV infection.

Therefore, I will now introduce the epidemiology, pathogenesis, and replication cycle of this rapidly emerging virus, and I will describe the rationale behind the work described in this thesis.

Epidemiology of chikungunya virus infections

CHIKV was first isolated from the forest of Tanzania, where the virus circulated

within transmission cycles of nonhuman primates and mosquito vectors. The first

outbreak of CHIKV in humans was recorded in 1952, after which several confined,

local epidemics of CHIKV were reported in the African continent

. In the first decade

of the 21

century CHIKV re-emerged in Africa, Asia and islands of the Indian Ocean

accompanied with large outbreaks

. From 2005, CHIKV continued to spread with

unprecedented speed in Southeast and East Asia. In 2013, CHIKV crossed the Atlantic

and spread rapidly throughout the Americas

. In the last decade, from 2010 till

2020, CHIKV outbreaks have been reported in >100 countries world-wide thereby

causing millions of human infections (Fig. 1)

.

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There are three main CHIKV lineages; (1) West African lineage, (2) Asian lineage and the (3) Eastern, Central, and Southern African (ECSA) enzootic lineage (Fig. 1)

. Recently, the ECSA lineage evolved into the Indian Ocean Lineage (IOL) and the acquired mutations increased the epidemic potential of the virus strain for human infections. In fact, due to the genetic adaptations, the virus was able to spread via the invasive mosquito species Aedes Albopictus, while beforehand the principal vector of CHIKV was the Aedes Aegypti mosquito. The Aedes Albopictus is also present in areas with moderate climates, enabling CHIKV to spread to new geographical areas, including Europe

. The first local transmission of CHIKV in Europe occurred in 2007 in northern Italy with several hundreds of infected individuals

. Recent vector competence studies showed that the Aedes Albopictus populations in southern European countries are highly susceptible for CHIKV. In these mosquito populations, a high number of virus particles was detected in mosquito-saliva, which is indicative for good transmission efficiency

. Importantly, with the ongoing global warming, it is not unlikely that the Aedes Albopictus population will be able to establish itself in other parts of Europe thereby further increasing the risk for future outbreaks

.

Figure 1 | World map with confirmed autochthonous chikungunya virus transmissions.

Overview of local transmissions worldwide of the different CHIKV strains transmitted via

Aedes mosquitoes A. aegypti or A. albopictus. Figure adapted from G. Rezza and S. Weaver

.

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Clinical features and pathogenesis of chikungunya virus infection

Most CHIKV-infected individuals develop Chikungunya Fever (CHIKF), as less than 15% of the infected individuals have an asymptomatic infection

. The clinical symptoms of CHIKF generally start 2-4 days after a mosquito bite and are characterized by an acute fever, flu-like symptoms, maculopapular rash, myalgia, headache, and rigors. However, the most characteristic clinical feature of CHIKF is the onset of a rheumatic disease, including (poly)arthralgia and polyarthritis. Typically, the acute phase of CHIKF lasts for 1 week after which most infected individuals recover while some develop chronic disease symptoms with debilitating joint pains that can last for months till years after infection

. Indeed, studies have shown that 57-60% of the inhabitants on Reunion Island experienced rheumatic symptoms for at least 15 months after the initial CHIKV infection in 2005-2006

. Besides the (poly)arthralgia, patients also showed other clinical symptoms including myalgia, asthenia, depression, sleeping and concentration disorders for several months after disease onset

. Generally, however, 30-40% of all infected individuals experience chronic disease symptoms

.

Upon a mosquito-bite of a CHIKV-infected mosquito, cells within the skin –including epithelial cells, dermal fibroblasts and Langerhans cells– become infected and the virus subsequently disseminates via the lymph nodes to different areas of the body including the bones, muscles, liver and spleen

. It is thought that the onset of myalgia, polyarthralgia and polyarthritis during the acute phase of CHIKV infection is a direct consequence of infected cell types in joints and muscles. The so-called cytopathic effect of an infected cell results in cell damage and the constitutive infiltration of mononuclear cells –predominantly macrophages and CD4+ T lymphocytes– in the affected areas

. CHIKV infection induces a robust innate type-I interferon-associated immune response early in infection

. Additionally, CHIKV infection stimulates the production of pro-inflammatory cytokines and chemokines (ea. IL-1β, IL-6, MCP-1, and TNF-α) that lead to a rapid antiviral state and the recruitment of adaptive immune cells to the site of infection

. After the first week of infection, clearance of the virus is controlled by the secretion of neutralizing IgM and IgG antibodies by B lymphocytes that leads to lifelong protection against CHIKV infection

.

It is thought that the chronic disease symptoms could be a consequence of

insufficient viral clearance in infected joint cells and macrophages. This notion is

based on studies demonstrating the presence of viral RNA in macrophages up to 90

days post-infection in a nonhuman primate animal model of CHIKV infection and in

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synovial macrophages isolated from humans

. In-depth studies showed that cells, mainly myofibers and dermal and muscle fibroblasts, which survived acute infection can persistently harbor viral RNA during the latter chronic stages

. Importantly, there is no evidence for active replication during the chronic phase. Thus far, no circulating virus or infectious virus can be detected in serum or isolated from tissues. Alternatively, chronic CHIKV disease could be a consequence of an aberrant immune response

. A high cytokine response during the acute phase is negatively correlated with the incidence of future chronic joint pain

. However, the precise mechanism on how CHIKV disease proceeds into the chronic phase is not clear.

Chikungunya virus – an enveloped positive-stranded RNA virus

CHIKV belongs to the Alphavirus genus which is part of the Togaviridae family.

Alphaviruses can be divided into two groups: New World and Old World alphaviruses

. The Old World viruses often cause clinical symptoms such as fever and arthritis and include CHIKV, O’Nyong-Nyong virus (ONNV), Semliki Forest virus (SFV) and Sindbis virus (SINV). The New World viruses are often associated with encephalitis in humans and include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV) and Western equine encephalitis virus (WEEV). All alphaviruses are small enveloped viruses with a diameter of 70 nm and contain a single-stranded positive-sense RNA (ss(+)RNA) genome of ~11,8 Kb in length

. Figure 2A/B shows a cryo-EM reconstruction of CHIKV virus-like particles.

The gRNA is packaged by 240 copies of the capsid protein in an icosahedral lattice, known as the nucleocapsid. The capsid protein (~35 kDa) of alphaviruses has a positive-charged N-terminal region with a specific RNA binding site and a ribosomal binding site

, while the C-terminal region contains a serine protease domain required for structural polyprotein processing. Additionally, the C-terminal region consists of a hydrophobic cleft to allow an interaction with the C-terminal domain of the E2 transmembrane protein located in the viral envelope.

The viral envelope is composed of lipids derived from the host cell and mainly

comprises cholesterol and phospholipids in a 1:1 ratio

. In addition, the viral envelope

contains 240 copies of two viral transmembrane glycoproteins: E1 and E2 (~40-45

kDa). On the outer surface, the E1 and E2 glycoproteins form heterodimers that are

associated as 80 trimeric spikes in an icosahedral lattice (Fig. 2C)

. E2 contains a

C-terminal tail with palmitoylated cysteines anchoring it to the membrane and an

N-terminal ectodomain with three immunoglobulin-like domains (domain A, B and

C, Fig. 2D). E2 domain A and B contain putative receptor-binding domains and an

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acid-sensitive β-ribbon connector that is important for the linkage of the different domains within the protein (Fig. 2D)

. E2 is glycosylated at two sites of the protein

. The E1 glycoprotein is composed of an ectodomain with three β-sheet-rich domains (domain I, II and III, Fig. 2D) and a small endodomain that consists of only five amino acids. E1 has one N-linked glycosylation site and it contains a hydrophobic fusion peptide that is covered by E2 in a mature virion (Fig. 2C/D, shown in white). Within infected cells, E2 is generated as a E2-E3 precursor to protect the acid-sensitive region of E2 during transport through the acidic environment of the secretory pathway (Fig. 2C, E3 is shown in orange)

. The structural protein, E3, is released at a late stage of the replicative cycle although it can sometimes be found on alphavirus virions.

Figure 2 | Cryo-EM reconstruction of a mature chikungunya virus-like particle. (A, left) Mature CHIK virus-like particle in a three-dimensional cryo-EM map. The white triangle with symbols represents the icosahedral asymmetric unit with the different icosahedral symmetry elements. (A, right) The icosahedral nucleocapsid containing 240 capsid proteins.

(B) A cross-section of CHIKV showing the E proteins, viral membrane, nucleocapsid, RNA

and transmembrane (TM) helix. The different colors represent the radial distance from the

center of the virus. Figure adapted from Sun et al

(C) Side- and top-view of trimeric E2–E1

heterodimer (PDB: 6JOG

). E2 is depicted in dark grey. E1 ectodomains I, II, and II are colored

red, yellow and blue, respectively. The E1 hydrophobic fusion peptide is shown in white. E3

is shown in orange. (D) Ribbon diagram showing the ectodomains of the CHIKV E1 and E2

glycoprotein (PDB: 3N42

). E1 is depicted in the same colors as in C. The structural domains

E2 are shown in cyan (domain A), green (domain B), and pink (domain C). The acid-sensitive

region is within the β-ribbon of E2 (dark purple). Figure C and D were prepared using the

program ChimeraX.

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Next to the above mentioned major structural proteins, chikungunya virions contain low copy numbers of the small accessory protein transframe (TF)

. TF is produced upon ribosomal frameshifting from the 6K gene. The 6K and TF proteins have an identical N-terminal transmembrane domain and a short cysteine-rich cytoplasmic loop that proceeds their unique C-terminal domains. The C-terminal domain of 6K is a second transmembrane domain, while the C-terminal domain of TF is a hydrophilic cytoplasmic extension. Palmitoylation of the N-terminal region of the TF protein leads to incorporation of the protein into newly produced virions, while 6K primarily stays in cellular membranes

.

Genomic RNA translation and replication

The genomic RNA (gRNA) of CHIKV encodes for two polyproteins, the non-structural polyprotein and the structural polyprotein (Fig. 3). The non-structural polyprotein is processed into 4 nonstructural proteins (nsPs); nsP1, nsP2, nsP3 and nsP4. The structural polyprotein is the precursor for the previously mentioned structural proteins; capsid, E3, E2, 6K/TF and E1.

Figure 3 | Genomic organization of the ss(+)RNA genome of chikungunya virus.

The gRNA of CHIKV contains two open reading frames (ORF). The first ORF encodes for the non-structural polyprotein which is cleaved into 4 nonstructural proteins (nsP) and the second ORF encodes for the structural polyprotein which is processed into 6 individual structural proteins. The gRNA is capped at the five prime untranslated region (5’UTR) and polyadenylated at the 3’UTR. Figure adapted from Pérez-Pérez et al

.

The ss(+)RNA genome of CHIKV has a 5’ cap and a 3’ poly(A) tail and acts in the cell as a eukaryotic mRNA (Fig. 3). In the cellular cytoplasm, the first open reading frame (ORF) of the viral gRNA is translated via the eukaryotic translation machinery that produces the two precursor nonstructural polyproteins nsP123 and nsP1234.

NsP1234 is a result of an opal stop codon and low-frequency read-through at the end of the viral nsP3 gene. NsP4 is cleaved from the polyprotein P1234 by proteolytic cleavage via nsP2 after which nsP4 and P123 form the early polymerase complex.

This early replication complex is required for the synthesis of full-length negative-

stranded viral RNA.

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Further proteolytical processing of the non-structural polyprotein into nsP1, nsP4 and nsP23 leads to the formation of the late intermediate replication complex. This replication complex is capable of synthesizing both minus and plus-stranded gRNA.

When nsP23 is cleaved into single nsP2 and nsP3 the final viral replicase is formed which is able to synthesize full-length gRNA and subgenomic plus-stranded RNAs (sgRNA), using the minus-strand as a template

. Initial RNA replication is believed to occur close to the plasma membrane where the replication compartments are membrane invaginations called spherules

. For some alphaviruses, these spherules become internalized and form large cytopathic vacuoles (CPV-I) later in infection.

For CHIKV however, most of the spherules remain at the plasma membrane (Fig. 4)

. The synthesized sgRNA is translated in the cytoplasm resulting in the production of the structural polyprotein capsid-E3-E2-6K-E1 or the smaller polyprotein capsid- E3-E2-TF due to ribosomal frameshifting

. The capsid protein cleaves itself from the rest of the polyprotein via autoproteolytic cleavage using its protease domain at the C-terminus. The residual polyprotein is directed towards the ER membrane via a signal sequence in E3. Shortly after the translocation of the polyprotein into the ER, the transmembrane glycoproteins E1 and the precursor E2 (pE2; which is E3-E2) are generated and form a stable interaction with each other to generate the acid-resistant pE2-E1 heterodimer

. This heterodimer undergoes posttranslational modification, including N-linked glycosylation and palmitoylation, as well as furin- mediated cleavage of pE2 to release E3 from E2, during transport via the secretory pathway towards the plasma membrane

.

Assembly and budding of progeny chikungunya virus particles

CHIKV assembly is initiated in the cytoplasm when the capsid proteins bind to specific nucleotide sequences in newly produced gRNA

. The exact cellular location of this interaction and packaging is not identified, but progeny nucleocapsids can be seen throughout the cytoplasm and can be found in association with membranous structures in the cell

. Next, the intact nucleocapsids interact with the E2’s endodomain of the membrane-associated glycoproteins. How the coalescences of the nucleocapsid with the envelope proteins is coordinated is not completely understood but there are two possible mechanisms of virion assembly;

(1) the nucleocapsid binds to E2 glycoproteins at the plasma membrane or (2)

the nucleocapsid is co-transported with E2-E1 proteins to the plasma membrane

via type II cytopathic vacuoles (CPV-II)

. CPV-II are large membranous structures

that can be found late in infection and are thought to be derived from the trans

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Golgi

. Interestingly, CPV-II contains highly structured helical tubular arrays of E2-E1 glycoproteins and nucleocapsids can be observed in association with the cytoplasmic site of these CPV-II

. At the end, the assembly step is coordinated at specific budding ‘patches’ at the plasma membrane, leaving the budded particles almost completely empty from cellular proteins

. Whether CPV-II structures play an important role in viral budding is not clear.

Host factors required for viral replication, assembly and budding

CHIKV requires the host cellular transcription machinery, as well as the secretory pathway to transcribe the incoming gRNA and to process the structural proteins.

Host proteins associated with these pathways were indeed found to be important for CHIKV infection, like the host proteins furin and other proprotein convertases that aid in the maturation of glycoproteins in the ER-Golgi secretory pathway

. Many more host factors have recently been identified that have a yet unknown mechanism of action on viral replication. For example, sphingosine kinase 2 (SK2) –a lipid kinase– colocalizes with CHIKV RNA and nonstructural proteins in the replication complex and FHL1 (four-and-a-half LIM domain protein 1) was identified as an important host factor for CHIKV replication. Details on how SK2 or FHL1 aids CHIKV replication remains to be determined

. Currently there are not many host factors known for budding and scission of progeny CHIKV particles: unlike other enveloped viruses, CHIKV does not make use of ESCRT (endosomal sorting complexes required for transport) proteins to mediate scission from the plasma membrane

.

Host proteins linked to the unfolded-protein response (UPR) are often associated

with virus infections due to ER-stress and a high concentration of misfolded proteins

during the production of viral proteins. Viruses employ these proteins for their own

benefit or they developed mechanisms to antagonize the UPR system to elongate

cell survival thereby stimulating viral replication and/or translation

. Indeed, the

UPR is also activated during CHIKV infection yet CHIKV antagonizes this response

using the viral nsP2

. Unfolded or misfolded proteins, that activate the UPR, are

generally re-folded or targeted for degradation by molecular chaperones called

heat shock proteins. Especially the family of heat shock proteins 70kDa (Hsp70s)

are important for controlling protein folding of nascent polypeptides, but they also

play a role in protein degradation and protein-protein interactions

. A Hsp70-

cycle regulates the interaction of Hsp70 with a ‘client’ protein in the peptide-binding

domain. Hsp70 cycles between an ATP- and ADP-bound state that is controlled via

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J proteins and nucleotide exchange factors (NEFs). J protein facilitate the ATPase activity of Hsp70, important for client binding, while NEFs regulate the nucleotide exchange reactions required for the dissociation of the client proteins from Hsp70

. This Hsp70 network is shown to be very important in the replication cycle of related arboviruses ZIKV and DENV, among others

. Although CHIKV has been associated with heat shock proteins

, it is currently unknown whether the molecular chaperones of the Hsp70 network are required for CHIKV infection.

Because most of the work presented in this thesis is directed towards the early stages of the CHIKV replication cycle, I will now describe the virus cell entry pathway and the virus:host interactions involved in this process. A schematic representation of the complete replication cycle is shown in Figure 4.

Virus:host interactions during chikungunya virus cell entry Attachment and binding

Cell entry of CHIKV starts with attachment and binding of the virion to the host cell;

two separate steps in virus cell entry. Generally non-specific host cell factors, that are common for many different types of viruses, are used for cellular attachment while for virus entry virus-specific receptors are utilized that can additionally activate intracellular signaling pathways required for the internalization of the virus

. The CHIKV E2 glycoprotein on the viral envelope is thought to be important for binding as it contains the putative receptor-binding domain

.

The attachment factors that are described for CHIKV include glycosaminoglycans (GAGs), T-cell immunoglobin and mucin receptor family (TIM), Dendritic Cell- Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN)

. Especially the GAGs, large carbohydrate molecules like heparan sulfate and keratan sulfate, are central factors in CHIKV attachment to mammalian host cells

(Fig.

4). They are ubiquitously expressed on numerous mammalian cell types and could therefore contribute to the wide cell tropism of CHIKV

.

The cell entry receptors that have been described for CHIKV include Prohibitin

(PHB) proteins 1 and 2 and, as of recently, the cell adhesion molecule Mxra8

.

PHB is an evolutionary conserved membrane protein that aids CHIKV cell binding

and is expressed on many different cell types

. Though, CHIKV infection can only

partly be inhibited by blocking this receptor with specific ligands like Flavaglines

.

The most recently discovered receptor for CHIKV and other alphaviruses is Mxra8,

which has been identified in 2018 as an important factor for CHIKV cell entry

.

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Figure 4 | Schematic representation of the replication cycle of chikungunya virus. (I) CHIKV particles attach to the host cell via interaction with attachment factors. Binding to a CHIKV-receptor leads to (II) clathrin-mediated endocytosis, (III) membrane fusion and (IV) nucleocapsid uncoating in the cytoplasm. (V) The genomic RNA is translated and processed into non-structural proteins (nsPs) that form the viral replication complex to produce negative- stranded RNA. (VI) Replication of genomic and subgenomic RNA occurs in spherules at the plasma membrane. (VI) Alphaviruses can internalize these spherules to form large cytopathic vacuoles (CPV-I). (VII) Translation of the subgenomic RNA in the cytoplasm produces the structural polyprotein. Autoproteolytic cleavage releases the capsid protein in the cytoplasm while the residual polyprotein is cleaved and processed in the ER-Golgi secretory pathway.

(VIII) The genomic RNA interacts with capsid proteins in the cytoplasm to form nucleocapsids.

(IX) The virion is assembled at the plasma membrane via interaction of the nucleocapsid with

E2-E1 heterodimers at the cell surface or (X) via CPV-II, that are formed late in infection, to

(XI) transport and assemble the structural proteins in mature virions to be released at the

cell surface. Reprinted from Silva et al

with permission from American Society for Clinical

Investigation through Copyright Clearance Center, In.

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Quickly after the discovery, the crystal structure in complex with the CHIKV glycoproteins was revealed and showed that Mxra8 receptors can have quaternary interactions with the glycoproteins within different trimeric spikes

. Figure 5 Mxra8 shows the antibody-binding sites of three Mxra8 molecules in a single E2-E1 spike. Subsequent in vivo studies in mice showed a marked reduction in CHIKV- infected tissues when treated with Mxra8-antibodies or when they had mutant Mxra8 alleles

. Importantly, a residual CHIKV infection could be observed indicating that Mxra8 is not the sole receptor for cell entry of CHIKV.

Figure 5 | Structure of the E2-E1 spike bound to Mxra8 molecules. Side- (A) and top- (B) view of trimeric E2–E1 heterodimer, colors are same as described in Figure 2 (PDB:

6JOG

). Three human Mxra8 molecules are shown in ribbon diagrams (lime green). This figure was prepared using the program ChimeraX.

In conclusion, several attachment factors and cellular receptors have been identified but there is not yet a consensus on the putative CHIKV binding factor or factors that are required for cell entry. Though, it is likely that the surface receptors used by CHIKV are ubiquitously expressed on eukaryotic cells due to the wide cellular tropism of CHIKV.

Clathrin-mediated endocytosis and endosomal sorting

Upon binding to the host cell, CHIKV particles are predominantly internalized via

clathrin-mediated endocytosis (CME)

, although other internalization routes have

been described as well

. CME is a conserved pathway that is triggered upon

stimulation of the receptor with the bound ligands from the plasma membrane

into the cytoplasm

. Till date, it is unknown via which receptor CME is initiated at

the CHIKV-bound site of the plasma membrane. CME starts with the recruitment

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of clathrin, clathrin-adaptor proteins (ea. heterotetrameric adaptor protein (AP2) complex, PICALM family and epsins), and scaffold proteins (epidermal growth factor receptor substrate 15 (EPS15) and intersectins) to form a clathrin-coat at the inner leaflet of the plasma membrane

. Next, the actin cytoskeleton network is polymerized surrounding the clathrin-coat to promote membrane bending. When the clathrin-coated pit is formed, a large GTPase called dynamin is recruited that assembles around the neck of the pit with the help of BAR proteins

. Hydrolysis of GTP drives membrane fission to form a clathrin-coated vesicle. Many of the described CME factors including the clathrin-heavy chain, dynamin and epsin15 were found important for CHIKV cell entry indicating that CME is the main pathway for endocytosis of CHIKV particles

.

During, or directly after dynamin-dependent fission, the uncoating of the clathrin- coated vesicle is mediated by heat shock 70 kDa proteins (Hsp70) and the J protein auxilin

. After coat-disassembly, the clathrin-coated vesicle is –dependent on the cargo– transported back to the plasma membrane or transported via endosomal sorting stations towards the trans-Golgi network or to the degradation pathway.

Sorting of this cargo is regulated in early endosomal vesicles, that mature into late

endosomes and lysosomes when cargo is selected for degradation. Internalized

CHIKV particles are targeted to these early endosomal vesicles

. When CHIKV

arrives in early endosomes they escape the degradative pathway via a membrane

fusion reaction with the endosomal membrane. This membrane fusion event is

dependent on the maturation status of the early endosomes, that is accompanied

with a gradual decrease in endosomal pH –mediated by the v-type vacuolar

H

-ATPase pump– and a change in lipid compositions and Rab GTPases on the

membrane bilayer

. The pH of the early endosomal lumen is slightly acidic, with a

pH range between ~6.8-5.9, and early endosomes have a high concentration of PI3P,

the Rab GTPase Rab5 and its Rab5 effector molecule EEA1

. We and others have

shown that >95% of the CHIKV particles fuse from within early endosomal Rab5-

positive compartments

. Additionally, we have shown that the threshold for CHIKV

membrane fusion to occur is at pH 6.0-6.2, with the presence of cholesterol and

sphingomyelin in the host-cell membrane. These characteristics indeed correspond

to the acidic environment and the lipid composition in early endosomes

.

Whether specific host-factors are required for targeting CHIKV for CME and

membrane fusion in the early endosomes is not yet known. Recent studies have

described the cellular factors fuzzy homologue (FUZ) and tetraspanin membrane

protein 9 (TSPAN9) as important proteins in the internalization of CHIKV, however

their precise role in the endocytic pathway is not yet defined

.

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Molecular events in viral membrane fusion

Membrane fusion in the early endosomes is a highly regulated process that can be divided into several separate steps (Fig. 6); (a) destabilization of the E2-E1 heterodimer, (b) exposure of the E1 fusion loop, (c) insertion of the fusion loop in the opposed target membrane, (d, e) formation of a hairpin-like homotrimer and re-folding of E1 to move opposing leaflets together, (f) merging of opposing membranes into a hemifusion intermediate and (g) fusion pore formation.

Figure 6 | Schematic model of alphavirus membrane fusion. (A) Alphavirus outer

membranes contain 240 copies of E2 and E1 glycoproteins that are arranged in 80 trimeric

E2-E1 heterodimers. (B) Membrane fusion is triggered by a low pH that destabilizes the E2-

E1 heterodimer. Dissociation of the acid-sensitive domain in E2 exposes the E1 fusion loop,

indicated by a red star. (C) Next, the fusion loop is inserted in the opposed target membrane

and full dissociation of E2 triggers the formation of stable E1 homotrimers (D,E) The re-

folding of E1 leads to the formation of a hairpin-like homotrimer that moves opposing leaflets

together. (F) Upon merging of opposing membranes, the hemifusion intermediate is formed

and (G) a complete fusion pore is formed. Reprinted from Kielian and Rey

with permission

obtained from Springer Nature through Copyright Clearance Center, In.

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The destabilization of the E2-E1 heterodimer is attributed to the highly conserved pH sensitive histidine residues in the glycoproteins of CHIKV. At low pH, the acid- sensitive region in the β-ribbon of E2, containing these hisitidines residues, becomes largely disordered resulting in conformational changes that guides the dissociation of the E2-E1 heterodimer thereby exposing the fusion loop in E1 (Fig. 7A). Indeed, CHIKV fusion can be blocked by diethylpyrocarbonate (DEPC) treatment of virus particles

. DEPC covalently modifies hisitidines and thereby abolishing the protonation of these residues

. Next, the fusion loop in E1, which is hydrophobic, is inserted into the endosomal membrane likely via E1-cholesterol interactions

. The lipids in the target membrane can ease this membrane insertion step, as sphingomyelin stimulates the cholesterol-accessibility in the target membrane

. The insertion of E1 in the target membrane and a further decrease in endosomal pH leads to the complete dissociation of the E2 with E1 proteins, which enables trimerization of E1

. This homotrimer of E1 molecules folds back towards the viral membrane in a hairpin-like structure (Fig. 7B). Due to this refolding, the opposing membranes are brought together into a fusion stalk, with the initial lipid connection between the outer membrane leaflets. Subsequently, they are forced to merge into the hemifusion intermediate state. Next, the inner membrane leaflets are merged, and a fusion pore is formed to release the nucleocapsid in the cytoplasm

.

Figure 7 | Structure of the E2-E1 spike and E1 trimer at low pH. (A) Trimeric E2-E1 heterodimer of Sindbis virus at low pH. The protein was crystallized at pH 5.6 (PDB: 3MUU

).

E2 (dark grey) is disordered thereby exposing the fusion loop (white) in E1. E1 domains I, II and

III are depicted in red, yellow and blue, respectively. (B) Ribbon structure of the homotrimer

of E1 glycoproteins from Semliki Forest Virus (PDB: 1RER

). E1 domains are depicted in the

same colors as described in A. This figure was prepared using the program ChimeraX.

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Nucleocapsid uncoating and genomic release

After the formation of the fusion pore, the genetic material of CHIKV is released in the cellular cytoplasm. The details of nucleocapsid release and uncoating are unknown, but it is thought that an intact, but primed, nucleocapsid is released into the cytosol upon membrane fusion

. Indeed, studies of alphavirus cores have shown that ribosomes in the cytoplasm facilitate the uncoating of the nucleocapsids, but the putative ribosomal binding site (RBS) is located at the inside of the intact nucleocapsid cores

. Therefore, it is likely that exposure of the RBS requires partial dissociation of the nucleocapsid. In other viruses, like Influenza A virus (IAV) or HIV-1, nucleocapsids are primed for nucleocapsid dissociation via viroporins, ion-permeable pores in the viral membrane

. It has been proposed that the alphavirus E1 glycoproteins –those that are not inserted in the opposing endosomal membrane during membrane fusion– could act as viroporins via back-insertion into the viral membrane

. However, there are not many studies that confirm this theory. Alternatively, TF proteins that are present in small number in a mature virion, could act as viroporins. The related 6K protein indeed functions as a viroporins, but has not been extensively found in mature CHIKV virions as it primarily remains associated with cellular membranes

. Because structural and functional data on TF is lacking, we cannot predict the function of TF in the viral membrane yet.

Recent comparison of cryo-EM reconstructions of immature (ie. CHIKV particles with pE2-E1 heterodimers) and mature CHIKV virus-like particles show that mature virus particles are less compact and have an extended nucleocapsid compared to immature particles

. This data implies that the nucleocapsids potentially are already primed during virus assembly

. Additionally, nucleocapsids obtained by treatment of virions with non-ionic detergents are fully infectious when microinjected into cells, indicating that additional priming of the nucleocapsid after its release in the cytoplasm is not required for nucleocapsid uncoating

. Though, more data is needed to fully understand the role of virus maturation and the cell entry pathway on the priming of the CHIKV nucleocapsid.

As mentioned before, ribosomes are thought to play an important role in

nucleocapsid uncoating of alphaviruses, although no details for CHIKV have been

described so far

. These studies showed that nucleocapsid binding to ribosomes

quickly leads to the dissociation of the capsid proteins

. Because this ribosomal-

mediated dissociation has only been shown in in vitro experiments without the

cellular environment of an infected cell

, it remains to be determined whether other

cellular factors are required for nucleocapsid uncoating.

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Therapeutic opportunities for chikungunya virus infections

Despite the public health emergency of CHIKV, infected individuals are currently treated with paracetamol or other analgesics because there are no antiviral treatments or vaccines available

. More than 15 CHIKV vaccines are in phase-I or phase-II of clinical trials

, but none have been tested in phase-III clinical trials.

This can partly be attributed to difficulties in the organization of clinical trials due to the unpredictability of future epidemics. Also, the convenience of production, transportation and administration of vaccines and the economical system of affected countries can be identified as obstacles in the development of safe and efficacious vaccines

. Next to vaccines, a few drug candidates have been tested in clinical trials but so far none of them have been proven effective

. The characteristics of a successful antiviral drug includes (I) reduction of viral load and dissemination, (II) alleviation of acute and/or chronic disease symptoms, (III) favorable pharmacodynamics and safety-profiles and (IV) a low risk to develop antiviral drug resistance.

There are two classes of antiviral drugs: (1) direct-acting antivirals and (2) host- directed antivirals. I will describe both classes of antiviral drugs below.

Direct-acting antivirals specifically target a viral protein or its enzymatic function

. The advantage of direct-acting antivirals is their specificity towards functional domains in the viral protein. Structural information of viral proteins have been proven helpful in the design of direct-acting antivirals

. This guides in silico development of potential ligands that bind and block the functions of viral non- structural or structural proteins

. Indeed, doxycycline has been docked to the E2 glycoprotein of CHIKV via molecular modelling and was found to exhibit inhibitory activity in vitro

. Computational modeling has revealed several other inhibitors of CHIKV nsPs which demonstrated significant inhibition in CHIKV infection in in vitro cellular assays

. These direct-acting antivirals can often be further optimized by using computational approaches and applying structural modifications

. Currently, inhibitors of the nsP2 of CHIKV have been developed by targeting the protease domain of nsP2

. Also, inhibitors targeting CHIKV nsP1 effectively disrupt the cellular functions of nsP1 within the infected host cells

. Yet, further advances in structural biology and functional assays are needed to improve the potency of direct-acting antivirals against CHIKV infections in humans.

Neutralizing antibodies can also be considered as direct-acting antivirals. In the past

decades, multiple specific monoclonal antibodies (mAbs) have been developed that

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block CHIKV at different stages of infection

. More specifically, mAbs were found to neutralize and block virus binding to the host cell or the membrane fusion reaction by inhibiting the conformational changes required for fusion. Several potent mAbs have been mapped in cryo-electron microscope (EM) structures in complex with their binding epitope. CHK-152, a humanized mAb produced by Pal and colleagues exerts its antiviral activity by cross linking E2 domains thereby potentially inhibiting membrane fusion activities

. Besides the potent effect in vitro and in mouse models it is not yet known what the efficiency of these therapeutic antibodies in humans is.

Host-directed antivirals target a cellular protein or molecule that is required in the virus replication cycle. The advantage of targeting host cell factors is that they have the potential to act as broad-spectrum antivirals, as they are often shared by different types of viruses, and that there is a limited risk for antiviral drug resistance.

There are several strategies that have been employed to identify host targets for

antiviral drug development. For example, by using high-throughput screening

methods strong antivirals against CHIKV infection like berberine, digoxin or pimozide

were identified

. Another strategy to identify host-directed antivirals entails the

screening of repurposing existing drugs. For example, chloroquine, an antimalarial

drug, raises the endosomal pH and strongly inhibits CHIKV infection in vitro by

preventing the membrane fusion step

. Chloroquine is one of the few drugs

that has been tested in human clinical trials as CHIKV-specific therapy. However, no

beneficial effect of chloroquine could be detected in patients infected with CHIKV

.

The therapeutic potential of other repurposed drugs, like the anticancer drug

obatoclax, the anti-trypanosomiasis drug suramin or the anti-hepatitis C virus drug

ribavirin (RBV), show promising results against CHIKV infections in vitro

.

Despite the recent advances in discovery of new direct-acting and host-directed

antivirals, most of the drugs have not yet been tested in animal models and humans

or do not enter the clinical trial phase for diverse reasons

. There is a strong need

to follow-up more and identify new molecules to combat CHIKV infection. For the

rational design of antiviral therapies, it is imperative to gain a better understanding

into the virus:host interactions that occur during CHIKV infection.

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Scope of the thesis

In this thesis, I focused on elucidating the virus:host interactions during CHIKV infection, with emphasis on the virus:host interactions that occur during the process of virus cell entry. Moreover, I studied the antiviral activity of novel host-directed antiviral compounds and the monoclonal antibody CHK-152 within the CHIKV replication cycle.

Previously, our research group showed that combining live-cell imaging of single virus particles with biochemical inhibitors and fluorescently-tagged cellular proteins provide valuable information about the cell entry pathway of arboviruses

. In Chapter 2, the role of Mxra8 during CHIKV cell entry is investigated. Zhang and colleagues recently revealed that Mxra8 functions as an important factor for CHIKV binding and cell entry

. Here, and in collaboration with Zhang and colleagues, we investigated the internalization pathway of Mxra8. Additionally, we used single- particle tracking to trace CHIKV-labeled virions in cells trans-complemented with Mxra8 protein fused with the fluorescent protein GFP. This way, we dissected the co-localization of Mxra8 with CHIKV during intracellular trafficking and membrane fusion.

In Chapter 3, the importance of the microtubule network in the early steps of CHIKV infection is evaluated. To this end, we performed live-cell microscopy in cells treated with nocodazole, a depolymerizing agent that disrupts the microtubule network, and analyzed the effect of this treatment on the intracellular trafficking and membrane fusion capacity of CHIKV particles. Also, the importance of microtubules in gRNA release to the cell cytoplasm is investigated. Thus, the experiments performed in this chapter dissect how microtubule-dependent trafficking is associated with CHIKV infection.

In Chapter 4, the anti-CHIKV activity of 5-nonyloxytryptamine (5-NT) and Methiothepin Mesylate (MM) is investigated. 5-NT is a serotonin receptor agonist that can activate serotonin receptors, while MM is a serotonin receptor antagonist.

Previously, 5-NT and MM have been described to have anti- and proviral activity

towards the dsRNA reovirus, respectively

. Furthermore, 5-NT was shown to

interfere with CHIKV infection

. In this chapter, we delineated the mode-of-action of

5-NT and MM within the replication cycle of CHIKV. More specifically, time-of-drug-

addition, cell entry bypass, virus cell binding, membrane fusion and nucleocapsid

delivery assays were performed. The experiments performed in this chapter delineate

the potential use of serotonergic drugs as antiviral therapy against CHIKV infection.

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In Chapter 5, the importance of the Hsp70 protein machinery in CHIKV infection is studied. Heat shock proteins have been associated with CHIKV infection and other arboviral infections, but their precise function in the infectious cycle of these viruses is not fully understood. I studied the antiviral activity of specific Hsp70 inhibitors against CHIKV infection and examined how Hsp70 proteins contribute to CHIKV replication. More specifically, I studied whether Hsp70 inhibitors influence virus cell entry, vRNA synthesis, viral protein expression and transport of the viral glycoproteins to the plasma membrane. Using this analysis, we obtained a better understanding of the involvement of the Hsp70 machinery in the replication cycle of CHIKV and identified the Hsp70 machinery as a new target for intervention.

Next to host-directed antivirals, therapeutic antibodies –or direct-acting antivirals–

can be very effective in neutralizing CHIKV infection. In Chapter 6 we investigated, in collaboration with Blijleven and colleagues from the Zernike Institute for Advanced Materials, the mechanism of inhibition of the monoclonal antibody CHK- 152. We studied the binding and fusion properties of CHIKV virions in presence of CHK-152. Also, we applied an in vitro single-particle fusion assay with fluorescently labeled CHK-152 to determine the rate and extent of membrane fusion in relation to antibody binding. Additionally, CHK-152 was used to calculate the potential cooperative properties of CHIKV glycoproteins during the process of membrane fusion. In conclusion, the experiments performed in this chapter delineate the molecular basis for CHK-152 neutralization and provide insights into the process of CHIKV membrane fusion.

In Chapter 7 I provide a summary and overall discussion of the work described in

this thesis.

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