Variability Between the Degrees of Maturation in Individual Dengue Virions
- Leonid V. Chernomordik, PhD, Head, Section on Membrane Biology
- Eugenia Leikina, DVM, Senior Research Assistant
- Kamram Melikov, PhD, Staff Scientist
- Elena Zaitseva, PhD, Staff Scientist
- Santosh K. Verma, PhD, Visiting Fellow
- Berna Uygur, PhD, Postdoctoral Intramural Research Training Award Fellow
- Harold Shin, BS, Postbaccalaureate Fellow
Disparate membrane remodeling reactions are tightly controlled by protein machinery but are also dependent on the lipid composition of the membranes. Whereas each kind of protein has its own individual personality, membrane lipid bilayers have rather general properties manifested by their resistance to disruption and bending. Our long-term goal is to understand how proteins break and reseal membrane lipid bilayers in important cell biology processes, such as membrane fusion and crossing cell membranes by water-soluble drugs on their way to intracellular targets. We expect that the analysis of the molecular mechanisms of different membrane rearrangements will clarify the generality of emerging mechanistic insights. Better understanding of these mechanisms will bring about new ways of controlling them and lead to new strategies for quelling diseases involving cell invasion by enveloped viruses, intracellular trafficking, and intercellular fusion.
In addition to our on-going work on poorly understood examples of cell-to-cell fusion processes, such as fusion stages of formation of multinucleated myotubes and osteoclasts, where even identities of the proteins involved remain to be clarified, in a recent study we focused on one of the best characterized fusion machineries, dengue virus envelope (E) protein. The important human pathogen dengue virus is a mosquito-borne single positive-stranded RNA virus, which enters cells via fusion between viral envelope and endosomal membrane. The protein network at the surface of the dengue viral particle is formed by the E protein and either membrane (M) protein in mature virus or prM, the uncleaved precursor of M in the immature virus. We examined the variability between the degrees of maturation in different virions within the same population.
The human pathogen dengue virus and its maturation
Dengue virus (DENV) causes the most prevalent mosquito-borne viral disease, with an estimated 3.6 billion people living in risk areas. Control of the growing threat of DENV is hindered by the lack of effective therapies and vaccines. Newly assembled DENV particles undergo maturation to become infectious. The proteins present on the surface of the dengue immature virus are the envelope (E) protein, which is responsible for viral entry, and prM, which consists of an N-terminal pr domain followed, via a furin cleavage site, by the M protein. Interactions between the pr portion of prM and E-protein prevent premature viral fusion or inactivation in the acidic intracellular compartments through which virions traffic. The mature DENV envelope contains E protein and M protein, a cleaved derivative of prM. The maturation is required for formation of pre-fusion conformation of E-protein. Following E-protein binding to cell surface receptors, the virus is internalized by endocytosis.
In earlier studies, we dissected the pathway of DENV entry and fusion and identified an anionic lipid specific for late endosomes as an essential lipid cofactor of E-protein fusion machinery. Our findings explained why DENV fuses not in early but in late endosomes, where a combination of acidic pH and anionic lipids facilitates fusogenic restructuring of E from homodimer to homotrimer, which brings about fusion between viral envelope and endosomal membrane and thus effectively delivers viral RNA into cytosol in the vicinity of the translation-replication sites. Our work also explained why earlier attempts by different laboratories to develop fusion assays for DV fusion failed. We proceeded to develop an arsenal of high-throughput assays for DENV cell-surface binding, internalization, and intracellular fusion.
In our most recent study, in collaboration with Leonid Margolis’s lab, we focused on the surface protein composition of DENV particles and developed an assay to characterize the maturation status of individual virions. We examined whether there are any DENV particles that are fully matured, i.e., in which all prM molecules are cleaved, or all the virions are either fully immature with all prM molecules in uncleaved form or “mosaic”, with the same virions carrying both M protein and prM. To answer this question, we analyzed these proteins on the surface of single virions using a novel high-throughput flow-virometry technique to characterize the degree of maturation of individual virions in a preparation of infectious DENV. We labeled DENV with a fluorescent lipid probe and then with fluorescent antibody to prM protein, and captured labeled virions (operationally defined as a membrane particle that carries E protein) with small MNPs coupled with an antibody against E protein. We then separated the DENV–MNPs complexes from unbound antibodies using magnetic columns and eluted DENV–MNPs complexes from the columns. Finally, we analyzed the eluted complexes with a flow cytometer. We found that, in the DENV population produced by BHK-21 cells, approximately half the virions are fully mature as no prM could be detected on their surface. In contrast, furin-deficient LoVo cells produced a DENV population in which only about 15% are mature virions. Detailed characterization of the maturation status of DENV released by infected cells is especially important given that immature DENV has been found to play a significant role in disease pathogenesis. It is worth noting that our approach characterizes the variability between virions within the same population and thus complements the existing bulk techniques, which describe the composition and properties of an average virion in the preparation. Given that the heterogeneity of viral particles may impact their biological properties, we expect that the high throughput approach we developed will help produce a vaccine and antivirals against DENV and possibly against other viruses.
Additional Funding
- United States - Israel Binational Science Foundation (BSF) grant “Machinery of myoblast fusion” 2015-2019
- NICHD FY16 NICHD Intramural DIR/DIPHR HIV/AIDS Research Award, 2016, 2017
Publications
- Kozlov MM, Chernomordik LV. Membrane tension and membrane fusion. Curr Opin Struct Biol 2015;33:61-67.
- Chernomordik LV, Kozlov MM. Myoblast fusion: playing hard to get. Dev Cell 2015;32:529-530.
- Zicari S, Arakelyan A, Fitzgerald W, Zaitseva E, Chernomordik LV, Margolis L, Grivel J-Ch. Evaluation of the maturation of individual dengue virions with flow virometry. Virology 2016;488:20-27.
- Leikina E, Defour A, Melikov K, Van der Meulen JH, Nagaraju K, Bhuvanendran S, Gebert C, Pfeifer K, Chernomordik LV, Jaiswal JK. Annexin A1 deficiency does not affect myofiber repair but delays regeneration of injured muscles. Sci Rep 2015;5:18246.
Collaborators
- Anush Arakelyan, PhD, Section on Intercellular Interactions, NICHD, Bethesda, MD
- Aurelia Defour, PhD, Children's National Medical Center, Washington, DC
- Claudia M. Gebert, PhD, Section on Genome Imprinting, NICHD, Bethesda, MD
- Jyoti Jaiswal, PhD, George Washington University School of Medicine and Health Sciences, Washington, DC
- Michael M. Kozlov, PhD, DHabil, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
- Leonid Margolis, PhD, Section on Intercellular Interactions, NICHD, Bethesda, MD
- Karl Pfeifer, PhD, Section on Genomic Imprinting, NICHD, Bethesda, MD
- Benjamin Podbilewicz, PhD, Technion-Israel Institute of Technology, Haifa, Israel
- Sonia Zicari, PhD, Section on Intercellular Interactions, NICHD, Bethesda, MD
Contact
For more information, email chernoml@mail.nih.gov or visit irp.nih.gov/pi/leonid-chernomordik.
