Fusion of a New World Alphavirus with Membrane Microdomains

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Fusion of a New World Alphavirus with Membrane Microdomains Involving Partially Reversible Conformational Changes in the Viral Spike Proteins Ivanildo P. Sousa Jr, Carlos A. Marques Carvalho, Ygara Silva Mendes, Gilberto Weissmuller, Andréa C. Oliveira, and Andre M. O. Gomes Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00650 • Publication Date (Web): 28 Sep 2017 Downloaded from http://pubs.acs.org on September 29, 2017

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Fusion of a New World Alphavirus with Membrane Microdomains Involving Partially Reversible

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Conformational Changes in the Viral Spike Proteins

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Ivanildo P. Sousa Jr.»,†,‡, Carlos A. M. Carvalho»,§,‡, Ygara S. Mendes»,#, Gilberto Weissmuller«,

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Andréa C. Oliveira», and Andre M. O. Gomes»,*

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»

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Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil

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«

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Instituto de Bioquímica Médica Leopoldo de Meis, Centro de Ciências da Saúde, Universidade

Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal do

Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil

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Corresponding Author

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*Phone: +55 (21) 2562-6756. E-mail: [email protected].

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ABBREVIATIONS

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Bis-ANS, bis-8-anilino-1-naphthalenesulfonate; DRMs, detergent-resistant membranes; EBOV, Ebola

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virus; LUVs, large unilamellar vesicles; MAYV, Mayaro virus; MBGV, Marburg virus; MBS, MES-

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buffered saline; PBS, phosphate-buffered saline; PFU, plaque-forming unit; pyrene, 12-(1-pyrene)-

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dodecanoic acid; SFV, Semliki Forest virus; SINV, Sindbis virus; SUVs, small unilamellar vesicles;

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SV40, Simian virus 40; Trp, tryptophan.

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ABSTRACT: Alphaviruses are enveloped arboviruses mainly proposed to infect host cells by

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receptor-mediated endocytosis followed by fusion between the viral envelope and the endosomal

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membrane. The fusion reaction is triggered by low pH and requires the presence of both cholesterol

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and sphingolipids in the target membrane, suggesting the involvement of lipid rafts in the cell entry

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mechanism. In this study, we show for the first time the interaction of an enveloped virus with

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membrane microdomains isolated from living cells. Using Mayaro virus (MAYV), a New World

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alphavirus, we verified that virus fusion to these domains occurred to a significant extent upon

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acidification, although its kinetics was quite slow when compared to that of fusion with artificial

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liposomes demonstrated on a previous work. Surprisingly, when virus was previously exposed to acidic

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pH, a condition previously shown to inhibit alphavirus binding and fusion to target membranes as well

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as infectivity, and then reneutralized, its ability of fusing with membrane microdomains at low pH was

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retained. Interestingly, this observation correlated with a partial reversion of low pH-induced

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conformational changes in viral proteins and retention of virus infectivity upon reneutralization. Our

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results suggest that MAYV entry into host cells could alternatively involve internalization via lipid

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rafts, and that the conformational changes triggered by low pH in the viral spike proteins during the

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entry process are partially reversible.

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Mayaro virus (MAYV) is an emerging arthropod-borne virus (arbovirus) belonging to the genus

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Alphavirus and related to sporadic endo-epidemic cycles naturally occurring in the Amazon region,

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where it is responsible for a chikungunya-like febrile illness accompanied by disabling arthralgia.1 As

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for other alphaviruses, MAYV is proposed to get into host cells through receptor-mediated endocytosis

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followed by fusion between the viral envelope and the endosomal membrane,2,3 although an alternative

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model involving direct penetration of the plasma membrane through a pore-like structure formed by

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virus and, possibly, host proteins has been proposed for alphavirus entry.4 In the endocytic model for

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alphavirus entry, the fusion process is induced by the low pH of the endosomal lumen, which triggers

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conformational changes in the viral surface glycoproteins E1 and E2.5 These conformational changes

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involve the dissociation of the E1/E2 heterodimer and the formation of E1 homotrimers, and it has been

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extensively demonstrated that prior exposure of alphavirus particles to low pH leads to the loss of the

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fusogenic activity.6,7

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In addition to the requirement of acidic pH, several studies have shown that alphavirus fusion

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reaction depends on the presence of specific lipids, such as cholesterol and sphingolipids, in the target

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membrane to occur.7–10 Cholesterol is proposed to help virus binding to the target membrane whereas

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sphingolipids are proposed to act as a cofactor for the subsequent fusion event.8 Such lipid

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requirements suggest an involvement of lipid rafts – specialized domains in cellular membranes

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enriched in cholesterol, sphingolipids and certain classes of membrane proteins11 – in the alphavirus

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fusion event. These membrane microdomains, originally defined as detergent-resistant membranes

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(DRMs) due to their relative insolubility to non-ionic detergents at low temperatures, function as

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platforms for signal transduction and are indeed hijacked by many enveloped viruses to gain access

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into host cells.12,13 In the specific case of alphaviruses, it has been already shown that although the viral

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fusion peptide associates with sterol-rich membrane domains,14 Semliki Forest virus (SFV) and Sindbis

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virus (SINV) are able to fuse with target membranes irrespective of the presence or absence of them.15 4 ACS Paragon Plus Environment

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However, these works were carried out using artificial model membranes, which does not reflect the

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heterogeneity of biological membranes.

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In the present study, using MAYV as a model, we show that alphavirus particles can interact

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efficiently with membrane microdomains isolated from living cells. Nevertheless, the kinetics of this

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interaction showed to be relatively slower than those described in early observations using synthetic

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liposomes. We also show that MAYV is still able to interact with DRMs after previous incubation at

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low pH, and that such process involves partially reversible conformational changes in the viral surface

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proteins.

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MATERIALS AND METHODS

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Cells and Virus. Baby hamster kidney (BHK-21) and African green monkey kidney (Vero)

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cells were cultured as monolayers at 37 °C and 5% CO2 in Dulbecco’s modified Eagle medium

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(DMEM) (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Cultilab,

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Campinas, SP, Brazil) and 3% tryptose phosphate (Difco, Detroit, MI, USA). Both cell lineages were

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maintained by subculturing twice a week using PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4,

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and 1.47 mM KH2PO4, pH 7.4) for washing and trypsin-versene (0.25% trypsin and 1 mM EDTA,

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PBS-based) for detaching. MAYV (VR-1277) was obtained from the American Type Culture

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Collection (Manassas, VA, USA).

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Virus Propagation, Purification, and Quantification. MAYV was propagated in BHK-21

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cells for 48 h at 37 °C. The virus was purified as described previously,16 with some modifications.

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After propagation, the supernatant was collected and cleared of cellular debris by centrifugation at

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8,000 rpm for 20 min at 4 °C in a Hitachi RPR 12-2 rotor. The supernatant was put on a PBS-based

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30% sucrose cushion and centrifuged in a Beckman 45 Ti rotor at 32,000 rpm for 1 h 40 min at 4 °C.

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The pellet was suspended in TNE buffer (10 mM Tris, 100 mM NaCl, and 1 mM EDTA, pH 7.5), 5 ACS Paragon Plus Environment

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layered onto a discontinuous 5–50% sucrose density gradient (PBS-based) and centrifuged at 30,000 rpm for 1 h 30 min at 4 °C in a Beckman SW 40 Ti rotor. Virus fraction was identified by reading the optical density of the fractions collected from the gradient at 260 and 280 nm, and corresponded to the 30% sucrose fraction. After harvesting this fraction by aspiration, viral protein concentration was determined by the Lowry assay,17 and infectivity was evaluated by plaque assay in Vero cells.18 To avoid the drawbacks of freeze-and-thawing, purified virions were maintained at 4 °C in the same PBSbased 30% sucrose solution in which they were recovered from the gradient and used within a couple of weeks. Fluorescent Labeling of MAYV. The biosynthetic labeling of MAYV with 12-(1-pyrene)dodecanoic acid (Molecular Probes, Eugene, OR, USA) was carried out essentially as described before.19 BHK-21 cells were cultured in medium containing a final concentration of pyrene-labeled fatty acid of 15 µg/mL. Upon confluence, cells were infected with MAYV at a multiplicity of 1 plaqueforming unit (PFU) per cell in medium without fluorescently labeled fatty acid. After propagation, virus was purified as indicated above. It is worth noting that although pyrene was already shown to inactivate some enveloped viruses upon irradiation,20 labeling itself with this fluorophore has practically no effect on the infectivity of alphaviruses such as SINV and does not interfere with cuvette-based vesicle fusion assays.19,21 Isolation and Detection of DRMs. DRMs were isolated as described before.22 Briefly, a monolayer of approximately 106 cells BHK-21 cells was washed twice with PBS and once with MBS (25 mM MES and 150 mM NaCl, pH 6.5) at ice bath temperature. After that, cells were incubated with 1 mL of lysis buffer (0.5% Triton X-100, 1 mM EDTA, 1 Mm Na3VO4, 1 mM PMSF, 10 µg/mL aprotinin, 25 mM MES, and 150 mM NaCl, pH 6.5) for 30 min at 4 °C. Cellular debris were then homogenized in a Potter-Elvehjem homogenizer, diluted with 1.5 mL of 85% sucrose and loaded to the bottom of an ultracentrifugation tube. The diluted sample was overlaid with 5 mL of 35% sucrose and 6 ACS Paragon Plus Environment

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4.5 mL of 5% sucrose and centrifuged at 34,000 rpm for 24 h at 4 °C in a Beckman SW 40 Ti rotor. The gradient fraction containing the DRMs was identified by GM1 ganglioside localization on a dotblot assay. Briefly, fractions of 1 mL were harvested from the sucrose gradient and an aliquot from each fraction was spotted directly onto a nitrocellulose membrane. GM1 was visualized using horseradish peroxidase-conjugated cholera toxin B subunit (CTB-HRP; Sigma) followed by enhanced chemiluminescence (ECL; Amersham Biosciences, Piscataway, NJ, USA) detection. Imaging of DRMs. Isolated DRMs were layered onto a freshly cleaved mica surface, where they were allowed to settle for 15 min at 21 °C, and then analyzed on an atomic force microscope MFP-3D (Asylum Research, Santa Barbara, CA, USA) using a standard nitrite tip with a 4 µm2 pyramidal base (Digital Instruments, Santa Barbara, CA, USA) mounted on a V-shaped CSC11 cantilever (MikroMasch, Watsonville, CA, USA) operating in contact mode with a nominal spring constant of 0.06 N/m. Images were acquired in liquid (PBS) at 21 °C under a scanning rate of 10 µm/s and a scanning force below 1 nN. Fusion and Conformational Change Assays. For the fusion assay, a previously described protocol19 was used. Pyrene-labeled MAYV and DRMs were mixed in a quartz cuvette to a final volume of 700 µL in HNE buffer (5 mM HEPES, 150 mM NaCl, and 0.1 mM EDTA, pH 7.4), and analyzed on a spectrofluorimeter PC1 (ISS, Champaign, IL, USA). The sample was stirred magnetically and thermostated at 37 °C. Fusion was triggered by the addition of a small pretitrated volume of 0.1 M MES and 0.2 M acetic acid to achieve a final pH of 5.5. When indicated, the mixture was neutralized with a pretitrated volume of 0.1 M NaOH to pH 7.5. The sample was excited at 340 nm, and the emission was scanned from 360 to 550 nm. Fusion was measured as a function of pyrene excimer/monomer fluorescence ratio (480/372 nm). The initial excimer/monomer ratio (F0) represented the initial state of the system and was taken as 0% fusion, while the ratio after the addition of Triton X100 to a final concentration of 1% (Fmax) represented the infinite dilution of the fluorophore and was 7 ACS Paragon Plus Environment

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taken as 100% fusion. Considering Ft the excimer/monomer ratio at a time t, the percentage of fusion at this time is given by the following equation: % fusion = [(Ft - F0)/(Fmax - F0)] x 100. The assays for conformational changes in viral surface proteins were carried out as described before.23 Intrinsic fluorescence from tryptophan (Trp) residues in the viral proteins was assessed by exciting the sample at 280 nm and scanning emission from 300 to 420 nm. Binding of bis-8-anilino-1naphthalenesulfonate (bis-ANS; Molecular Probes) was monitored by exciting the samples at 360 nm and collecting the emission in the range of 400 to 600 nm. The concentration of bis-ANS was 2 µM.

RESULTS Identification and Visualization of DRMs. To investigate if MAYV is able to fuse with biological membrane microdomains, we first isolated DRMs from living BHK-21 cells by flotation in a sucrose gradient. To determine the localization of DRMs in the gradient after ultracentrifugation, 12 fractions of the gradient were collected, starting from the top, and a dot-blot assay was performed to identify GM1, a ganglioside whose partition is exclusively within lipid rafts.24 As shown in Figure 1A, GM1 was found in the interface of the low-density fractions, consistent with its behavior as a raft marker.24,25 To assess their morphological characteristics, the isolated DRMs were imaged by atomic force microscopy, with the scanning being performed in an aqueous environment to preserve the hydrophobic interactions involved in the assembly of biological membranes. As shown in Figure 1B, these microdomains presented a pleomorphic vesicular structure, with a diameter ranging from 50 to 500 nm.


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A

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Figure 1. Identification of GM1 ganglioside in DRMs isolated from living cells and imaging of the preparation. (A) Extracts of BHK-21 cells were spun in a sucrose density gradient and the collected gradient fractions were assayed for the distribution GM1 ganglioside by dot blot analysis. An aliquot from each fraction was spotted directly onto a nitrocellulose membrane and the presence of GM1 was visualized using HRP-conjugated cholera toxin B subunit, followed by ECL detection. Percentages denote the sucrose concentration to which the collected fractions correspond in the density gradient. (B) Isolated DRMs were layered onto a freshly cleaved mica surface and imaged by atomic force microscopy in contact mode immersed in a buffered aqueous environment at 21 °C under a scanning rate of 10 µm/s and a scanning force below 1 nN. Scale bar = 500 nm. Analysis of MAYV Fusion with DRMs. After the identification of the sucrose gradient fraction enriched in DRMs, pyrene-labeled MAYV was incubated with the isolated DRMs and the 9 ACS Paragon Plus Environment

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pyrene fluorescence was monitored. The pyrene excimer-to-monomer fluorescence ratio is proportional to the concentration of the probe.26 Upon fusion of a pyrene-labeled donor membrane with an unlabeled target membrane, the pyrene phospholipids are diluted, resulting in a decrease in the excimer-tomonomer ratio. Figure 2 shows the fusion kinetics of pyrene-labeled MAYV with DRMs. After lowering the pH, we observed a mild increase in the fusion extension up to approximately 50% at 3 h post-acidification (line with circles in Figure 2). When the interaction between pyrene-labeled MAYV and DRMs was analyzed without lowering pH, the maximal extension of fusion was approximately 15% (line with triangles in Figure 2), much lower than that observed at low pH. This indicates that fusion of MAYV with DRMs is dependent on low pH. No fusion was observed in the absence of DRMs under the same conditions (line with diamonds in Figure 2). Surprisingly, when we pre-incubated MAYV at low pH for 20 min and then reneutralized it immediately before the addition of DRMs, the fusion activity was retained upon reacidification of the environment and the fusion kinetics seemed to be a little faster when compared to what was observed upon direct acidification (line with squares in Figure 2).

Figure 2. Kinetics of fusion between MAYV and DRMs at low and neutral pH. Pyrene-labeled MAYV was incubated with DRMs in TNE buffer (pH 7.5) and the mixture was acidified to pH 5.5 (line with circles). Alternatively, pyrene-labeled MAYV was pre-acidified to pH 5.5 for 20 min and then 10 ACS Paragon Plus Environment

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neutralized to pH 7.5. After that, DRMs were added and the mixture was acidified again to pH 5.5 (line with squares). Fusion was measured at 37 °C as a function of pyrene excimer/monomer fluorescence ratio (480/372 nm) upon excitation at 340 nm until 3 h post-acidification. Fusion of MAYV with DRMs at neutral pH was also evaluated (line with triangles). No changes in pyrene fluorescence were observed when labeled virus was incubated at 37 °C at neutral pH in the absence of DRMs (line with diamonds). Investigation of pH-Induced Conformational Changes of MAYV Proteins. To get insight into the structural basis behind the unexpected preservation of MAYV fusogenic activity upon previous acidification followed by reneutralization, we next evaluated the conformational changes induced in viral proteins by the pH variations. To achieve that, we followed the changes in intrinsic fluorescence from Trp residues. Trp fluorescence spectrum shifts to shorter wavelengths as the polarity of the environment surrounding this residue decreases, thus correlating with the degree of solvent exposure.27 Analysis of intrinsic fluorescence emission spectra revealed that a dramatic change in MAYV structure occurred when the virus was subjected to low pH in both the presence or the absence of DRMs (dashed lines in Figures 3A and 3B), expressed by a large blue-shift in fluorescence emission maximum in comparison to that of control virus (solid lines in Figures 3A and 3B). Upon reneutralization, the structural changes were more pronounced in the absence than in the presence of DRMs (dotted lines in Figures 3A and 3B), even though to an extent lesser than that observed upon acidification. Addition of a high concentration of urea as a control caused a remarkable red-shift in Trp fluorescence emission, suggesting complete unfolding of the viral proteins (dot-dashed lines in Figures 3A and 3B). In a further assessment of the pH-induced structural changes in MAYV proteins, we performed a binding assay with the fluorescent probe bis-ANS. The fluorescence yield of bis-ANS increases up to 100-fold when it is transferred from water to a non-polar environment, as may occur when the probe binds to hydrophobic structured domains in proteins.28 When a protein is partially disorganized, 11 ACS Paragon Plus Environment

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hydrophobic segments may be exposed allowing bis-ANS binding with consequent increase in its fluorescence. It has been shown that bis-ANS is useful in providing an additional and simple way to assess protein conformational changes in enveloped viruses as they assume the fusion-active state.19,29– 31

We observed that binding of bis-ANS was more pronounced when MAYV was exposed to low pH in

the absence than in the presence of DRMs (dashed lines in Figures 3C and 3D) in comparison to that of control virus (solid lines in Figures 3C and 3D). When pH was reneutralized, we verified a decrease of bis-ANS fluorescence in both the presence and the absence of DRMs (dotted lines in Figures 3C and 3D). Despite this decreasing, binding of bis-ANS was still higher than the baseline, indicating a partial reversion of structural changes induced by low pH. Addition of a high concentration of urea as a control led to a negligible binding of bis-ANS, suggesting complete unfolding of the viral proteins (dotdashed lines in Figures 3C and 3D). A

B


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Figure 3. Conformational changes in MAYV proteins due to pH transitions assessed by Trp fluorescence and bis-ANS binding. Virus suspension was excited at 280 nm in the presence (A) or absence (B) of DRMs and fluorescence emission of Trp residues was scanned from 300 to 420 nm. Alternatively, virus suspension was incubated with bis-ANS in the presence (C) or absence (D) of DRMs, excited at 360 nm and scanned for fluorescence emission from 400 to 600 nm. Spectra represent normalized fluorescence intensity of control (solid lines), acidified (dashed lines), acidifiedand-neutralized (dotted lines) and urea-denatured (dot-dashed lines) samples. Acidification alone and acidification followed by neutralization were performed as in Figure 2. Effect of pH Transitions on MAYV Infectivity. Finally, we attempted to evaluate the effect of pH transitions on virus infectivity. MAYV was subjected to acidification followed by reneutralization as before and assayed for its infectivity in Vero cells. No changes in the order of magnitude of virus infectious titer were observed in comparison to control MAYV (1010 PFU/mL for both samples), indicating significant retention of infectivity of acid-exposed and reneutralized virus (Figure 4).


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Figure 4. Effect of acidification followed by reneutralization on MAYV infectivity. Virus particles were acidified and reneutralized (Treated) or incubated with a corresponding volume of PBS (Control), and then subjected to plaque assay in Vero cells for assessment of the infectious titer. Acidification followed by neutralization were performed as in Figure 2. Bars: mean ± range. As assessed by unpaired t test, difference between the experimental conditions was not significant (P = 0.1171).

DISCUSSION The present work addresses important questions about the current knowledge on the interactions of alphaviruses with lipid rafts, using membrane microdomains isolated from living cells. We also investigated the nature of the conformational changes undergone by MAYV glycoproteins during fusion of the viral envelope with these microdomains. Our results reveal that MAYV, a New World alphavirus, can efficiently interact and fuse its envelope with DRMs. Although isolation of membrane microdomains based on their detergent-insolubility at low temperatures can somewhat alter their real composition,32 this system still resembles the heterogeneity of lipid rafts better than artificial membrane models and comprises an excellent model to study virus-membrane microdomain interactions. Although the fusion extension maximum of MAYV with DRMs was very similar to that with liposomes,31 its kinetics was much slower. Interestingly, slow kinetics of interaction with membrane 14 ACS Paragon Plus Environment

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microdomains were previously shown for Simian virus 40 (SV40), Ebola virus (EBOV) and Marburg virus (MBGV).33,34 This can be an alternative pathway that should be taken into consideration together with other well-characterized endocytic and also non-endocytic mechanisms for alphavirus entry into host cells. Regarding other endocytic routes, a previous work by our group suggested that alphavirus exploitation of membrane microdomains for entry into the host cell may occur in parallel with clathrinmediated endocytosis, and even overlap with it, relying on low pH for fusion with the endosomal membrane.3 Concerning non-endocytic routes, a previous work by Vancini and coworkers provided compelling evidence that alphaviruses may also get into the host cell by direct penetration of the plasma membrane at neutral pH through a pore-like structure.4 In the former scenario, low pH would be the trigger to the conformational changes of the viral envelope proteins required for membrane fusion, while, in the latter scenario, receptor binding would be the trigger to the conformational changes required for pore formation. To accommodate both views, it is possible that the endocytic and the nonendocytic scenarios occur in parallel during alphavirus entry. Since the identity of the receptors that mediate alphavirus entry into host cells has been elusive, the member species of this virus genus may attain its very wide host range by using more than one protein as a receptor, as previously suggested.35 If this is true, interaction with different receptors could allow for one or another virus entry scenario, through distinct kinetics. With regard to size compared to liposomes, DRMs are presumably small in situ (i.e., associated with bulk cell membranes), ranging from 5 to 20 nm in diameter,36 but, when isolated, they coalesce to form larger vesicular structures with diameters ranging from 50 to 500 nm, as revealed by our atomic force microscopy analysis. Such dimensions resemble those of small unilamellar vesicles (SUVs, 100 nm), respectively – the major types of liposomes with a single lipid bilayer.37 Thus, for equivalence purposes, if a liposome preparation can support multiple virus fusions per vesicle, our DRM preparation should be able to do it as well. Indeed, a previous study 15 ACS Paragon Plus Environment

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revealed that low-pH induced fusion of MAYV with liposomes occurs to a maximal extent of ~40%,31 similar to what we observed for fusion of MAYV with DRMs – however, the viral fusion kinetics was much faster with liposomes than with DRMs. Since our fusion assays were based on pyrene excimer/monomer fluorescence ratio (480/372 nm), light scattering variations due to an eventual aggregation or dissolution of the system components would affect the fluorescence intensities as stray light (light with other wavelengths than the targeted) at the same time. Adding 0.5 mm slits to the light pathways and calculation of the pyrene excimer/monomer fluorescence ratio results in a significant attenuation of such possible artifact. Moreover, constant stirring of the samples allowed for a homogeneous distribution in the cuvette, avoiding an eventual precipitation. Another interesting finding of this work regards to the capability of MAYV to fuse with a target membrane after pre-exposure of the virus to low pH, a condition known to lead to a rapid and complete loss of the fusion activity of other alphaviruses.6,7 It is believed that this loss of fusion activity is due to conformational changes in the viral envelope proteins when exposed to an acidic environment, which would irreversibly render an inactive protein conformation in the absence of a target membrane.38 Neutralization of acid-exposed MAYV, however, rendered a virus that was still able to fuse with DRMs at an extent and kinetics very similar to that of control virus. This observation is in agreement to what was previously shown for SFV, an Old World alphavirus, using liposomes.39 As for SFV, also overall MAYV infectivity was retained after the pH transitions. While previous works on Old World alphaviruses focused on the formation of oligomers involved in the fusion process during pH transitions, here we used fluorescence spectroscopy to assess the conformational changes undergone by the glycoproteins of a New World alphavirus. Our assays for protein conformational changes suggested that upon exposure to low pH in the absence of a target membrane, MAYV undergoes striking structural changes involving exposure of structured hydrophobic sites in its surface proteins. These changes are likely to represent the well-known conformational 16 ACS Paragon Plus Environment

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Biochemistry

rearrangements that occur in the viral surface glycoproteins when in an acid environment and comprise dissociation of the E1/E2 dimer, exposure of the fusion peptide, and E1 trimer formation.5 Interestingly, these changes seemed to be softer when a target membrane was present, suggesting the hindering of several hydrophobic sites amongst membrane lipids. When pH was reneutralized in the presence of DRMs, no return of Trp fluorescence was observed, while bis-ANS binding showed still some reversibility. This suggests that, when pH transitions occur in the presence of a target membrane, part of the conformational changes are stabilized by binding to such membrane. This stabilization probably leads to the formation of E1 trimers and consequently to fusion, but the energetic barrier for such trimerization reaction may be high enough to prevent its occurrence in the absence of membrane binding. Although other alphavirus envelope proteins were previously shown to undergo irreversible conformational changes at low pH,38 we observed that neutralization of MAYV suspension led to partial restoration of viral protein structure in both presence and absence of target membranes. Consistently with what was shown previously for SFV,39 after the initial acid treatment and neutralization, the original virus structure does not appear to be restored, even though the low pHinduced structural changes seemed to be partially reversible. In a previous study with SINV,40 cryoelectron microscopy also revealed dramatic changes in the virus structure as it was moved to pH 5.3 and then returned to pH 7.2, involving the appearance of a prominent protruding structure at the virus fivefold axis upon exposure to acid pH and its disappearance upon return to neutral pH. Since the return to neutral pH also eliminated the tendency for SINV to clump at low pH, it was suggested that such protruding structure contained hydrophobic domains – which correlates with the data from our bis-ANS binding assay. Similar to what we observed for MAYV, SINV reneutralization also caused the virus structure to revert partially to the structure seen before its acidification, except that large fissures were created along the virus twofold axis. 17 ACS Paragon Plus Environment

Biochemistry

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It is possible that bis-ANS stabilizes some regions of E1 to remain in the hydrophobic state, even though binding of bis-ANS to proteins is non-covalent and involves a combination of electrostatic and hydrophobic modes41. Moreover, some investigators have noted that the dye-binding event itself may induce protein conformational changes, indicating the advisability of correlating bis-ANS fluorescence measurements with data obtained using other physical techniques42. However, our analysis of the intrinsic fluorescence from Trp residues, which was performed in the absence of bisANS, also revealed an incomplete reversibility of the conformational changes of viral envelope proteins upon reneutralization following acidification in the absence of DRMs, thus suggesting that they were not stuck in an intermediate hydrophobic state due to bis-ANS binding. As suggested by our data, the final state of MAYV envelope proteins after the acidificationneutralization procedure presumably does not represent neither the acid-induced homotrimer nor the native dimer, but a structured intermediate state that retains the fusogenic ability. It is noteworthy that induction of fusogenic intermediate states in viral proteins was already achieved using high hydrostatic pressure, but they did not retain the ability of the particles to efficiently infect cells.19,30 Our results show that MAYV pre-exposed to acidic pH and returned to neutral pH not only remains infectious, but presents non-significant changes in the infectious titer as compared to the native non-exposed virus. Interestingly, subsequent reacidification is still required for the viral fusion reaction with target membranes, showing that low pH remains the cue for triggering the virus entry program. Regarding this low pH dependence for fusion, a previous study revealed that MAYV is still able to efficiently fuse with liposomes at pH values as low as 4.5 – only from this point down there is a dramatic drop in virus fusion ability, an event probably related to protein denaturation.31 Membrane binding seems to be an essential step to direct the reaction to the formation of the final fusogenic conformation of the viral envelope proteins. Based on our data with MAYV, we propose that, when a target membrane is not present, the trimerization of E1 upon acidification does not 18 ACS Paragon Plus Environment

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Biochemistry

occur and the protein is trapped in an energy minimum (Figure 5). As described before, this would be neither the native heterodimeric (E1/E2) nor the final homotrimeric (E13) state, but a stable intermediate state of the fusion reaction.39 A previous work with SFV suggested that such intermediate state would present a different conformation from monomeric E1,43 and a previous work with SINV suggested that it might involve a pentameric E1 structure.40 Here we show that reneutralization of the pH leads to conformational changes that hide some of the hydrophobic sites exposed by acidic pH. Under these conditions, E1 is in a non-native, non-fusogenic state (E1') that retains the characteristics necessary for binding, inducing fusion and thus infecting host cells, but there would still be an energy barrier between E1' and E13. Low pH is necessary, but not enough, to overcome this barrier and achieve the fusogenic E13 conformation – binding to the target membrane is the other requirement to achieve homotrimer formation. Acidified E1 is different from E1', and both are different from E1/E2, but only E1' and E1/E2 are able to form E13.

Figure 5. Proposed model for the energetics of the conformational changes in alphavirus glycoproteins. Energy transitions when the virus particle is directly allowed for membrane binding (MB) at low pH (solid line) are substantially different from those when the virus particle is first exposed to low pH, 19 ACS Paragon Plus Environment

Biochemistry

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reneutralized, and then allowed for membrane binding at low pH (dotted line). In the latter case, E1 is first trapped in an energy minimum between the native heterodimeric (E1/E2) and the final homotrimeric (E13) structure, and then move to a non-native, non-fusogenic state (E1') before reaching the fusion-competent state (see body text for further details). It is proposed in the literature that the role of E2 is to protect E1 from the acidic environment during transport inside the host cell.44 However, E2 remains bound to E1 even after virus particle assembly ad release – i.e., when this function is no longer necessary. During the cell entry process, the low pH leads to the dissociation of the E1/E2 heterodimer and allows the interaction of E1 with the target membrane. E2, which is responsible for receptor binding, may serve to position the virus particle precisely in relation to the target membrane to optimize such interaction.40 Dissociation is a necessary step in the process, but not a determinant of the fusogenic function.44 Our data suggest that an E2dissociated form of E1 is perfectly competent for the membrane fusion process, but E1 only triggers the fusion process if bound to the target membrane and after its acidification. It is not possible through our data to determine whether membrane binding must necessarily precede acidification, but binding of bis-ANS in the presence of DRMs showed that hydrophobic segments of the viral envelope proteins are isolated from interaction with the probe when the particles interact with the target membrane. This suggests that only acidification in the presence of membrane interactions results in triggering of the process necessary for the fusion of the envelope to occur efficiently. Indeed, using a supported lipid bilayer assay instead of a bulk solution-based fusion assay, a previous work showed that SINV does not bind receptor-free membranes at neutral pH, but, under acidic conditions, the delay between membrane binding and lipid mixing is less than half a second.45 The successful crystallization of low pH conformations of trimers of SFV fusion proteins was shown to require the presence of liposomes and lipid-like detergents,46 also suggesting that the virus might require exposure to lipids before the acidiﬁcation. Further investigations are required to determine if 20 ACS Paragon Plus Environment

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Biochemistry

previous binding to the membrane already causes some conformational change in the viral envelope proteins. Unlike class I and class II viral fusion proteins (e.g., HA protein found on orthomyxoviruses and E1 protein found on alphaviruses, respectively), for which the structural rearrangement is proposed to be effectively irreversible, class III viral fusion proteins (e.g., G protein found on rhabdoviruses) can undergo a reversible conformational change.47 Altogether, our data suggest that class II viral fusion proteins may, however, undergo partially reversible structural rearrangements, thus behaving as a middle ground between class I and class III viral fusion protein in relation to such conformational changes. This would prevent the fusion protein to act as a "suicide enzyme" (which undergoes an irreversible priming step and acts only once), allowing it to recover its fusogenic capacity if triggered prematurely.

AUTHOR INFORMATION Present Addresses †

Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ 21040-900, Brazil. §Seção de

Arbovirologia e Febres Hemorrágicas, Instituto Evandro Chagas, Secretaria de Vigilância em Saúde, Ministério da Saúde, Ananindeua, PA 67030-000, Brazil. #Instituto de Tecnologia em Imunobiológicos, Fundação Oswaldo Cruz, Rio de Janeiro, RJ 21040-900, Brazil. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources This work was supported by an international grant from International Centre for Genetic Engineering and Biotechnology (ICGEB) and by Brazilian grants from Coordenação de Aperfeiçoamento de 21 ACS Paragon Plus Environment

Biochemistry

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Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Programa de Apoio ao Desenvolvimento Científico e Tecnológico (PADCT), and Programa de Apoio a Núcleos de Excelência (PRONEX). Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We are grateful to Emerson R. Gonçalves for competent technical assistance. Authors thank Prof. Jerson Lima Silva for his support and insightful discussions of the work.

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For Table of Contents Use Only

Fusion of a New World Alphavirus with Membrane Microdomains Involving Partially Reversible Conformational Changes in the Viral Spike Proteins Ivanildo P. Sousa Jr., Carlos A. M. Carvalho, Ygara S. Mendes, Gilberto Weissmuller, Jerson L. Silva, Andréa C. Oliveira, and Andre M. O. Gomes


Fusion of a New World Alphavirus with Membrane Microdomains

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