Dengue and Zika virus capsid proteins bind to membranes and self-assemble into liquid droplets with nucleic acids

Abstract

Dengue virus (DENV) and Zika virus (ZIKV) capsid proteins efficiently recruit and surround the viral RNA at the endoplasmic reticulum (ER) membrane to yield nascent viral particles. However, little is known either about the molecular mechanisms by which multiple copies of capsid proteins assemble into nucleocapsids (NCs) or how the NC is recruited and wrapped by the ER membrane during particle morphogenesis. Here, we measured relevant interactions concerning this viral process using purified DENV and ZIKV capsid proteins, membranes mimicking the ER lipid composition, and nucleic acids in in vitro conditions to understand the biophysical properties of the RNA genome encapsidation process. We found that both ZIKV and DENV capsid proteins bound to liposomes at liquid-disordered phase regions, docked exogenous membranes, and RNA molecules. Liquid–liquid phase separation is prone to occur when positively charged proteins interact with nucleic acids, which is indeed the case for the studied capsids. We characterized these liquid condensates by measuring nucleic acid partition constants and the extent of water dipolar relaxation, observing a cooperative process for the formation of the new phase that involves a distinct water organization. Our data support a new model in which capsid–RNA complexes directly bind the ER membrane, seeding the process of RNA recruitment for viral particle assembly. These results contribute to our understanding of the viral NC formation as a stable liquid–liquid phase transition, which could be relevant for dengue and Zika gemmation, opening new avenues for antiviral intervention. Dengue virus (DENV) and Zika virus (ZIKV) capsid proteins efficiently recruit and surround the viral RNA at the endoplasmic reticulum (ER) membrane to yield nascent viral particles. However, little is known either about the molecular mechanisms by which multiple copies of capsid proteins assemble into nucleocapsids (NCs) or how the NC is recruited and wrapped by the ER membrane during particle morphogenesis. Here, we measured relevant interactions concerning this viral process using purified DENV and ZIKV capsid proteins, membranes mimicking the ER lipid composition, and nucleic acids in in vitro conditions to understand the biophysical properties of the RNA genome encapsidation process. We found that both ZIKV and DENV capsid proteins bound to liposomes at liquid-disordered phase regions, docked exogenous membranes, and RNA molecules. Liquid–liquid phase separation is prone to occur when positively charged proteins interact with nucleic acids, which is indeed the case for the studied capsids. We characterized these liquid condensates by measuring nucleic acid partition constants and the extent of water dipolar relaxation, observing a cooperative process for the formation of the new phase that involves a distinct water organization. Our data support a new model in which capsid–RNA complexes directly bind the ER membrane, seeding the process of RNA recruitment for viral particle assembly. These results contribute to our understanding of the viral NC formation as a stable liquid–liquid phase transition, which could be relevant for dengue and Zika gemmation, opening new avenues for antiviral intervention. Proteins in solution can phase separate into liquid compartments by intermolecular interactions with nucleic acids. Such transitions occur with a first seed formed either in bulk or at the interface of substrates/soluble components, leading to a new liquid entity that displays different physical properties compared with those of its surroundings. The nucleolus is the paradigm of these liquid compartments, nowadays known as membrane-less organelles, but already described in the 1830s (see Refs. (1Uversky V. Finkelstein A. Life in phases: Intra- and inter- molecular phase transitions in protein solutions.Biomolecules. 2019; 9: 842Crossref Scopus (19) Google Scholar, 2Alberti S. Gladfelter A. Mittag T. Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates.Cell. 2019; 176: 419-434Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar) and references therein). It is accepted that one of the in vivo–in vitro interaction that triggers liquid–liquid phase separation is the association of RNA with proteins. One mechanism is by enabling multimerization of the RNA-binding protein (3Rhine K. Vidaurre V. Myong S. RNA droplets.Annu. Rev. Biophys. 2020; 49: 247-265Crossref PubMed Scopus (28) Google Scholar). Furthermore, there are several reports indicating that proteins can also self-phase separate into liquid phases at the lipid bilayer interface (4Feng Z. Chen X. Wu X. Zhang M. Formation of biological condensates via phase separation: Characteristics, analytical methods, and physiological implications.J. Biol. Chem. 2019; 294: 14823-14835Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The capsid proteins of dengue virus (DENV) and Zika virus (ZIKV) interact with viral RNA, forming a nucleocapsid (NC), in the process of viral genome packaging. This process is called encapsidation, and up to date, a clear notion concerning the molecular mechanisms behind this phenomenon is not fully understood (5Byk L.A. Gamarnik A.V. Properties and functions of the dengue virus capsid protein.Annu. Rev. Virol. 2016; 3: 263-281Crossref PubMed Scopus (72) Google Scholar, 6Tan T.Y. Fibriansah G. Kostyuchenko V.A. Ng T.S. Lim X.X. Zhang S. Lim X.N. Wang J. Shi J. Morais M.C. Corti D. Lok S.M. Capsid protein structure in Zika virus reveals the flavivirus assembly process.Nat. Commun. 2020; 11: 895Crossref PubMed Scopus (31) Google Scholar). DENV and ZIKV are relevant mosquito-borne human pathogens of the Flavivirus genus. Upon infection, the positive-stranded RNA genome is directly used as a messenger for translating a large polyprotein, which is proteolytically processed at the endoplasmic reticulum (ER) membrane. The viral capsid is the first protein encoded in the open reading frame and remains inserted into the ER membrane by a transmembrane anchor peptide that links capsid to the pre-Membrane (prM) protein. Two successive cleavages exerted by cellular and viral proteases are necessary for capsid release. The viral protease NS2B/NS3 is responsible for cleaving the anchor peptide, permitting the release of the mature capsid protein at the cytoplasmic side (5Byk L.A. Gamarnik A.V. Properties and functions of the dengue virus capsid protein.Annu. Rev. Virol. 2016; 3: 263-281Crossref PubMed Scopus (72) Google Scholar, 6Tan T.Y. Fibriansah G. Kostyuchenko V.A. Ng T.S. Lim X.X. Zhang S. Lim X.N. Wang J. Shi J. Morais M.C. Corti D. Lok S.M. Capsid protein structure in Zika virus reveals the flavivirus assembly process.Nat. Commun. 2020; 11: 895Crossref PubMed Scopus (31) Google Scholar). Capsid proteins recruit the newly synthesized viral genome to form the NC complex, which subsequently buds into the ER lumen gaining the lipid bilayer together with the viral proteins envelope (E) and prM (5Byk L.A. Gamarnik A.V. Properties and functions of the dengue virus capsid protein.Annu. Rev. Virol. 2016; 3: 263-281Crossref PubMed Scopus (72) Google Scholar). How NC is recruited to the ER membrane is under extensive scrutiny. So far, high-resolution microscopy techniques such as cryo-EM do not provide compelling evidence that the capsid or NC is physically associated to the inner leaflet of the viral lipid membrane (7Prasad V.M. Miller A.S. Klose T. Sirohi D. Buda G. Jiang W. Kuhn R.J. Rossmann M.G. Structure of the immature Zika virus at 9 A resolution.Nat. Struct. Mol. Biol. 2017; 24: 184-186Crossref PubMed Scopus (95) Google Scholar, 8Kuhn R.J. Zhang W. Rossmann M.G. Pletnev S.V. Corver J. Lenches E. Jones C.T. Mukhopadhyay S. Chipman P.R. Strauss E.G. Baker T.S. Strauss J.H. Structure of dengue virus: Implications for flavivirus organization, maturation, and fusion.Cell. 2002; 108: 717-725Abstract Full Text Full Text PDF PubMed Scopus (1137) Google Scholar, 9Kostyuchenko V.A. Lim E.X. Zhang S. Fibriansah G. Ng T.S. Ooi J.S. Shi J. Lok S.M. Structure of the thermally stable Zika virus.Nature. 2016; 533: 425-428Crossref PubMed Scopus (332) Google Scholar). To attempt to answer these questions, we use fluorescence methods to measure the interaction of both DENV and ZIKV capsids with ER-mimicking lipid membranes in the absence or in the presence of nucleic acids. We describe how ZIKV and DENV capsid proteins interact with liposomes and RNA molecules. We measured the protein–membrane binding and the ability of DENV and ZIKV capsids to recruit ssDNA or RNAs and liposomes onto the interface of giant unilamellar vesicles (GUVs). The capsid–RNA–membrane association was found at regions corresponding to liquid disordered (Ld) phase. Also, the viral protein–RNA interaction generated phase-separated liquid droplets, with clear-cut changes on water organization within the droplets respect to their surroundings. We further characterized this process by computing an apparent dissociation constant for the nucleic acids–protein interaction, together with the determination of the cooperativity of the process. To investigate whether DENV and ZIKV capsid proteins interact with membranes, we used different fluorescence methods. We first monitored the characteristics of the fluorescence emission spectra of a single tryptophan (W) contained in the proteins (highlighted in Fig. 1, A and D) in the absence and presence of ERmix large unilamellar vesicles (LUVs). W fluorescence emission properties are well known to depend on the polarity of the environment (10Lakowicz J.R. Principles of Fluorescence Spectroscopy.3rd Ed. Springer, New York; Berlin2006Crossref Scopus (16527) Google Scholar). The W emission spectra of both DENV and ZIKV capsids show a shift toward shorter wavelengths (Fig. 1, B and E) in the presence of LUVs, which is quantified by the ratio of the fluorescence intensities at 337 and 346 nm (Fig. 1, C and F). This indicates that upon membrane interaction, the W senses a less polar milieu. The polarity change can be associated to a protein structural rearrangement, to a new environment because of the proximity of the amino acid to the membrane, or both. To answer this question, we took advantage of the well-known energy transfer effect between W and the nitrobenzoxadiazole (NBD) dye, represented in Figure 1G (11Clausell A. Rabanal F. Garcia-Subirats M. Asuncion Alsina M. Cajal Y. Membrane association and contact formation by a synthetic analogue of polymyxin B and its fluorescent Chem. PubMed Scopus Google Scholar). The fluorescence of upon of ERmix is at the interface of the with of DENV and ZIKV capsid proteins (Fig. 1, and These data that proteins bind to membranes, and their are to the lipid bilayer interface nm A. J. M. and lipid of protein in Biophys. PubMed Scopus Google to transfer energy to the interaction was either by measuring shift of ERmix The of and nucleic acid interaction in the partition properties of to Biophys. 2020; PubMed Scopus Google Scholar) or by lipid the a change in the of the was that protein interaction with the liposomes a To further whether this process lipid between different liposomes or of we a using the as in Figure (see for Upon of either DENV or ZIKV capsid proteins to the an of fluorescence about the to was indicating lipid this change on of the with respect to (Fig. These data support the that is upon protein–membrane to about this interaction, we used fluorescence microscopy to directly the membrane between ERmix and ERmix with in the absence or the presence of viral capsid proteins. the absence of proteins, there was at the membrane of (Fig. However, upon of DENV (Fig. or ZIKV (Fig. there is a clear recruitment and of at the membrane a of the was DENV and ZIKV capsids form the NC by binding to the viral RNA is how the NC is recruited into the nascent viral particle at the ER membrane. The NC in contact with ER membranes for particle but is whether membrane interaction is a in this To further the of the interaction with lipid membranes of the DENV and ZIKV capsid proteins, we an ERmix with a ssDNA or in the presence or the absence of such proteins (Fig. their the nucleic acid was (Fig. in their the nucleic acid was recruited at the membrane interface of (Fig. C and in the the recruitment was not on the of the and to either bound to or by membranes (Fig. C and when and ssDNA the nucleic acid and liposomes both to the interface of by the capsid proteins (Fig. and protein Furthermore, to the the capsid proteins to the of ERmix RNA and LUVs, the at the interface of that the viral proteins and are contained in these (Fig. and These are relevant because that DENV and ZIKV capsid proteins can recruit nucleic acids and membranes onto the interface of a membrane. when the are at regions corresponding to phases Fig. a membrane association when capsid proteins recruit The that the process involves a large of as a or a and is when nucleic acids are These liquid condensates as for several when positively charged and charged interact in the of 2020; PubMed Google Scholar, interaction of PubMed Scopus Google Scholar, Scopus Google Scholar). nucleic acids interact with the viral capsid proteins, we an of and to the those to be associated to either or RNA can be the fluorescence a (Fig. A and and DENV with an of in the ZIKV an of their and these of condensates to nucleic liquid droplets (3Rhine K. Vidaurre V. Myong S. RNA droplets.Annu. Rev. Biophys. 2020; 49: 247-265Crossref PubMed Scopus (28) Google Scholar). we further the process and the and properties of the new phase. the assembly can be by the of the This is in the at the in Figure A and at the of the either DENV or ZIKV capsid proteins and is indicating the presence of that can this effect is this with a well-known liquid S. J. D. E. M. phase separation of the of the protein Commun. 2017; PubMed Scopus Google are in with the of the phase separation by RNA or these can recruit an protein. This is the case when is into a (Fig. by the fluorescent of the capsid (Fig. and but also newly formed droplets together or an already seed (Fig. This of is a for such to be a of several (3Rhine K. Vidaurre V. Myong S. RNA droplets.Annu. Rev. Biophys. 2020; 49: 247-265Crossref PubMed Scopus (28) Google Scholar). We also whether the extent of water dipolar is different in this new phase compared with that in bulk this we used the fluorescent properties depend on the dipolar of water in their L.A. The use of as of biological on A to Google Scholar, G. and properties of a fluorescent PubMed Scopus Google a to the of Figure and the change of the fluorescence emission of at the of DENV and ZIKV with respect to the measured by fluorescence is a shift of the emission (Fig. nm regions of the is the to nm (DENV) and (ZIKV) regions of the is at the indicating an change of the extent of water in the is the of the found for ZIKV compared with those DENV a to water by capsid the To into the of the liquid–liquid phase for the nucleic interaction, we took advantage of fluorescence of an in the of the can a in to Scopus Google an effect that can our results in the are Figure the change upon with DENV or ZIKV capsids. these the of nucleic acid into the new phase is This not to relevant of the process constant and but also to understand its cooperativity by using the (see data to the of formation upon protein (Fig. and E) that the association and the process is a cooperative are (Fig. C and F). We apparent dissociation constants in the of in with those for for of proteins by the S. J. Y. D. Z. on the interface of nucleic acid and binding PubMed Google Scholar). these the energy of the process both for and RNA with DENV and ZIKV can be (Fig. C and F). Our data show an process in both of dengue and Zika viral at the membrane of the ER is a viral RNA is by capsid proteins and wrapped by the ER membrane the So far, the NC and ER association is to be by interaction with prM and proteins at this (5Byk L.A. Gamarnik A.V. Properties and functions of the dengue virus capsid protein.Annu. Rev. Virol. 2016; 3: 263-281Crossref PubMed Scopus (72) Google Scholar, Y. A. interactions and RNA structure within virus using 2020; PubMed Scopus Google Scholar). data using ZIKV virus that the transmembrane anchor peptide the capsid protein be on at the ER membrane, interaction with transmembrane regions of prM and proteins, and of particle assembly T.Y. Fibriansah G. Lok S.M. Capsid protein is to the of flavivirus 2020; PubMed Scopus Google Scholar). the viral particle a and liquid phase as the of the on how this phase is and how is at the is to virus and in the viral can be this we studied how DENV and ZIKV capsids can interact with membrane model that the ER lipid and the effect of the presence of and liposomes this We that both viral capsids can bind liposomes to membrane as a of membrane this membrane recruitment is together with binding onto the interface of giant The associated capsids on the lipid bilayer both and nucleic acids, a membrane at the of giant is that the at the of a lipid physical for a viral particle we describe how DENV and ZIKV capsids phase separate when bind to or this we that the new phases a that can recruit such as the fluorescent of DENV capsid or nucleic acids. the extent of water dipolar these droplets with we a between DENV and ZIKV the extent of water by in the case of DENV droplets is with respect to ZIKV These be to structural capsid (7Prasad V.M. Miller A.S. Klose T. Sirohi D. Buda G. Jiang W. Kuhn R.J. Rossmann M.G. Structure of the immature Zika virus at 9 A resolution.Nat. Struct. Mol. Biol. 2017; 24: 184-186Crossref PubMed Scopus (95) Google Scholar, 8Kuhn R.J. Zhang W. Rossmann M.G. Pletnev S.V. Corver J. Lenches E. Jones C.T. Mukhopadhyay S. Chipman P.R. Strauss E.G. Baker T.S. Strauss J.H. Structure of dengue virus: Implications for flavivirus organization, maturation, and fusion.Cell. 2002; 108: 717-725Abstract Full Text Full Text PDF PubMed Scopus (1137) Google Scholar, 9Kostyuchenko V.A. Lim E.X. Zhang S. Fibriansah G. Ng T.S. Ooi J.S. Shi J. Lok S.M. Structure of the thermally stable Zika virus.Nature. 2016; 533: 425-428Crossref PubMed Scopus (332) Google Scholar) in of these proteins, which in their ability to structure water A new for the of water and for of water Chem. Google Scholar). 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