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

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University of Groningen

Virus:host interactions during chikungunya virus infection

Bouma, Ellen Marleen

DOI:

10.33612/diss.171018969

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Publication date: 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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replication cycle requires

the Hsp70 network

Ellen M. Bouma

1

_{, Denise P.I. van de Pol}

1

_,

Heidi H. van der Ende-Metselaar

1

_{, Harrie H. Kampinga}

2

_,

Jolanda M. Smit

1

1_{Department of Medical Microbiology and Infection Prevention, University Medical}

Center Groningen, University of Groningen, The Netherlands

2_{Department of Biomedical Sciences of Cells and Systems, University Medical Center}

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Abstract

Chikungunya virus (CHIKV) is an enveloped positive-stranded alphavirus that is transmitted to humans via mosquitoes. CHIKV has emerged rapidly around the world and is causing debilitating disease manifestations such as the development of chronic polyarthritis. Currently, there are no vaccines or antiviral treatments available to treat the acute or chronic symptoms of CHIKV infection. The molecular chaperone system, which controls the protein homeostasis in cells, has been shown important for the infectious cycle of a diverse range of viruses by aiding viral entry, replication, viral protein synthesis or virion assembly. Members of the heat shock protein (HSP) family of chaperones are upregulated in stressed conditions, including several viral infections, to assist in the efficient protein folding of key viral components. Here, we show that the activity of the cellular Hsp70 machine is required for the replication cycle of CHIKV. In presence of Hsp70 inhibitors, progeny CHIKV production is severely hampered and subsequent analysis revealed that the inhibitors mainly act at post-entry stages of the virus replication cycle. While the Hsp70 inhibitors did not influence the number of intracellular viral RNA copies during infection, a strong antiviral effect was seen for viral protein expression. Intriguingly, however, the transport of structural proteins to the plasma membrane was not compromised. Together, this study demonstrates that Hsp70 plays an important role in the late stages of the CHIKV replication cycle and opens new avenues to potentially combat CHIKV infections in the future.

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Introduction

Chikungunya virus (CHIKV) is an alphavirus from the Togaviridae family and is transmitted to humans via mosquitoes1,2_{. Since 2005, CHIKV has emerged across the}

globe which resulted in substantial medical and economic burden in the countries affected3–5_{. More than 90% of the CHIKV-infected individuals develop flu-like}

symptoms with rash, headache, fever and myalgia. However, the most characteristic and debilitating disease manifestations are polyarthralgia and polyarthritis that, in 30-40% of the infected individuals, can reside for months till years after infection1,5–7_.

Currently, there are no vaccines or antiviral treatments available to treat the acute or chronic symptoms of CHIKV infection8,9_.

CHIKV has a positive-sense single-stranded RNA genome of ~12 Kb in length that is packaged in a small icosahedral nucleocapsid shell via interaction with 240 copies of the viral capsid protein. The nucleocapsid is surrounded by a viral envelope composed of a host-derived lipid bilayer and 240 copies of the transmembrane E1 and E2 glycoproteins which are arranged as 80 trimeric spikes of E2/E1 heterodimers1,2,10,11_{. The viral proteins E1 and E2 are required for virus cell entry, i.e.}

attachment/binding of the particle to the host cell, internalization of the virion via clathrin-mediated endocytosis and subsequent viral membrane fusion from within early endosomes10,12,13_{. Next, the viral RNA (vRNA) is translated to produce}

non-structural proteins (nsP) that, together with host factors, form a viral replication complex in the cell cytoplasm14_{. The viral replicase synthesizes full-length}

negative-stranded vRNA that serves as a template for the transcription of the genomic vRNA or the subgenomic vRNA1,2_{. The subgenomic RNA encodes for a polyprotein}

that is cleaved into the structural proteins of the virus. The capsid protein is autoproteolytically cleaved from the nascent polyprotein in the cytoplasm, after which the rest of the polyprotein is translocated to the ER. In the ER, the structural envelope proteins E1 and E2 are processed and folded. The envelope proteins undergo further maturation while passing through the Golgi apparatus and are expressed at the plasma membrane of infected cells. The genomic vRNA interacts with capsid proteins to form a nucleocapsid and progeny virions are assembled via budding at the plasma membrane15_.

As obligatory parasites, viruses require cellular host proteins to initiate and complete their replication cycle. The heat shock proteins 70kDa (Hsp70s) are molecular chaperones that play important roles in protein homeostasis during stressed conditions, including viral infections. They regulate protein folding, degradation and influence protein-protein interactions16,17_{. Here, Hsp70 proteins bind unfolded}

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client proteins via controlled cycles of ATP binding, hydrolysis, nucleotide exchange and substrate binding17,18_{. Specificity is provided via co-chaperones of the J-domain}

protein (JDP) family and nucleotide exchange factors (NEFs). JDP recruit Hsp70 to client proteins and NEFs regulate the processivity of the cycle and may (co)determine client fate in terms of folding or degradation17,19_{. To study the Hsp70 network in}

the cellular environment, several Hsp70 inhibitors have been developed. One is VER-15500820_{, a drug that competes with ATP binding and arrests the nucleotide}

binding domain (NBD) in a half-open conformation and prevents allosteric control within Hsp70 whereby its refolding capacity is reduced21_{. Other well-characterized}

compounds are the MKT-077 analogues (JG-98 and JG-34522,23_{) that affect the}

interaction of Hsp70 with NEFs causing the stalling of the cycle and hence Hsp70 dysfunction. These Hsp70 inhibitors have been shown to have a strong impact on the viral replication cycle of several viruses. For example, the replication cycle of the closely related arthropod-borne flaviviruses (arboviruses), dengue virus (DENV) and Zika virus (ZIKV) was found strongly impaired by Hsp70 inhibitors24,25_{. Hsp70}

inhibitors have been suggested to act at various levels including viral entry and vRNA synthesis26_{. Hsp70 and its co-chaperones were also found to be involved in}

the stabilization and/or folding of the DENV/ZIKV capsid protein24,25_.

In this study, we investigated the role of Hsp70 during CHIKV infection using the allosteric Hsp70 inhibitors VER-155008, JG-98 and JG-345. We show that CHIKV infection is strongly impaired in cells treated with these inhibitors, with a major effect in the late steps of the virus replication cycle, after cell entry and vRNA synthesis. These results pave the path to study the molecular mechanism of the Hsp70 network in the CHIKV replication cycle and to exploit the Hsp70 network as a host-directed antiviral strategy.

Results

Chikungunya virus infection is restricted by Hsp70 inhibitors in human bone osteosarcoma epithelial cells

To study the role of the Hsp70 network in CHIKV infection, we tested the effect of Hsp70 inhibitors VER-155008 and the MKT-077 analogs JG-98 and JG-345 on CHIKV infectivity. We used human osteosarcoma epithelial U-2 OS cells as these cells are highly permissive to CHIKV infection. First, we determined the cellular cytotoxicity of the inhibitors using the commercially available ATPlite luminescence assay system (Fig. S1A) and a trypan blue exclusion test (Fig. S1B). A clear dose-dependent effect on cell viability was seen and for further experiments the maximum concentration for JG-98, JG-345 and VER-155008 was set on 2 μM, 1 μM and 20 μM, respectively.

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At these concentrations, no major cellular cytotoxicity was observed (>80 % survival, Fig. S1A) and no change in the number of viable cells could be detected (Fig. S1B). We next evaluated the antiviral effect of the Hsp70 inhibitors on CHIKV infection. To this end, U-2 OS cells were infected with CHIKV LR2006 OPY1, a clinical isolate derived from La Réunion Island in the Indian Ocean, in presence of the Hsp70 inhibitors or with the vehicle control DMSO. At 1.5 hours post infection (hpi), the cells were washed to remove unbound virus particles and new medium containing the inhibitor was added. At 9 and 16 hpi, the supernatant was harvested, and infectious virus particle production was assessed using a standard plaque assay on Vero-WHO cells and the number of secreted GEC was determined by measuring extracellular vRNA copies by qRT-PCR.

Figure 1 | Progeny CHIKV production is impaired by Hsp70 inhibitors. U-2 OS cells

were infected with CHIKV at a multiplicity of infection (MOI) 1 in presence of 2 μM JG-98, 1 μM JG-345, 20 μM VER-155008, the vehicle control DMSO or without any compound (non-treated; NT). (A) Infectious virus particle production in cell supernatant was measured using a standard plaque assay on Vero-WHO cells at 9 and 16 hpi. (B) Extracellular vRNA presented as genome equivalent copies (GEC) at 9 and 16 hpi of the same supernatants described in A.

(C) GEC/PFU ratio at 9 and 16 hpi presented as fold-change to vehicle control DMSO. Data

is presented as mean ± SEM from at least three independent experiments. Each symbol represents a single experiment. Dotted line indicates the threshold of detection. Student T-test was used to evaluate statistical differences to vehicle control DMSO and a p value ≤ 0.05 was considered significant with *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 or non-significant (ns).

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Harvesting was done at multiple time-points to assess the inhibitory effect over 1 and 2 CHIKV replication cycles; one CHIKV replication cycle takes approximately 8 h in these cells27_.

At 9 hpi, a clear antiviral effect was seen in presence of the Hsp70 inhibitors (Fig 1A). The largest effect was observed in JG-98-treated cells, with a reduction in infectious virus particle production of 2.1 Log (99%). For JG-345 and VER-155008 a 1.0 Log (91%) and 1.4 Log (96%) reduction was observed, respectively. Collectively, these results show that the production of infectious virus particles is reduced by more than 90% in presence of the Hsp70 inhibitors. For the 16 h time point we analyzed the effect of multiple Hsp70 inhibitor concentrations and a clear dose-dependent inhibitory effect was seen (Fig. S2). At the highest non-toxic concentration, we still observed a reduction of 1.0 Log (91%) in JG-98- and VER-155008-treated cells, and 0.9 Log (88%) in JG-345-treated cells.

Next, we determined the number of secreted genome equivalent copies (GEC) in the supernatant of cells treated the Hsp70 inhibitors. Following treatment with VER-155008, the number of GEC was reduced with 1.8 Log and 1.2 Log at 9 hpi and 16 hpi, respectively (Fig. 1B). The number of secreted GEC in JG-98 and JG-345-treated cells were reduced with 1.0 Log (JG-98 and JG-345) at 9 hpi and with 0.5 Log (JG-98) and 0.6 Log (JG-345) at 16 hpi (Fig. 1B). To determine whether the infectious potential of the secreted particles is affected in presence of the compounds, we next calculated the GEC to PFU ratio and compared it to the vehicle control DMSO. Interestingly, the GEC/PFU ratio is strongly increased in 98-treated cells at both 9 hpi (10 fold) and 16 hpi (4 fold) (Fig. 1C). In JG-345-treated cells, the GEC/PFU ratio was found increased at 16 hpi (2 fold) when compared to the vehicle control DMSO. This implies that these inhibitors specifically affect the production of infectious viral particles. Strikingly, VER-155008 reduced the GEC/PFU ratio at 9 hpi by 2 fold and there was no significant difference in GEC/ PFU ratio at 16 hpi (Fig. 1C), suggesting that VER-155008 exerts different effects on CHIKV infectious particle production compared to the MKT-077 analogues.

Hsp70 inhibitors mainly affect the chikungunya virus replication cycle in late stages of infection

To assess whether the Hsp70 inhibitors act early or late in the viral replication cycle, we performed a time-of-drug-addition experiment. The experimental set-up is depicted in Figure 2A. Supernatant was collected at 9 hpi and the number of secreted infectious virus particles was determined by plaque assay. We used Bafilomycin A1 (BafA1), an inhibitor of the vacuolar H+_{-ATPase as a control, as this compound is}

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known to interfere with CHIKV membrane fusion and thus acts early in the viral replication cycle. Indeed, BafA1, severely hampered virus particle production when added prior to and during incubation with the CHIKV inoculum (conditions 1-5). No inhibition was seen when BafA1 was added at late in the virus replication cycle (conditions 6-8) (Fig. S3).

Figure 2 | The Hsp70 network is mainly important during late steps in the CHIKV replication cycle. (A) Schematic representation of the time-of-drug-addition assay. U-2 OS cells are treated with the

Hsp70 inhibitors at indicated time-points (condition 1-8) with vehicle control DMSO, 2 µM JG-98 (B), 1 µM JG-345 (C) or 20 μM VER-155008 (D). Virus inoculum was present for 1.5 h after which the inoculum was removed, and new medium was added in presence or absence of the inhibitor. Supernatants were collected at 9 hpi and infectious virus particle production was measured using a standard plaque assay on Vero-WHO cells. Data is presented as mean ± SEM from at least three independent experiments. Each symbol represents a single experiment. For clarity, the symbols of the DMSO control are not shown. Student T-test was used to evaluate statistical differences to the non-treated control and a p value ≤ 0.05 was considered significant with *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.

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In line with Figure 1, a stark reduction in infectious virus particle production was seen for all Hsp70 inhibitors when present throughout the experiment. When the JG-98 and JG-345 inhibitors were present prior to (condition 2), during (condition 4) or prior to and during (condition 3) incubation with the CHIKV inoculum a significant albeit lower antiviral effect was observed. For VER-155008 no antiviral effect was seen when added during the early stages of CHIKV infection (condition 2-4). Largely similar effects on virus particle production were seen when the compounds were added simultaneously with the virus inoculum and remained present during the complete course of infection (condition 5), indicating that pre-incubation with the compound is not strictly required (condition 1). Interestingly, for all inhibitors, a potent antiviral effect was still seen when the drugs were added after the removal of the virus inoculum (condition 6). Even more strikingly, all Hsp70 inhibitors still exerted antiviral activity when added late in the virus replication cycle (condition 8). In conclusion, Hsp70 inhibitors are mainly targeting late steps in the CHIKV replication cycle.

Hsp70 inhibitors do not have a major effect on the number of intracellular CHIKV vRNA copies

Previously, Hsp70 inhibitors were shown to inhibit infections by DENV and ZIKV and this was partly attributed to a reduction in the number of intracellular DENV and ZIKV vRNA copies. Based on these findings, it was postulated that the Hsp70 network is responsible for the proper folding of the non-structural viral proteins required for the formation of the viral replication complex25,28_{. To test this in the}

context of CHIKV infection, we next investigated the number of intracellular vRNA copies in CHIKV-infected cells at 4, 6 and 9 hpi in the presence or absence of JG-98 or JG-345. Because JG-98 showed the strongest reduction in CHIKV infection, we continued with the MKT-077 analogues in the subsequent experiments.

The total number of vRNA copies increased over time with from 7.6 Log at 4 hpi to 10.4 Log copies of vRNA at 9 hpi in DMSO-treated control cells (Fig. 3). In contrast to DENV, no statistically significant effects on vRNA synthesis were observed in the presence of JG-98 and JG-345 at 4 hpi and a slight yet statistical significant decrease was observed at 6 hpi (Fig. 3). At 9 hpi, no statistical difference in vRNA synthesis in cells treated with JG-98 and JG-345 was observed. Thus, unlike for DENV and ZIKV, the effects of Hsp70 inhibition on CHIKV infection cannot be explained by an effect on the production of new vRNA copies.

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Figure 3 | Hsp70 inhibitors have a minor effect on the number of intracellular vRNA copies in chikungunya virus infected cells. The intracellular vRNA copy number

was assessed at 4, 6 and 9 hpi in U-2 OS cells infected with CHIKV at MOI 10 and treated with vehicle control DMSO, 2 μM JG98 or 1 μM JG345. Total vRNA was determined by RT-qPCR. Data is presented as mean ± SEM from three independent experiments. Each symbol represents a single experiment. Student T-test was used to evaluate statistical differences to vehicle control DMSO and a p value ≤ 0.05 was considered significant with *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.

The expression level of viral proteins is impaired by Hsp70 inhibitors

The above results clearly demonstrate that the Hsp70 inhibitors JG-98 and JG-345 do not play a major role in CHIKV RNA replication. Therefore, we next analyzed the expression level of multiple viral proteins at 9 hpi. We observed a strong decrease in the levels of the capsid protein (83% with JG-98; 88% with JG-345), the E1 protein (67% with 98; 80% with 345) and the nsP2 (55% with 98; 49% with JG-345) (Fig. 4A, left panel; quantification in Fig. 4B). Also when the inhibitors were added late in the virus replication cycle (at 6 hpi, condition 8, Fig. 2) a significant albeit less strong decrease in protein levels of capsid and E1 was observed (Fig. 4A, right panel; quantification in Fig. 4C). This is consistent with the findings that these inhibitors are still effective when added late in the replication cycle (Fig. 2). Under this condition, no significant decrease was seen in the nsP2 protein levels, which is likely explained by the kinetics of virus replication cycle as non-structural proteins are mainly produced early in infection.

The observed decrease in viral protein levels upon Hsp70 inhibition could be a consequence of impaired protein synthesis, folding and/or accelerated proteasomal degradation of the translated viral proteins. We aimed to test whether an increase in viral protein levels is observed when proteasomal degradation is inhibited, however due to the anti-CHIKV effect of the proteasomal MG-132 inhibitor itself this was impossible29_{. Indeed, the CHIKV structural proteins levels were strongly}

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Figure 4 | Viral protein expression is decreased by Hsp70 inhibitors. (A) Representative

western blot of capsid, E1, nsP2 or vinculin expression from protein lysates of U-2 OS cells infected with CHIKV at MOI 10 and treated for 9 h (0-9hpi) or 3 h (6-9hpi) with Hsp70 inhibitor JG-98 (2μM), JG-345 (1μM) or vehicle control DMSO. (B, C) Quantification of western blots from three independent experiments. Protein levels are normalized to vinculin and are expressed as relative protein level to vehicle control DMSO. (B) Cells treated for 9 h (0-9hpi) or (C) 3 h (6-9hpi) with Hsp70 inhibitors. (D) Plasma membrane expression of E1 glycoprotein was assessed at 9 hpi in U-2 OS cells infected with CHIKV at MOI 10 and treated with vehicle control DMSO, 2μM JG-98 or 1μM JG-345. Cells were harvested at 9 hpi and stained with E1-specific antibodies to assess E1 expression on the plasma membrane.

(E) Mean fluorescence intensity (MFI) of the infected population at 9 hpi normalized

to the vehicle control DMSO. Data is presented as mean ± SEM from three independent experiments. Student T test was used to evaluate statistical differences and a p value ≤ 0.05 was considered significant with *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 or non-significant (ns).

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reduced in cells treated with the proteasomal inhibitor MG-132 (Fig. S4). Upon RNA translation and processing of the structural proteins, the envelope protein E1 and E2 are transported from the ER to the plasma membrane via the secretory pathway. To investigate whether JG-98 and JG-345 also interfere with the transport of viral structural proteins to the plasma membrane, we determined the protein levels of the E1 glycoprotein at the plasma membrane of CHIKV-infected cells. Hereto, U-2 OS cells were infected with CHIKV at MOI 10 and treated with Hsp70 inhibitors JG-98 and JG-345 or the vehicle control DMSO. The cells are harvested at 6 hpi and 9 hpi and subsequently stained with E1-specific antibodies to determine the E1 expression levels at the plasma membrane. At 6 hpi, the extent of E1-expressing cells is relatively low (3%) (Fig. S5). At 9 hpi, 19% cells express the viral E1 protein at the cell surface (Fig. 4D). To our surprise, no significant reduction (JG-345) and only a minor statistically significant reduction (JG-98) in total numbers of E1-expressing cells (Fig. 4D) was seen. Also, no alterations in mean fluorescent intensity (MFI) in E1-expressing cells treated with the Hsp70 inhibitors compared to the DMSO-treated cells was seen. MFI represents the number of E1 proteins expressed on the plasma membrane of E1-expressing cells (Fig. 4E, see Fig. S6 for the gating strategy). Collectively, this data indicates that JG-98 and JG-345 strongly affect total CHIKV protein levels including E1 within the target cells. Surprisingly, the transport and expression levels of E1 at the plasma membrane are largely unaffected.

Discussion

In this study, we investigated the importance of Hsp70 in the replication cycle of CHIKV by use of Hsp70 inhibitors. We showed that both VER-155008 and the MKT-077 analogs, JG-98 and JG-345, have a strong dose-dependent effect on progeny virion production in human epithelial cells. For JG-98, the infectivity of secreted particles was strongly impaired. None of the MKT-077 analogs had an effect on viral RNA replication as no major differences were observed in RNA copy number in cells with and without Hsp70 inhibitor treatment. JG-98 and JG-345 strongly reduced the intracellular expression of nsP2, capsid, E1 yet only minor differences in E1 expression were observed at the plasma membrane of the cell.

Our findings show that Hsp70 inhibitors do not have a major impact on CHIKV cell entry and do not affect the number of intracellular RNA copies in infection. This is in contrast to DENV and ZIKV25,28,32_{. Indeed, Taguwa and colleagues showed}

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Although further research is required to understand these differences, it might be related to differences in the replication cycle of these viruses. Flavivirus cell entry is dependent on membrane fusion from within late endosomal membranes and replicate in replication complexes close the endoplasmic reticulum33–35_{. CHIKV on}

the other hand, fuses from within early endosomal membranes and replicate in membrane invagination at the plasma membrane1,12_{. The differences in viral protein}

characteristics, including location and structure, may influence which Hsp70 co-chaperone bind and aids in the Hsp70-protein interactions17,19_{. Several co-chaperones}

of the JDP family have been shown to play a role in the replication cycle of DENV. For example, DnaJB6b is important for DENV particle biogenesis and is mostly located in the cytosol, while DnaJB11 is most likely involved in vRNA synthesis and is localized at the viral replication complexes close to the endoplasmic reticulum31_{. These JDPs}

bring specificity to the Hsp70 system and to better understand the Hsp70-CHIKV interactions we should aim to identify specific interactions of JDP family members with CHIKV proteins.

We observed that Hsp70 inhibitors JG-98 and JG-345 strongly perturbed the levels of CHIKV structural and non-structural proteins in infected cells. Even when the inhibitors were added at 6 hpi, a significant albeit lower reduction in the level of capsid and E1 was observed, indicating an important role for Hsp70 in the synthesis and/or stability of CHIKV structural proteins. The inhibitors JG-98 and JG-345 bind an allosteric binding site in the nucleotide binding domain of Hsp70 thereby blocking the Hsp70 interaction with members of the BAG family of NEFs23_{. This inhibition can}

lead to protein dysregulation due to non-productive folding of the client protein by Hsp70 and/or to degradation of bound client proteins via the proteasomal degradation system. Indeed, viral proteins of the closely related arboviruses ZIKV and DENV were found to interact with Hsp70 and can be rescued from proteasomal degradation using the proteasomal inhibitor MG-13224,31_{. For CHIKV, Hsp70 has been}

described as a CHIKV-binding protein30_{, however, it is unknown which CHIKV protein}

binds to Hsp70 during the virus replication cycle. In this study, we were unable to assess proteasomal degradation of CHIKV proteins due to the antiviral activity of proteasomal inhibitors themselves29_{. Future studies should elucidate whether the}

reduced protein expression levels is due to impaired synthesis or stability of the viral proteins.

The secretion of progeny virus particles is drastically reduced in cells treated with Hsp70 inhibitors. For JG-98 an even starker reduction in the infectious properties of secreted infectious virus particles was seen. Trafficking of E1 towards the plasma

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membrane was however largely unaltered, suggesting that E1 protein trafficking is independent of the Hsp70 network. What then causes the significant drop in virion secretion and infectivity? First, Hsp70 might play an important role in proper processing, folding and maturation of the viral spike proteins in the secretory pathway. Second, the secretion/infectivity of newly produced virions may be hampered due to defects in nucleocapsid formation in the cellular cytoplasm. Indeed, we observed a strong reduction in capsid protein levels in this study. Misassembled nucleocapsids, due to defects in capsid-capsid interactions or capsid-vRNA interactions may influence virus assembly and/or could alter the stability or function of incoming vRNA during virus cell entry36–38_{. Future structural and virological assays are needed}

to understand the precise function of Hsp70 proteins in the CHIKV replication cycle. This study highlights the importance of the Hsp70-CHIKV interaction in eukaryotic, human cells and opens a new avenue to gain a better understanding of the involvement of molecular chaperones in the post-entry stages of the CHIKV replication cycle. The Hsp70 inhibitors used in this study originate from drug development strategies for anticancer therapy yet several limitations have to be overcome before these compounds can be used as antivirals23,39_{. Indeed, phase I clinical trial with MKT-077}

showed that this compound had poor pharmacokinetic properties and was rapidly metabolized in vivo22,40_{. The MKT-077 analogs JG-98 and JG-345 were generated}

with the aim to improve stability and efficacy of the compounds yet require better pharmacokinetic properties to be used in the clinic23_{. Thus, continuous efforts in}

understanding the interaction between Hsp70 chaperones and CHIKV viral proteins are needed and the development of more specific Hsp70 inhibitors might represent a promising strategy to alleviate the disease burden of CHIKV.

Materials and Methods

Cells, inhibitors, and plasmids

Human bone osteosarcoma epithelial U-2 OS cells (a gift from Mario Mauthe, University Medical Center Groningen, Groningen, The Netherlands) were maintained in DMEM, high glucose, glutaMAX supplemented with 10% FBS, penicillin (100 U/ ml), and streptomycin (100 U/ml). Green monkey kidney Vero-WHO cells (ATCC CCL-81) were maintained in DMEM supplemented with 5% FBS, penicillin (100 U/ ml), and streptomycin (100 U/ml). Baby hamster kidney cells (BHK-21 cells; ATCC CCL-10) were maintained in RPMI medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 U/ml). All cells were Mycoplasma negative and cultured at 37°C under 5% CO₂.

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HSP70 inhibitors JG-345 and VER-155008 were a kind gift of Jason Gestwicki (University of California, San Francisco, USA). JG-98 was purchased from MedChemExpress (USA). All compounds are dissolved in dimethyl sulfoxide to a working stock of 10mM (DMSO; BioAustralis, Australia) and stored according to the manufacturer's instructions. Bafilomycin A1 was diluted to a 200mM stock in DMSO.

The infectious clone based on CHIKV strain La Réunion (LR)2006 OPY1 was kindly provided by prof. Andres Merits (University of Tartu, Tartu, Estonia).

Cellular cytotoxicity

Cytotoxicity of the Hsp70 inhibitors was tested using an ATPlite Luminescence Assay System (PerkinElmer, United States), according to the manufacturer’s instructions. Trypan Blue exclusion test was used to determine the number of viable cells. Supernatant and cells were collected and diluted with trypan blue dye (Sigma-Aldrich, United States) in a 1:1 dilution. Cell viability was determined via trypan blue exclusion with an TC20 automated cell counter (Bio-rad, United States)

Virus production and quantification

Virus production was performed as described previously12_{. Briefly, CHIKV strain}

LR2006 OPY1 was produced by electroporation of in vitro-transcribed RNA transcripts into BHK-21 cells. Virus working stocks were produced by subsequent passages in Vero-WHO cells. The infectious virus titer in plaque forming units (PFU) was determined by a standard plaque assay using Vero-WHO cells. Plaques were counted 2 days after infection. The number of genome equivalent copies (GECs) were determined by reverse transcriptase PCR (RT-PCR) followed by quantitative PCR (qPCR) on a Biorad CFX (Bio-rad), as described previously26_{. Data were analyzed}

using Biorad CFX Maestro. The number of GEC was determined using a standard curve (correlation coefficient >0.995) of a quantified cDNA plasmid containing the CHIKV E1 sequences (pCHIKV-LS3 1B).

Antiviral assays in U-2 OS cells

U-2 OS cells were infected with CHIKV at the indicated multiplicity of infection (MOI). Hsp70 inhibitors were added to the virus inoculum. Infection was done in cell culture medium containing 2% FBS. After 1.5 h the virus inoculum was removed and the cells were washed 3 times with plain DMEM. Fresh DMEM containing 10% FBS with or without compounds was added and incubation was continued. Supernatants were collected at 9 hours post inoculation (hpi) or 16 hpi, centrifuged to clarify

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from cell debris and subjected to plaque assay on Vero-WHO cells to determine the infectious particle production. The number of secreted GEC was determined by measuring extracellular vRNA copies by qRT-PCR, as mentioned above.

Time-of-drug-addition assay

The cells were treated with Hsp70 inhibitors at the indicated concentrations prior, during or post-incubation with the virus inoculum. A schematic representation of the time points can be found in Figure 2A. For conditions prior to CHIKV infection, (Fig. 2A, condition 2) U-2 OS cells were incubated with the compounds for 2h and washed 3 times with plain DMEM before the addition of the virus inoculum in cell culture medium containing 2% FBS. After 1.5h the virus inoculum was removed, and the cells were washed 3 times with plain DMEM. Fresh DMEM containing 10% FBS without compounds was added and incubation was continued. In the during condition, the inhibitors were solely present during the 1.5 h infection period by addition of the compounds to the virus inoculum. For the post-infection conditions, the inhibitors were added to the culture medium at 1.5, 4 and 6 h after removal of the virus inoculum. Supernatants were collected at 9 hpi, centrifuged to clarify from cell debris and subjected to plaque assay on Vero-WHO cells to determine viral titers.

Quantification of intracellular vRNA copies by RT-qPCR

To determine the intracellular levels of vRNA copies, U-2 OS cells were infected with CHIKV at MOI 10 with or without Hsp70 inhibitors, as described above. Briefly, at 4, 6 or 9 hpi the U-2 OS cells were washed with PBS and RNA was extracted using the RNAeasy mini kit according to manufacturer’s instructions (Qiagen, Germany). The samples were subjected to RT-qPCR as described above.

Expression of viral proteins by Western Blotting

To determine the viral protein levels in U-2 OS cells treated with or without Hsp70 inhibitors, U-2 OS cells were infected with CHIKV at MOI 10 and treated with Hsp70 inhibitors or MG-132 at the indicated time-points. At 6 or 9 hpi U-2 OS cells were washed with PBS. Next, protein extraction was performed using the RIPA lysis buffer system (Santa Cruz, United States) containing protease inhibitors, according to manufacturer’s instructions. Total protein levels were determined using the Bradford Ultra reagent assay (Abcam, United Kingdom). Samples were diluted in 4X SDS sample buffer (Merck, Germany) and heated at 95°C for 5 min before separation by SDS PAGE. The proteins were transferred onto PVDF membranes (immobilon-P,

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5

Millipore, United States). Antibodies used were anti-vinculin (1:1000, mouse monocolonal, hVIN1, Merck), anti-nsP2 (1:1000, mouse monoclonal, ABM3F3.2E10, Abgenex, India), anti-capsid (1:1000, rabbit polyclonal, kindly provided by G. Pijlman (Wageningen University, the Netherlands)) and anti-E1 (1:1000, Rabbit polyclonal, kindly provided by G. Pijlman). Secondary HRP-conjugated antibodies donkey rabbit HRP (1:5000, Alpha Diagnostic International, United States) and goat anti-mouse HRP (1:2000, A8924, Merck) were used. Quantification of protein signal intensities was done using ImageQuant TL 8.1 software.

Flow cytometry analysis of E1 surface expression

To determine the protein levels of the E1 glycoprotein at the plasma membrane of CHIKV-infected cells U-2 OS cells were infected with CHIKV at MOI 10 with or without Hsp70 inhibitors as described previously. At 6 or 9 hpi, the U-2 OS cells were washed with PBS, trypsinized and fixed with 4% PFA (Alfa Aesar, United States). Fixed cells were stained with rabbit anti-E1-stem antibody (1:1000; obtained from G. Pijlman, Wageningen University, The Netherlands) and Alexa Fluor 647-conjugated chicken anti-rabbit antibody (1:300; Life Technologies, United States). Subsequently, cells were analyzed by flow cytometry. Flow cytometry was performed with a FACSVerse instrument (BD Biosciences, USA) and analyzed with FlowJo vX.0.7.

Statistical analysis

All data were analyzed in GraphPad Prism software. Data are presented as mean ± SEM. Non-paired Student T test was used to evaluate statistical differences. P value ≤0.05 was considered significant with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p≤ 0.0001.

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Supplementary figures

Figure S1 | Cytotoxicity of Hsp70 inhibitors in U-2 OS cells. (A) U-2OS cells are treated

with increasing concentrations of JG-98, JG-345 or VER-155008 (in µM) for 24 h or with vehicle control DMSO. Survival rate normalized to the non-treated control was assessed using the ATPlite luminescence assay system. (B) U-2 OS cells are treated with different concentrations of JG-98, JG-345 or VER155008 (in µM) for 24 h or with vehicle control DMSO. Cell viability normalized to vehicle control DMSO was assessed with the Trypan Blue exclusion test. Data is presented as mean ± SEM from three independent experiments.

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Figure S2 | Dose-dependent antiviral effect of Hsp70 inhibitors on CHIKV infection.

U-2 OS cells are infected with CHIKV at a multiplicity of infection (MOI) 1 in presence of increasing concentrations (µM) of JG-98, JG-345 or VER-155008, the vehicle control DMSO or without any compound (non-treated; NT). Infectious virus particle production in cell supernatant was measured using a standard plaque assay on Vero-WHO cells at 16 hpi. Data is presented as mean ± SEM from at least three independent experiments. Each symbol represents a single experiment. Dotted line indicates the threshold of detection. Student T-test was used to evaluate statistical differences to vehicle control DMSO and a p value ≤ 0.05 was considered significant with *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 or non-significant (ns).

Figure S3 | Bafilomycin A1 inhibits chikungunya virus infection early in the replication cycle. (A) Schematic representation of the time-of-drug-addition assay. U-2 OS cells are

treated with Bafilomycin A1 (BafA1) at indicated time-points (condition 1-8) with vehicle control DMSO or with 100nM BafA1. Virus inoculum was present for 1.5 h after which the inoculum was removed by careful washing with DMEM. Supernatants are collected at 9 hpi and virus production was measured using a standard plaque assay on Vero-WHO. (B) Data is presented as mean ± SEM from three independent experiments. Student T test was used to evaluate statistical differences and a p value ≤ 0.05 was considered significant with *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.

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Figure S4 | Viral protein production in U-2 OS cells treated with or without MG-132.

Representative western blot of 3 experiments is shown. Capsid, E1, nsP2 or vinculin expression from protein lysates of U-2 OS cells infected with CHIKV at MOI 10 at 9 hpi. The cells were treated for 3 h with the proteasomal inhibitor MG-132 (6-9hpi, column 1, 2 and 3). Column 4 represents protein expression at 6 hpi in U-2 OS cells infected with CHIKV.

Figure S5 | Percentage E1-expressing cells at 6 hpi in U-2 OS cells treated with Hsp70 inhibitors. Plasma membrane expression of the E1 glycoprotein was assessed at 6 hpi in U-2

OS cells infected with CHIKV at MOI 10 and treated with vehicle control DMSO, 2μM JG-98 or 1μM JG-345. Data is presented as mean ± SEM from three independent experiments.

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Figure S6 | Gating strategy of flow cytometry analyses. (A-C) Gating strategy to

determine MFI of U-2 OS cells infected with CHIKV at MOI of 10 and treated with vehicle control DMSO (A), 2μM JG-98 (B) or 1μM JG-3459hpi (C).

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Processed on: 7-6-2021 PDF page: 134PDF page: 134PDF page: 134PDF page: 134

Cooperative chikungunya

virus membrane fusion

and its sub-stoichiometric

inhibition by CHK-152

antibody

Jelle S. Blijleven

1

_{, Ellen M. Bouma}

2

_{, Mareike K.S. van Duijl}

2

_,

Jolanda M. Smit

2

_{, Antoine M. van Oijen}

3

1_{Zernike Institute for Advanced Materials, University of Groningen, Groningen, the}

Netherlands

2_{Department of Medical Microbiology and Infection Prevention, University Medical}

Center Groningen, University of Groningen, Groningen, the Netherlands

3_{llawarra Health and Medical Research Institute, University of Wollongong,}